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NIR Spectroscopic Properties of Aqueous Acids Solution
Acid content is one of the important quality attributes in determining the maturity index of agricultural product, particularly fruits. Despite the fact that much research on the measurement of acidity in fruits through non-destructive spectroscopy analysis at NIR wavelengths between 700 to 1,000 nm has been conducted, the same response towards individual acids is not well known. This paper presents NIR spectroscopy analysis on aqueous citric, tartaric, malic and oxalic solutions through quantitative analysis by selecting a set of wavelengths that can best be used to measure the pH of the solutions. The aquaphotomics study of the acid solutions has generated R 2 above 0.9 for the measurement of all acids. The most important wavelengths for pH are located at 918-925 nm and 990-996 nm, while at 975 nm for water.
# Introduction
Acidity and soluble solids content are the common quality attributes that are associated with the maturity index of agricultural products, especially fruits. The measurement of these attributes assists in determining appropriate harvesting time, methods for postharvest handling and supply chain management. Non-destructive spectroscopy measurements of these attributes have been applied widely by researchers worldwide. Nicolai et al. [bib_ref] Nondestructive measurement of fruit and vegetable quality by means of NIR spectroscopy:..., Nicolai [/bib_ref] have summarised spectroscopic measurement results and analysis that have been conducted on various types of fruits and vegetables over the last two decades.
## Open access
The following are the outline of the advantages of wavelengths between 700 nm and 1,100 nm (the range of NIR wavelengths that have been used in this research) for quality measurements of intact fruits [bib_ref] Measurement of Soluble Solids in Cherry and Apricot by PLS Regression and..., Carlini [/bib_ref] [bib_ref] Firmness, dry matter and soluble solids assessment of postharvest kiwifruit by NIR..., Mcglone [/bib_ref] [bib_ref] Application of commercially available, low-cost, miniaturized NIR spectrometers to the assessment of..., Walsh [/bib_ref] [bib_ref] Practical NIR spectroscopy with applications, Osborne [/bib_ref] [bib_ref] Distinguishing Carrot's Characteristics by Near Infrared (NIR) Reflectance and Multivariate Data Analysis, Abu-Khalaf [/bib_ref].
(1) Penetration of the radiation into sample is deeper than other ranges. [bib_ref] Measurement of Soluble Solids in Cherry and Apricot by PLS Regression and..., Carlini [/bib_ref] Water absorbance peaks are less strong and broad than in other ranges and the risk of masking spectral information correlated to low concentration constituents is low. The cost of instrumentation for this range is relatively low, it is portable and suitable for process control and for in situ field measurements. [bib_ref] Application of commercially available, low-cost, miniaturized NIR spectrometers to the assessment of..., Walsh [/bib_ref] The bands are ascribed to the third and forth overtones of O-H and C-H stretching modes and are expected to be separated due to anharmonicity (approximately around 700-800 nm [bib_ref] Theory and application of near infrared reflectance spectroscopy in determination of food..., Cen [/bib_ref] [bib_ref] Non-destructive texture analysis of farmed Atlantic salmon using visual/near-infrared reflectance spectroscopy, Isaksson [/bib_ref] [bib_ref] Prediction of gasoline octane numbers from near-infrared spectral features in the range..., Kelly [/bib_ref]. [bib_ref] Practical NIR spectroscopy with applications, Osborne [/bib_ref] Lower absorbance at these wavelengths allows transmission optics. [bib_ref] Distinguishing Carrot's Characteristics by Near Infrared (NIR) Reflectance and Multivariate Data Analysis, Abu-Khalaf [/bib_ref] Moreover, there is strong evidence, that the range from 700-900 nm constitutes a "diagnostic window" in which chemical compositions of samples can be investigated.
For soluble solids content in fruits, the major measurand is the mixture of sugars (sucrose, glucose and fructose) and water. There are various NIR spectroscopy experiments that have been conducted for characterising water-sugar solutions such as those by Bakier [bib_ref] Application of NIR Spectroscopy for the Analysis of Water-Carbohydrate Interactions in Water..., Bakier [/bib_ref] , Giangiacomo [bib_ref] Study of water-sugar interactions at increasing sugar concentration by NIR spectroscopy, Giangiacomo [/bib_ref] , Buning-Pfaue [bib_ref] Analysis of water in food by near infrared spectroscopy, Buning-Pfaue [/bib_ref] , Rodriguez-Saona [bib_ref] Rapid analysis of sugars in fruit juices by FT-NIR spectroscopy, Rodriguez-Saona [/bib_ref] , Berentsen et al. [bib_ref] Analysis of aqueous solutions by near-infrared spectrometry (NIRS) IV. One-and two-component systems..., Berentsen [/bib_ref] , Rambla et al. [bib_ref] PLS-NIR determination of total sugar, glucose, fructose and sucrose in aqueous solutions..., Rambla [/bib_ref] and Luck [bib_ref] Infrared overtone region, Luck [/bib_ref]. However, despite the fact acidity is one of the most important attributes for the agricultural industry and there are a wide range of spectroscopy experiments that have been conducted in evaluating food acidity, the NIR spectroscopy characteristics of simple water-acid solutions are not well known. For instance, Gonzalez-Caballero et al. [bib_ref] First steps towards the development of a non-destructive technique for the quality..., Gonzalez-Caballero [/bib_ref] have conducted spectroscopy experiments for the measurement of grape acidity (pH). According to them, the NIR wavelengths with the greatest weight for acid measurement are 768 nm, which is associated with the C-H stretch fourth overtones, and 986 nm, which is associated to the second overtone of O-H in sugars. In another experiment, Shao and He [bib_ref] Nondestructive measurement of the internal quality of bayberry juice using Vis/NIR spectroscopy, Shao [/bib_ref] claimed that 910-925 nm is an important wavelength range for acidity prediction in bayberry juice. Liu et al. [bib_ref] Feasibility of the use of visible and near infrared spectroscopy to assess..., Liu [/bib_ref] have applied NIR spectroscopy to assess the pH of rice wines. From their work, 920 nm and 990-995 nm are the most suitable wavelengths for pH measurement. On the other hand, there are also several studies on the measurement of organic acids that have been conducted on fruit juices such as a research conducted by Xie et al. [bib_ref] Application of principal component-radial basis function neural networks (PC-RBFNN) for the detection..., Xie [/bib_ref] where they have applied high performance liquid chromatography (HPLC) and NIR spectroscopy between 800-2,400 nm for the measurement of citric and malic acids to detect water-adulterated bayberry juice. Likewise, Li et al. [bib_ref] Quantitative analysis of individual sugars and acids in orange juices by near-infrared..., Li [/bib_ref] have applied standard enzymatic assays techniques and NIR spectroscopy between 1,100-2,500 nm for the measurement of citric acid in orange juices.
In this paper, the NIR spectroscopy analysis has been conducted for the measurement of pH of aqueous acid solutions. The main objective of this research is in identifying the combination of wavelengths that can best be used for the measurement of various acids that are commonly found in fruits, which for this research are citric (C 6 H 8 O 7 ), tartaric (C 4 H 6 O 6 ), malic (C 4 H 6 O 5 ) and oxalic (C 2 H 2 O 4 ) acid. lists the common types of organic acids that are naturally present in fruits. Acids which occur in considerable quantities are shown in italics. In grape juice, for instance, the predominant organic acids are tartaric and malic acids, while succinic and citric acids are present in minor proportions. Organic acids with low molecular weight are an important group of compounds in juices for their influence on organoleptic properties such as flavour, colour and aroma and in the stability and microbiologic control of fruits beverages [bib_ref] A review of the analytical methods to determine organic acids in grape..., Mato [/bib_ref]. The changes in fruits organics acids are useful for checking their maturation processes [bib_ref] Distribution and effect of grape maturity on organic acid content of red..., Lamikanra [/bib_ref]. . Organic acids that are naturally present in select fruits.
## Fruits
Acids Fruits Acids Apples
Malic, Citric According to Tsenkova [bib_ref] Aquaphotomics: Water in the biological and aqueous world scrutinised with invisible light, Tsenkova [/bib_ref] NIR spectra are highly influenced by pH and contain physical information such as contributions from scattering. NIR application in biological systems with high water contents such as fruits (above 80%) are strongly dominated by the spectral signature of water and will thus have peak absorptions at 760, 970, 1,190, 1,450 and 1,940 nm [bib_ref] Application of NIR Spectroscopy for the Analysis of Water-Carbohydrate Interactions in Water..., Bakier [/bib_ref] [bib_ref] Analysis of water in food by near infrared spectroscopy, Buning-Pfaue [/bib_ref]. The behaviour of water at a molecular level can be explained through spectral evaluation of a whole biological sample or aqueous system under diverse conditions. The NIR spectrum of water (solvent) carries information about its solutes since they are very sensitive to the configuration and charges of the solvated molecules or clusters [bib_ref] Aquaphotomics: Water in the biological and aqueous world scrutinised with invisible light, Tsenkova [/bib_ref]. This paper will implement an aquaphotomics study, an area of research that is introduced to systematize the readily rich information related to the interaction between light at various wavelengths, especially within the NIR region, with aqueous systems. This will be done through Water Absorbance Pattern (WAP) by characterizing the spectral patterns of aqueous and biological systems that represent water absorbance bands which corresponds to the respective perturbations and the weight of each band in the regression equation due to the different pH values of acid solutions [bib_ref] Aquaphotomics: Water in the biological and aqueous world scrutinised with invisible light, Tsenkova [/bib_ref]. The robustness of wavelength selection in quantifying acids pH will be evaluated through the accuracy and precision of the predicted algorithm.
# Materials and methods
The value of absorbance was measured using a Jaz spectrometer with effective wavelength from 650 nm to 1,100 nm. Other custom setup prior to the experiment includes integration time = 3 msec, spectra averaged = 30 and boxcar smoothing = 1. Light source used was a tungsten halogen lamp with spectral emission between 360 nm and 2,000 nm and a colour temperature of 2,960 K. The reference spectrum for the absorbance measurement was collected through an empty quartz cuvette. The overall experimental setup conducted using spectroscopic instrumentations was from Ocean Optics and is illustrated in [fig_ref] Figure 1: Experimental setup for absorbance measurement [/fig_ref]. The chemicals (citric, tartaric, malic and oxalic acids in powder form) was diluted using pure (reverse osmosis) water and their pH was calibrated using an ExStik pH meter (PH100) from Extech Instruments (Waltham, MA, USA) with a range of measurement between 0.00-14.00 pH, resolution of 0.01 pH and accuracy of ±0.01 pH. The pH meter was calibrated using buffer solutions with pH values of 7 and 4. The measurement unit, pH is used (and will be used through the entire research) as the measurement unit to standardise the unit of measurement for the entire research since it is one of the scientifically used units in representing acidity in fruits besides "g/100 mL" which is commonly used for titratable acidity. In this work, the response was only due to the mixtures of water with citric (Unilab Chemical), tartaric (Bendosen Laboratory Chemicals), malic and oxalic (supplied by Sinaran Saintifik Enterprise, Penang) acids. The characteristics of the acids samples used in this work are listed in [fig_ref] Table 2: Sample characteristics [/fig_ref]. The first set of 50 acid samples was used for the development of the calibration equation. The next set of 50 samples was used to test the prediction accuracy of the developed equation. The linear relationship between optical parameters (absorbance at selected wavelengths) with the chemical composition was calculated using multiple linear regression (MLR) employing the Minitab (version [bib_ref] Analysis of aqueous solutions by near-infrared spectrometry (NIRS) IV. One-and two-component systems..., Berentsen [/bib_ref] software to obtain the value of coefficient of determination, R 2 , where the value of R 2 = 1 represents the theoretically perfect fit.
# Results and analysis
Light that is transmitted through an aqueous sample will attenuate in its intensity due to the absorption of light energy and also as the result of light scattered by any particles in the solution. [fig_ref] Figure 2: Linear relationship between absorbance and pH of aqueous citric, tartaric, malic and... [/fig_ref] shows the linear relationship between absorbance and pH of aqueous citric, tartaric, malic and oxalic solutions at a wavelength of 950 nm. One of the most critical observations from the response produced by each acid in [fig_ref] Figure 2: Linear relationship between absorbance and pH of aqueous citric, tartaric, malic and... [/fig_ref] is that the responsivity of pH measurement which can be represented by the slope (m) of the graph is highly correlated to the molecular size of the acids. The steepest response is produced by citric (m = 0.0131) with a single molecule that consist of 21 atoms, tartaric (m = 0.0079) 16 atoms, malic (m = 0.005) 15 atoms and oxalic (m = 0.0017) eight atoms. The "absorbance" notation in the graph does not only refer to the attenuation of light due to absorption, but also could be due to scattered light as has been stated earlier. Therefore, acid solutions with larger molecular size appear to scatter more light away from the receiving ends of the cuvette holder (to the spectrometer), thus producing a steeper response slope. Cox et al. [bib_ref] Feasibility of the use of visible and near infrared spectroscopy to assess..., Liu [/bib_ref] have conducted an experiment in determining the light scattering cross section of particles suspended in water. Particles with radii of 0.0285, 0.0605 and 0.2615 µm have been studied and the results show that particles with larger size produce higher scattering cross sections. Nonetheless, unlike the results presented by Cox et al. [bib_ref] An experiment to measure Mie and Rayleigh total scattering cross sections, Cox [/bib_ref] where they claimed that absorption for the particles studied was negligible, in this research, the NIR attenuation could be also due to absorbance by the acid molecules. Lower pH (higher acids concentration) can be associated to higher absorption (higher loss of NIR radiation).
Technically, light at all wavelengths will be scattered when it interacts with particles. The efficiency of scattering will decay exponentially from highly scattering ability in the UV to less significant when approaching NIR. The scattered intensity is proportional to 1/λ 4 that is based on a function of wavelength. Since scattering is subjugated by physical-optical phenomenon if compared to absorption which is dominated by chemical-optical phenomena, it is inadequate for scattering effect to cause a peak response for a wavelength that lies in between non-effective wavelengths unless it is being contributed to by absorption from the molecular bonds within the sample. This is imperative in understanding and elaborating the response shown in [fig_ref] Figure 3: Coefficient of determination generated at different wavelengths for pH measurement of aqueous... [/fig_ref] where the peak response wavelength for citric acid is 850 nm (R 2 = 0.9233), tartaric is 900 nm (R 2 = 0.9243) and 960 nm (R 2 = 0.9222), malic is 965 nm (R 2 = 0.8711) and oxalic is 850 nm (R 2 = 0.6337). For citric and tartaric, the R 2 measured for all examined wavelengths for the measurement of pH produced high R 2 (above 0.85) with some wavelengths producing a better result than the others. Both acids have relatively similar magnitude and shape of response. The high efficient R 2 produced for the measurement of pH for both acids could be due to two factors:
i. Low absorptive with considerable peak at certain wavelengths while accompanied by high scattering for all wavelengths. ii. High absorptive for all examined wavelengths with higher absorption peaks at certain wavelengths. Malic acid produces a higher uncertainty in its response, where a peak response with R 2 above 0.75 occurs at 700 nm, 850 nm and the rest for wavelengths between 930 nm and 1,000 nm. For oxalic acid, the highest response produced is at 850 nm with a very weak R 2 of only 0.6337. The response produced by malic and oxalic acids show a strong indication that the responses are due to absorbance by acid molecules with no significant scattering effect.
The following discussion will dictate the combination of NIR wavelengths (between 700 nm and 1,000 nm) that can produce the best calibration algorithm in predicting the pH of aqueous acid solutions. An individual wavelength that can generate a high correlation with pH of the acid does not necessarily contribute to an increased R 2 of the calibration algorithm when combined with other wavelengths. Therefore, the combination of wavelengths selected for the development of calibration algorithm is based on the wavelength that has a sizeable contribution in producing higher R 2 with low RMSE. The calibration equation for all acids are represented by Equations (1)-(4) for citric, tartaric, malic and oxalic acid, respectively. The wavelength 975 nm, which is one of the peak absorptions for water, appears to be important in the development of the calibration algorithm for all types of acids examined in this research, There are two ranges of wavelengths that are seen to contribute significantly for the quantification of pH of all acids. These wavelengths are 918-925 nm and 990-996 nm. The wavelength of 850 nm is observed to contribute for citric and oxalic solutions. Other wavelengths such as 700 nm, 800 nm and 900 nm for malic acid and 755 nm for oxalic acid appear to uniquely contribute only to those individual acids. [fig_ref] Figure 4: Predicted vs [/fig_ref] shows the relationship between predicted and actual pH for (a) citric (b) tartaric (c) malic (d) oxalic acids. [fig_ref] Table 3: Calibration results from MLR using wavelengths from water and pH absorbance bands [/fig_ref] lists the summary of the combinations of important wavelengths in quantifying pH of citric, tartaric, malic and oxalic acids. The squared column in the table indicates the combination of wavelengths that produces the best result for the respective acid.
# Conclusions
This study has managed to quantify the pH of aqueous acids solutions, i.e., citric, tartaric, malic and oxalic, through the application of NIR spectroscopy between 700 nm and 1,000 nm. The most important water absorbance wavelength for this study is 975 nm. For the pH measurement of all acids Oxalic Acid Prediction R 2 = 0.909 RMSEP = 0.206pH solution high measurement accuracy with R 2 above 0.9 (R 2 above 0.95 for citric, tartaric and malic acids) have been produced through the wavelengths selected for every acid. Besides, it is sufficient to state the significance of NIR range of wavelengths at 918-925 nm and 990-996 nm in determining acids' pH since the relatively similar range of wavelengths have been used by other researchers in food acidity measurements.
[fig] Figure 1: Experimental setup for absorbance measurement. [/fig]
[fig] Figure 2: Linear relationship between absorbance and pH of aqueous citric, tartaric, malic and oxalic acid solutions at λ = 950 nm. [/fig]
[fig] Figure 3: Coefficient of determination generated at different wavelengths for pH measurement of aqueous citric, tartaric, malic and oxalic acid solutions. [/fig]
[fig] Figure 4: Predicted vs. actual pH of ( [/fig]
[table] Table 2: Sample characteristics. [/table]
[table] Table 3: Calibration results from MLR using wavelengths from water and pH absorbance bands. [/table]
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Quartz Crystal Microbalance-Based Aptasensors for Medical Diagnosis
# Introduction
Aptamers have attained popularity as a promising molecular recognition element bearing values of the dissociation constant (K d ) from picomolar to nanomolar ranges in sensing applications such as environmental safety, food safety, and healthcare [bib_ref] Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4..., Tuerk [/bib_ref] [bib_ref] In vitro selection of RNA molecules that bind specific ligands, Ellington [/bib_ref] [bib_ref] Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA, Robertson [/bib_ref] [bib_ref] Detection of food-born allergens with aptamer-based biosensors, Khedri [/bib_ref] [bib_ref] Potentialities of aptasensors in cancer diagnosis, Sharma [/bib_ref]. Aptamers were first reported in 1990 [bib_ref] Aptamer based tools for environmental and therapeutic monitoring: A review of developments,..., Kudłak [/bib_ref]. They are short-chain oligonucleotides of RNA or single-stranded DNA commonly chosen via in vitro operation, namely, systematic evolution of ligands by exponential enrichment (SELEX). As such, they are described as artificial antibodies due to their high affinity and high-resolution molecular discrimination to their target analytes [bib_ref] Recent advances on aptamer-based biosensors for detection of pathogenic bacteria, Li [/bib_ref]. Unlike antibodies, aptamers are formed through chemical synthesis, and compared with natural antibodies their high surface density leads to less steric hindrance, enhancing their efficiency in recognizing targets [bib_ref] Molecular diagnostic and drug delivery agents based on aptamer-nanomaterial conjugates, Lee [/bib_ref]. In addition, aptamers maintain their properties during storage at room temperature and in different reactionary environments, are readily labeled with various reporters [bib_ref] Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA, Robertson [/bib_ref] [bib_ref] Aptamer and Its Potential Applications for Food Safety, Dong [/bib_ref] , and costs for their synthesis are relatively low, which is an important feature in diagnostic analysis [bib_ref] Quantifying small numbers of antibodies with a "near-universal" protein-DNA chimera, Burbulis [/bib_ref]. A diverse type of aptamer has been reported to recognize targets such as metal ions [bib_ref] Functionalized magnetic composites based on the aptamer serve as novel bio-adsorbent for..., Li [/bib_ref] , small organic compounds [bib_ref] Label-free and rapid detection of ATP based on structure switching of aptamers, Ji [/bib_ref] , toxins [bib_ref] Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1, Sun [/bib_ref] , nucleotides [bib_ref] Adaptive recognition by nucleic acid aptamers, Hermann [/bib_ref] , peptides [bib_ref] An electrochemical sense array based on aptamer and biotin-avidin system for the..., Wu [/bib_ref] , amino acids [bib_ref] An electrochemical aptamer-based sensor for the rapid and convenient measurement of l-tryptophan, Idili [/bib_ref] , enzymes [bib_ref] Impedimetric aptasensor for lysozyme detection based on carbon nanofibres enriched screen-printed electrodes, Senturk [/bib_ref] , proteins [bib_ref] Aptamer functionalized hydrophilic polymer monolith with gold nanoparticles modification for the sensitive..., Chen [/bib_ref] , hormones [bib_ref] Aptamer based ratiometric electrochemical sensing of 17β-estradiol using an electrode modified with..., Liu [/bib_ref] , bacteria [bib_ref] Optical fiber aptasensor for label-free bacteria detection in small volumes, Janik [/bib_ref] , and whole cells [bib_ref] Aptamer-based electrochemical cytosensors for tumor cell detection in cancer diagnosis: A review, Sun [/bib_ref] with high sensitivity and affinity.
Aptamer biomolecules consist of short single-stranded DNA/RNA nucleic acid/ oligonucleotide sequences chosen from random nucleic acid libraries by a SELEX process [bib_ref] DNA Aptamers in the Detection of Leukemia Cells by the Thickness Shear..., Poturnayová [/bib_ref] [bib_ref] Diazonium-based impedimetric aptasensor for the rapid label-free detection of Salmonella typhimurium in..., Bagheryan [/bib_ref] [bib_ref] Surface plasmon resonance aptasensor for detection of human activated protein C, Koyun [/bib_ref] [bib_ref] Preparation of Surface Plasmon Resonance Aptasensor for Human Activated Protein C Sensing, Akgönüllü [/bib_ref] [bib_ref] Recent Development of Aptasensor for Influenza Virus Detection, Kim [/bib_ref]. Nucleic acids are more resistant to changes in physical conditions, including pH differences and high temperature, than proteins, and they have smaller sizes than antibodies. In addition, chemical modifications can be made easily. Finally, when considering obtaining antibodies, the preparation of aptamers has several advantages including quicker synthesis [bib_ref] Rapid and label-free detection of Brucella melitensis in milk and milk products..., Bayramoglu [/bib_ref] [bib_ref] Recent advances in optical aptasensor technology for amplification strategies in cancer diagnostics, Kou [/bib_ref]. Aptamers are one of the most employed biorecognition materials for proteomics, medical diagnostics, and applications. They are of great interest as an alternative Herein, unique features such as higher chemical stability, easy low-cost mass manufacturability, and longer self-time compared to natural biorecognition elements, namely, antibodies, enzymes, and proteins, enable the use of aptamers in biosensors as excellent biorecognition elements in sensing applications. Biosensors are analytical tools employed to detect target analytes in biological or chemical reactions via measurable signals proportional to the analyte level in an interested medium in a wide range of concentrations at the ng/mL or even fg/mL level. They combine a biorecognition material with a chemical, physical, or physicochemical transducer [bib_ref] Review of diverse optical fibers used in biomedical research and clinical practice, Keiser [/bib_ref] [bib_ref] Amperometric biosensors, Heller [/bib_ref] [bib_ref] Principles and applications of thermal biosensors, Ramanathan [/bib_ref] [bib_ref] Electrochemical biosensors: Towards point-of-care cancer diagnostics, Wang [/bib_ref] [bib_ref] Recent developments in potentiometric biosensors for biomedical analysis, Koncki [/bib_ref]. The target analytes may be of various kinds, including ions [bib_ref] A SERS DNAzyme biosensor for lead ion detection, Wang [/bib_ref] , gases [bib_ref] Performance analysis of graphene-based surface plasmon resonance biosensor for blood glucose and..., Panda [/bib_ref] , drugs [bib_ref] A novel colorimetric biosensor based on non-aggregated Au@Ag core-shell nanoparticles for methamphetamine..., Mao [/bib_ref] , oligonucleotides [bib_ref] Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based..., Jafari [/bib_ref] , or proteins [bib_ref] Peptide-based electrochemical biosensors utilized for protein detection, Vanova [/bib_ref] , and there may be different kinds of transducer as well, such as plasmonic-optical [bib_ref] Development of Gold Nanoparticles Decorated Molecularly Imprinted-Based Plasmonic Sensor for the Detection..., Akgönüllü [/bib_ref] , mass-sensitive piezoelectric [bib_ref] Flexible hydrogel non-enzymatic QCM sensor for continuous glucose monitoring, Liu [/bib_ref] , thermal calorimetric thermometric [bib_ref] Thermal sensor based on a polymer nanofilm, Culebras [/bib_ref] , and chemical-electrochemical-electrical [bib_ref] The synergy of chemical immobilization and electrical orientation of T4 bacteriophage on..., Xu [/bib_ref]. Biosensors offer comparable sensitivity and selectivity while enabling online monitoring and real-time detection [bib_ref] Simultaneous detection of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol in corn..., Wei [/bib_ref] [bib_ref] Surface plasmon resonance for cell-based clinical diagnosis, Yanase [/bib_ref]. In recent years, use of such sensors has played a noteworthy role in critical research topics. Different types of aptamer-based biosensors, namely, aptasensors, are classified differently according to their transduction mechanisms, including electrochemical, optical, field effect transistor, calorimetric, and piezoelectric [bib_ref] Potentialities of aptasensors in cancer diagnosis, Sharma [/bib_ref] [bib_ref] Highly sensitive protein sensor based on thermally-reduced graphene oxide field-effect transistor, Mao [/bib_ref] [bib_ref] Selective detection of lysozyme biomarker utilizing large area chemical vapor deposition-grown graphene-based..., Ghosh [/bib_ref] [bib_ref] Label-Free Biosensors Based on Aptamer-Modified Graphene Field-Effect, Ohno [/bib_ref]. A quartz crystal microbalance as a mass-sensitive Herein, unique features such as higher chemical stability, easy low-cost mass manufacturability, and longer self-time compared to natural biorecognition elements, namely, antibodies, enzymes, and proteins, enable the use of aptamers in biosensors as excellent biorecognition elements in sensing applications. Biosensors are analytical tools employed to detect target analytes in biological or chemical reactions via measurable signals proportional to the analyte level in an interested medium in a wide range of concentrations at the ng/mL or even fg/mL level. They combine a biorecognition material with a chemical, physical, or physicochemical transducer [bib_ref] Review of diverse optical fibers used in biomedical research and clinical practice, Keiser [/bib_ref] [bib_ref] Amperometric biosensors, Heller [/bib_ref] [bib_ref] Principles and applications of thermal biosensors, Ramanathan [/bib_ref] [bib_ref] Electrochemical biosensors: Towards point-of-care cancer diagnostics, Wang [/bib_ref] [bib_ref] Recent developments in potentiometric biosensors for biomedical analysis, Koncki [/bib_ref]. The target analytes may be of various kinds, including ions [bib_ref] A SERS DNAzyme biosensor for lead ion detection, Wang [/bib_ref] , gases [bib_ref] Performance analysis of graphene-based surface plasmon resonance biosensor for blood glucose and..., Panda [/bib_ref] , drugs [bib_ref] A novel colorimetric biosensor based on non-aggregated Au@Ag core-shell nanoparticles for methamphetamine..., Mao [/bib_ref] , oligonucleotides [bib_ref] Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based..., Jafari [/bib_ref] , or proteins [bib_ref] Peptide-based electrochemical biosensors utilized for protein detection, Vanova [/bib_ref] , and there may be different kinds of transducer as well, such as plasmonic-optical [bib_ref] Development of Gold Nanoparticles Decorated Molecularly Imprinted-Based Plasmonic Sensor for the Detection..., Akgönüllü [/bib_ref] , mass-sensitive piezoelectric [bib_ref] Flexible hydrogel non-enzymatic QCM sensor for continuous glucose monitoring, Liu [/bib_ref] , thermal calorimetric thermometric [bib_ref] Thermal sensor based on a polymer nanofilm, Culebras [/bib_ref] , and chemicalelectrochemical-electrical [bib_ref] The synergy of chemical immobilization and electrical orientation of T4 bacteriophage on..., Xu [/bib_ref]. Biosensors offer comparable sensitivity and selectivity while enabling online monitoring and real-time detection [bib_ref] Simultaneous detection of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol in corn..., Wei [/bib_ref] [bib_ref] Surface plasmon resonance for cell-based clinical diagnosis, Yanase [/bib_ref]. In recent years, use of such sensors has played a noteworthy role in critical research topics. Different types of aptamer-based biosensors, namely, aptasensors, are classified differently according to their transduction mechanisms, including electrochemical, optical, field effect transistor, calorimetric, and piezoelectric [bib_ref] Potentialities of aptasensors in cancer diagnosis, Sharma [/bib_ref] [bib_ref] Highly sensitive protein sensor based on thermally-reduced graphene oxide field-effect transistor, Mao [/bib_ref] [bib_ref] Selective detection of lysozyme biomarker utilizing large area chemical vapor deposition-grown graphene-based..., Ghosh [/bib_ref] [bib_ref] Label-Free Biosensors Based on Aptamer-Modified Graphene Field-Effect, Ohno [/bib_ref]. A quartz crystal microbalance as a mass-sensitive based sensor (QCM) establishes the mass per unit area by measuring the change in frequency (∆ƒ), which is related to the mass accumulated on the quartz crystal resonator electrode [bib_ref] Quartz crystal microbalance based histidine sensor, Sönmezler [/bib_ref]. The rapid, accurate, real-time, label-free, and even on-site detection capability of the QCM technology, and especially its high sensitivity, have gained attention in the design of novel diagnostic tools integrating the significant advantages of aptamers [bib_ref] Quartz crystal microbalance-based biosensors as rapid diagnostic devices for infectious diseases, Lim [/bib_ref]. In this review, we briefly point out several typical aptamer biosensors used for medical diagnostic analysis. We focus on introducing novel methods for improving the potential of current analytical sensing approaches in terms of sensitivity and specificity.
## Qcm biosensor
Biosensors are tools that detect and determine specific target molecules and convert recognition of target molecules into the measurable signals [bib_ref] Development of Gold Nanoparticles Decorated Molecularly Imprinted-Based Plasmonic Sensor for the Detection..., Akgönüllü [/bib_ref] [bib_ref] Aptasensors: A review, Lim [/bib_ref] [bib_ref] Nanomaterials for biosensing applications: A review, Holzinger [/bib_ref]. This detectable signal is easily induced by specific molecular interactions between target molecules and recognition materials (i.e., receptors) [bib_ref] Molecularly imprinted polymer based sensors for medical applications, Saylan [/bib_ref]. Typically, a biosensor contains two major units, a receptor and a transducer, as shown in [fig_ref] Figure 2: Working principle of a biosensor [/fig_ref]. The optimal recognition material must be highly sensitive and specific for the target analyte of interest. It should adequately recognize and specifically capture the target analyte, resulting in a quick reply and strong performance. Recognition elements, including nucleic acids, antibodies, cells, and enzymes, can now be easily produced in an experimental laboratory. A transducer converts the biomolecular binding events into measurable signals, including optical and electrochemical signals as well as mass changes [bib_ref] A review: Recent advances in ultrasensitive and highly specific recognition aptasensors with..., Yan [/bib_ref] [bib_ref] Recent Advances and Progress in Development of the Field Effect Transistor Biosensor:..., Wadhera [/bib_ref]. The label-free surface-sensitive technique is perfect for monitoring interaction processes in liquid samples without the need to label related molecules, as they only generate signals by their physical existence on the recognition surface [bib_ref] Data evaluation for surface-sensitive label-free methods to obtain real-time kinetic and structural..., Saftics [/bib_ref]. In addition to the low cost of the label-free technique, it offers the capability of detecting the kinetic behavior of biological interactions even at the submolecular level, providing real-time monitoring [bib_ref] In situ viscoelastic properties and chain conformations of heavily hydrated carboxymethyl dextran..., Saftics [/bib_ref]. Several label-free transduction-based studies have been reported and have proven suitable in medical areas such as the pharmaceutical industry or healthcare for point-of-care testing and applications in basic research [bib_ref] Bulk and surface sensitivity of a resonant waveguide grating imager, Orgovan [/bib_ref].
based sensor (QCM) establishes the mass per unit area by measuring the change in frequency (Δƒ), which is related to the mass accumulated on the quartz crystal resonator electrode [bib_ref] Quartz crystal microbalance based histidine sensor, Sönmezler [/bib_ref]. The rapid, accurate, real-time, label-free, and even on-site detection capability of the QCM technology, and especially its high sensitivity, have gained attention in the design of novel diagnostic tools integrating the significant advantages of aptamers [bib_ref] Quartz crystal microbalance-based biosensors as rapid diagnostic devices for infectious diseases, Lim [/bib_ref]. In this review, we briefly point out several typical aptamer biosensors used for medical diagnostic analysis. We focus on introducing novel methods for improving the potential of current analytical sensing approaches in terms of sensitivity and specificity.
## Qcm biosensor
Biosensors are tools that detect and determine specific target molecules and convert recognition of target molecules into the measurable signals [bib_ref] Development of Gold Nanoparticles Decorated Molecularly Imprinted-Based Plasmonic Sensor for the Detection..., Akgönüllü [/bib_ref] [bib_ref] Aptasensors: A review, Lim [/bib_ref] [bib_ref] Nanomaterials for biosensing applications: A review, Holzinger [/bib_ref]. This detectable signal is easily induced by specific molecular interactions between target molecules and recognition materials (i.e., receptors) [bib_ref] Molecularly imprinted polymer based sensors for medical applications, Saylan [/bib_ref]. Typically, a biosensor contains two major units, a receptor and a transducer, as shown in [fig_ref] Figure 2: Working principle of a biosensor [/fig_ref]. The optimal recognition material must be highly sensitive and specific for the target analyte of interest. It should adequately recognize and specifically capture the target analyte, resulting in a quick reply and strong performance. Recognition elements, including nucleic acids, antibodies, cells, and enzymes, can now be easily produced in an experimental laboratory. A transducer converts the biomolecular binding events into measurable signals, including optical and electrochemical signals as well as mass changes [bib_ref] A review: Recent advances in ultrasensitive and highly specific recognition aptasensors with..., Yan [/bib_ref] [bib_ref] Recent Advances and Progress in Development of the Field Effect Transistor Biosensor:..., Wadhera [/bib_ref]. The label-free surface-sensitive technique is perfect for monitoring interaction processes in liquid samples without the need to label related molecules, as they only generate signals by their physical existence on the recognition surface [bib_ref] Data evaluation for surface-sensitive label-free methods to obtain real-time kinetic and structural..., Saftics [/bib_ref]. In addition to the low cost of the label-free technique, it offers the capability of detecting the kinetic behavior of biological interactions even at the submolecular level, providing real-time monitoring [bib_ref] In situ viscoelastic properties and chain conformations of heavily hydrated carboxymethyl dextran..., Saftics [/bib_ref]. Several label-free transduction-based studies have been reported and have proven suitable in medical areas such as the pharmaceutical industry or healthcare for point-of-care testing and applications in basic research [bib_ref] Bulk and surface sensitivity of a resonant waveguide grating imager, Orgovan [/bib_ref]. The piezoelectric crystal materials of an electromechanical transducer are fitted for employment as biosensors and actuators in devices and structures [bib_ref] Highly Selective Gas Sensor Based on Hydrophobic Silica Decorated with Trimethoxyoctadecylsilane, Xie [/bib_ref] [bib_ref] Centimeter-Scale Pillared-Layer Metal−Organic Framework Thin Films Mediated by Hydroxy Double Salt Intermediates..., Kim [/bib_ref]. These piezoelectric devices provide quick and label-free detection of the target molecules, resulting in specific and/or non-specific binding on the resonator surface. QCM biosensors are an effective analytical platform, and have been broadly applied to monitor interactions between biomolecules [bib_ref] Applicability of QCM-D for Quantitative Measurements of Nano-and Microparticle Deposition Kinetics: Theoretical..., Adamczyk [/bib_ref] [bib_ref] Hydrodynamic Solvent Coupling E ff ects in Quartz Crystal Microbalance Measurements of..., Adamczyk [/bib_ref] [bib_ref] Effects of Electrokinetic Phenomena on Bacterial Deposition Monitored by Quartz Crystal Microbalance..., Shan [/bib_ref]. Compared to traditional methods, they provide label-free and realtime detection, easy use with modern technologies, portable size, high sensitivity, low cost, and basic data analysis [bib_ref] GC-like Graphene-Coated Quartz Crystal Microbalance Sensor with Microcolumns, Son [/bib_ref] [bib_ref] Construction of Self-Assembled Polyelectrolyte/Cationic Microgel Multilayers and Their Interaction with Anionic Dyes..., Wu [/bib_ref] [bib_ref] Self-Assembled Monolayers Generated from Unsymmetrical Partially Fluorinated Spiroalkanedithiols, Chinwangso [/bib_ref]. QCM is nanogram-sensitive, and physical technologies can determine changes in resonance frequency (Δf) of the electrically driven quartz crystal by changes in thickness or mass per unit field (Δm). A relationship between the mass loaded The piezoelectric crystal materials of an electromechanical transducer are fitted for employment as biosensors and actuators in devices and structures [bib_ref] Highly Selective Gas Sensor Based on Hydrophobic Silica Decorated with Trimethoxyoctadecylsilane, Xie [/bib_ref] [bib_ref] Centimeter-Scale Pillared-Layer Metal−Organic Framework Thin Films Mediated by Hydroxy Double Salt Intermediates..., Kim [/bib_ref]. These piezoelectric devices provide quick and label-free detection of the target molecules, resulting in specific and/or non-specific binding on the resonator surface. QCM biosensors are an effective analytical platform, and have been broadly applied to monitor interactions between biomolecules [bib_ref] Applicability of QCM-D for Quantitative Measurements of Nano-and Microparticle Deposition Kinetics: Theoretical..., Adamczyk [/bib_ref] [bib_ref] Hydrodynamic Solvent Coupling E ff ects in Quartz Crystal Microbalance Measurements of..., Adamczyk [/bib_ref] [bib_ref] Effects of Electrokinetic Phenomena on Bacterial Deposition Monitored by Quartz Crystal Microbalance..., Shan [/bib_ref]. Compared to traditional methods, they provide label-free and real-time detection, easy use with modern technologies, portable size, high sensitivity, low cost, and basic data analysis [bib_ref] GC-like Graphene-Coated Quartz Crystal Microbalance Sensor with Microcolumns, Son [/bib_ref] [bib_ref] Construction of Self-Assembled Polyelectrolyte/Cationic Microgel Multilayers and Their Interaction with Anionic Dyes..., Wu [/bib_ref] [bib_ref] Self-Assembled Monolayers Generated from Unsymmetrical Partially Fluorinated Spiroalkanedithiols, Chinwangso [/bib_ref]. QCM is nanogram-sensitive, and physical technologies can determine changes in resonance frequency (∆f ) of the electrically driven quartz crystal by changes in thickness or mass per unit field (∆m). A relationship between the mass loaded on the quartz crystal surface and the resonant frequency is derived by the Sauerbrey Equation (1):
According to Sauerbrey effect [bib_ref] Sauerbrey and anti-Sauerbrey behavioral studies in QCM sensors-Detection of bioanalytes, Latif [/bib_ref] [bib_ref] Enhanced mass sensitivity of ZnO nanorod-grown quartz crystal microbalances, Lee [/bib_ref] ,
[formula] ∆ f = − 2 f 0 2 A √ ρ q µ q ∆m (1) [/formula]
where f 0 is the resonant frequency, ∆f is the change in resonant frequency (Hz) due to mass loaded per unit area (∆m) on the surface, ρ is the quartz density (2.648 g/cm 3 ), and µ is the shear modulus of quartz (2.947 × 10 11 g·cm −1 ·s 2 ). Quartz is the acoustic resonator and experiences the piezoelectric effect that induces acoustic waves by applying an alternating current to the quartz crystal. The QCM is known as a thickness shear mode resonator or bulk acoustic wave transducer. A typical AT-cut quartz crystal with gold electrode photographs is shown in [fig_ref] Figure 3: A quartz crystal with gold electrodes [/fig_ref]. The sole design criterion of thickness-shear mode resonators for frequency control is frequency stability. The AT-cut is most suitable [bib_ref] Role of mass accumulation and viscoelastic film properties for the response of..., Lucklum [/bib_ref] [bib_ref] Application of QCM in Peptide and Protein-Based Drug Product Development, Migoń [/bib_ref]. AT-cut quartz crystals are typically employed as sensor components, although the needs of sensor applications are more complicated [bib_ref] A review of interface electronic systems for AT-cut quartz crystal microbalance applications..., Arnau [/bib_ref] [bib_ref] Interface Circuits for QCM Sensors, Lucklum [/bib_ref]. The full physical definition of a viscoelastic charge in contact with a quartz crystal resonator has allowed the study of the mechanical characteristics of different materials coated on the sensor surface, such as the viscoelastic characteristics of polymers. The "acoustically thin" or "acoustically thick" coatings are of main significance [bib_ref] Role of mass accumulation and viscoelastic film properties for the response of..., Lucklum [/bib_ref] [bib_ref] A review of interface electronic systems for AT-cut quartz crystal microbalance applications..., Arnau [/bib_ref]. AT-QCM sensors are becoming a good alternative analytical technique in numerous applications. They are widely used as QCM sensors in gaseous mediums [bib_ref] QCM nanocomposite gas sensors-Expanding the application of waterborne polymer composites based on..., Trajcheva [/bib_ref] [bib_ref] Gas and humidity sensing with quartz crystal microbalance (QCM) coated with graphene-based..., Fauzi [/bib_ref]. Following the first studies showing that QCM can be used for the liquid phase, the use of crystal resonators has been reported for a great number of applications in various areas. Biorecognition materials undergo important changes in physical or chemical features in response to surrounding stimuli, including solvents, temperature, pH, magnetic fields, chemical agents, and electrical fields. Recently, pH-responsive materials have increasingly been used in different fields [bib_ref] PH-Responsive Copolymer Films Prepared by Surface-Initiated Polymerization and Simple Modification, Deng [/bib_ref] [bib_ref] Morphogen-driven self-construction of covalent films built from polyelectrolytes and homobifunctional spacers: Buildup..., Rydzek [/bib_ref] [bib_ref] Determination of isoelectric points and the role of pH for common quartz..., Cuddy [/bib_ref].
On the other hand, QCM technology provides use to the physical parameters of the sample by measuring the dissipation factor or another equivalent electrical parameter [bib_ref] Advanced Impedance Spectroscopy for QCM Sensor in Liquid Medium, Burda [/bib_ref]. Quartz crystals have been broadly employed to analyze mass, molecular interaction, membrane structure, and viscoelasticity changes on the surface of the electrodes [bib_ref] A QCM humidity sensor based on fullerene/graphene oxide nanocomposites with high quality..., Ding [/bib_ref]. Viscoelastic and conformational properties of the sample are monitored depending on the dispersion parameter [bib_ref] Advanced Impedance Spectroscopy for QCM Sensor in Liquid Medium, Burda [/bib_ref]. QCM with dissipation monitoring (QCM-D) is a powerful device employed to sensitively analyze the real-time and label-free responses of polymer films to external responses. The QCM-D technique is widely utilized to monitor film growth, material adsorption, thin film swelling, and ion exchange. QCM-D, similar to a QCM tool, utilizes the inverse piezoelectric effect, which results in vibrational oscillations of a quartz crystal when an alternating potential is applied. The difference between these two methods is that QCM-D measures the change in both the resonant frequency (∆f ) and the dissipation of oscillations (∆D), while QCM only measures the change in the resonant frequency [bib_ref] Morphogen-driven self-construction of covalent films built from polyelectrolytes and homobifunctional spacers: Buildup..., Rydzek [/bib_ref] [bib_ref] A practical guide to quartz crystal microbalance with dissipation monitoring of thin..., Easley [/bib_ref]. QCM-D has the ability to sensitively monitor mass changes on small time scales. Recently, there have been many reports of the QCM-D technique being employed as a powerful device to understand a variety of phenomena such as fouling [bib_ref] In situ real-time investigations on adsorptive membrane fouling by thermomechanical pulping process..., Rudolph [/bib_ref] , swelling [bib_ref] The Key Role of Side Chain Linkage in Structure Formation and Mixed..., Schmode [/bib_ref] , adsorption [bib_ref] Reducing anionic surfactant adsorption using polyacrylate as sacrificial agent investigated by QCM-D, Liu [/bib_ref] , and ion exchange [bib_ref] In situ investigation of lysozyme adsorption into polyelectrolyte brushes by quartz crystal..., You [/bib_ref].
QCM is a highly nanogram-sensitive technology of mass variations on the electrode surface. In this technique, a specific bioreceptor (for example, an antibody or aptamer) for the target biomolecule can be attached to the electrode surface. Thus, bioreceptors on the QCM electrode surface can interact with target molecules, which can be detected as a result of the detectable frequency changes [bib_ref] Rapid and label-free detection of Brucella melitensis in milk and milk products..., Bayramoglu [/bib_ref]. Several methods can be applied to the design of QCM-based biosensor surfaces for various application areas. Many uses of QCM biosensors have been published for detection of various molecules, such as amino acids [bib_ref] Chiral Recognition on Bare Gold Surfaces by Quartz Crystal Microbalance, Xiao [/bib_ref] , proteins [bib_ref] Development of an open-source thermally stabilized quartz crystal microbalance instrument for biomolecule-substrate..., Meléndrez [/bib_ref] , enzymes [bib_ref] Sensing of digestive proteins in saliva with a molecularly imprinted poly(ethylene-co-vinyl alcohol)..., Lee [/bib_ref] , drugs [bib_ref] Hollow molecularly imprinted polymer based quartz crystal microbalance sensor for rapid detection..., Zhao [/bib_ref] , vitamins [bib_ref] Improved piezoelectricity of polylactide using vitamin B2 for poling-free mechanical and acoustic..., Li [/bib_ref] , metals [bib_ref] A new hydrazone-linked covalent organic framework for Fe(III) detection by fluorescence and..., Li [/bib_ref] , pesticides [bib_ref] High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) immunosensor for pesticide detection in..., Cervera-Chiner [/bib_ref] , biomarkers [bib_ref] Detection of cancer biomarkers by piezoelectric biosensor using PZT ceramic resonator as..., Su [/bib_ref] , antibiotics [bib_ref] Escriche, I. High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCMD) Immunosensor for detection..., Cervera-Chiner [/bib_ref] , bacteria [bib_ref] Detection of the shrimp pathogenic bacteria, Vibrio harveyi, by a quartz crystal..., Buchatip [/bib_ref] , etc. Mass-sensitive QCM biosensors are commonly utilized for the detection of biomarkers. biomarkers [bib_ref] Detection of cancer biomarkers by piezoelectric biosensor using PZT ceramic resonator as..., Su [/bib_ref] , antibiotics [bib_ref] Escriche, I. High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCMD) Immunosensor for detection..., Cervera-Chiner [/bib_ref] , bacteria [bib_ref] Detection of the shrimp pathogenic bacteria, Vibrio harveyi, by a quartz crystal..., Buchatip [/bib_ref] , etc. Mass-sensitive QCM biosensors are commonly utilized for the detection of biomarkers. It is very important to utilize accurate and timely diagnostic methods to prevent the progress of a disease and stop the chain of transmission through early detection. Traditional devices are often time-consuming and costly. It is necessary to develop clinically sensitive, quick, and cost-effective clinical diagnostic methods. QCM biosensors are one of these technologies. QCM technologies are used as strong sensing devices because of their label-free properties, which provide the detection and determination of a large variety of biomolecules [bib_ref] Quartz crystal microbalance-based biosensors as rapid diagnostic devices for infectious diseases, Lim [/bib_ref]. It is quite useful to combine aptamers with QCM as a transducer. The relatively small size of aptamers is a positive advantage for mass-based sensing devices and other transducer applications. QCM devices have been broadly employed in several fields [bib_ref] Piezoelectric biosensors for virus detection, Akgönüllü [/bib_ref] , such as analytical chemistry [bib_ref] QCM-based immunosensor for rapid detection of Salmonella Typhimurium in food, Fulgione [/bib_ref] , immunology [bib_ref] Quartz crystal microbalance biosensor for label-free MDA MB 231 cancer cell detection..., Bakhshpour [/bib_ref] , and drug development [bib_ref] Antiproliferative drug-loaded multi-functionalized intraocular lens for reducing posterior capsular opacification, Huang [/bib_ref] , because of their high-quality features and high sensitivity [bib_ref] Uniform Mass Sensitivity Distribution of Elliptically Designed Electrodes Based on a Quartz..., Jiang [/bib_ref]. The following critical review of recent progress on QCM aptasensors for the medical diagnosis is intended to present researchers with a detailed understanding of their development and design while providing useful foundations for further practical biomedical applications.
## Application of qcm aptasensors for medical diagnostics
This section reviews QCM aptasensors for the selective recognition and detection of various biological molecules such as viruses, bacteria, proteins, and cells. In addition, immobilization techniques of aptamers, preparation of QCM detection electrodes/chips, and performance in terms of detection limit, selectivity, and sensitivity are examined.
## Viruses
Viruses are pathogenic microorganisms that are the reason for many infectious diseases. Viruses can live and multiply in the human body and spread easily and rapidly from infected people to healthy people. Therefore, timely detection of disease-causing viruses is the most important way to prevent the unwanted spread of infectious diseases and ensure timely medical treatment [bib_ref] Piezoelectric biosensors for virus detection, Akgönüllü [/bib_ref]. Aptamers are produced for a broad variety of viruses such as EBOV, HIV, HBV, severe acute respiratory syndrome (SARS), influenza viruses. dengue virus, rabies virus, norovirus, and vaccinia virus.
Avian influenza viruses (AIV) have created worldwide concern because of their potential pandemic threat to public health and major economic losses [bib_ref] A target-responsive and size-dependent hydrogel aptasensor embedded with QD fluorescent reporters for..., Xu [/bib_ref] [bib_ref] An impedance aptasensor with microfluidic chips for specific detection of H5N1 avian..., Lum [/bib_ref]. Wang et al. developed an ssDNA crosslinked polymeric hydrogel-coated QCM aptasensor for quick and selective detection of AIV H5N1. The chosen specific aptamer and single-stranded It is very important to utilize accurate and timely diagnostic methods to prevent the progress of a disease and stop the chain of transmission through early detection. Traditional devices are often time-consuming and costly. It is necessary to develop clinically sensitive, quick, and cost-effective clinical diagnostic methods. QCM biosensors are one of these technologies. QCM technologies are used as strong sensing devices because of their label-free properties, which provide the detection and determination of a large variety of biomolecules [bib_ref] Quartz crystal microbalance-based biosensors as rapid diagnostic devices for infectious diseases, Lim [/bib_ref]. It is quite useful to combine aptamers with QCM as a transducer. The relatively small size of aptamers is a positive advantage for mass-based sensing devices and other transducer applications. QCM devices have been broadly employed in several fields [bib_ref] Piezoelectric biosensors for virus detection, Akgönüllü [/bib_ref] , such as analytical chemistry [bib_ref] QCM-based immunosensor for rapid detection of Salmonella Typhimurium in food, Fulgione [/bib_ref] , immunology [bib_ref] Quartz crystal microbalance biosensor for label-free MDA MB 231 cancer cell detection..., Bakhshpour [/bib_ref] , and drug development [bib_ref] Antiproliferative drug-loaded multi-functionalized intraocular lens for reducing posterior capsular opacification, Huang [/bib_ref] , because of their high-quality features and high sensitivity [bib_ref] Uniform Mass Sensitivity Distribution of Elliptically Designed Electrodes Based on a Quartz..., Jiang [/bib_ref]. The following critical review of recent progress on QCM aptasensors for the medical diagnosis is intended to present researchers with a detailed understanding of their development and design while providing useful foundations for further practical biomedical applications.
## Application of qcm aptasensors for medical diagnostics
This section reviews QCM aptasensors for the selective recognition and detection of various biological molecules such as viruses, bacteria, proteins, and cells. In addition, immobilization techniques of aptamers, preparation of QCM detection electrodes/chips, and performance in terms of detection limit, selectivity, and sensitivity are examined.
## Viruses
Viruses are pathogenic microorganisms that are the reason for many infectious diseases. Viruses can live and multiply in the human body and spread easily and rapidly from infected people to healthy people. Therefore, timely detection of disease-causing viruses is the most important way to prevent the unwanted spread of infectious diseases and ensure timely medical treatment [bib_ref] Piezoelectric biosensors for virus detection, Akgönüllü [/bib_ref]. Aptamers are produced for a broad variety of viruses such as EBOV, HIV, HBV, severe acute respiratory syndrome (SARS), influenza viruses. dengue virus, rabies virus, norovirus, and vaccinia virus.
Avian influenza viruses (AIV) have created worldwide concern because of their potential pandemic threat to public health and major economic losses [bib_ref] A target-responsive and size-dependent hydrogel aptasensor embedded with QD fluorescent reporters for..., Xu [/bib_ref] [bib_ref] An impedance aptasensor with microfluidic chips for specific detection of H5N1 avian..., Lum [/bib_ref]. Wang et al. developed an ssDNA crosslinked polymeric hydrogel-coated QCM aptasensor for quick and selective detection of AIV H5N1. The chosen specific aptamer and single-stranded DNA (ssDNA) were used to form the crosslinker in a polymeric hydrogel. The aptamerattached polymeric hydrogel was coated homogeneously onto a QCM electrode's gold surface using a self-assembled monolayer (SAM) technique. The different molar ratios of three polymeric hydrogels were synthesized with acrylamide and aptamer. The hydrogel swelling was monitored with a QCM device of decreasing frequency. The authors reported that the 1:1 hydrogel-aptamer coated QCM electrode provided the best sensitivity. The limit of detection (LOD) was found to be 0.0128 HAU. The detection time for detection of AIV H5N1 was only 30 min with the designed aptamer-attached hydrogel-coated QCM aptasensor [bib_ref] Hydrogel based QCM aptasensor for detection of avian influenza virus, Wang [/bib_ref]. The preparation of this QCM sensor design and sensorgram is shown in [fig_ref] Figure 4: Preparation of aptamer hydrogel-coated QCM aptasensor and QCM sensorgram [/fig_ref]. DNA (ssDNA) were used to form the crosslinker in a polymeric hydrogel. The ap attached polymeric hydrogel was coated homogeneously onto a QCM electrode surface using a self-assembled monolayer (SAM) technique. The different molar ra three polymeric hydrogels were synthesized with acrylamide and aptamer. The hy swelling was monitored with a QCM device of decreasing frequency. The auth ported that the 1:1 hydrogel-aptamer coated QCM electrode provided the best sens The limit of detection (LOD) was found to be 0.0128 HAU. The detection time for de of AIV H5N1 was only 30 min with the designed aptamer-attached hydrogel-coated aptasensor [bib_ref] Hydrogel based QCM aptasensor for detection of avian influenza virus, Wang [/bib_ref]. The preparation of this QCM sensor design and sensorgram is sh [fig_ref] Figure 4: Preparation of aptamer hydrogel-coated QCM aptasensor and QCM sensorgram [/fig_ref]. A label-free QCM aptasensor based on nanowell material for quick and sensit tection of H5N1 AIV was designed by Wang et al. The design process of the nan based electrode for the nanoporous gold film included immobilization onto the go trode surface through a bifunctional dithiol 1,6-hexanedithiol. For th pose, a mixture solution was prepared of 1% dithiol 1,6-hexanedithiol (HDT) and MHDA (16-mercaptohexadecanoic acid) at a ratio of 1:1. The thickness of the na was reported as 120 nm. The pore size of the nanoporous film synthesized using a m corrosion technique was ~20 nm. After characterization studies, nanoporous fil coated onto a nanowell-based QCM gold chip surface using the SAM technique ( 5). Then, the specific aptamer was attached to a QCM aptasensor through covalent ing. The mechanism of the NH2-aptamer immobilization is displayed inA label-free QCM aptasensor based on nanowell material for quick and sensitive detection of H5N1 AIV was designed by Wang et al. The design process of the nanowellbased electrode for the nanoporous gold film included immobilization onto the gold electrode surface through a bifunctional dithiol 1,6-hexanedithiol. For this purpose, a mixture solution was prepared of 1% dithiol 1,6-hexanedithiol (HDT) and 20 mM MHDA (16-mercaptohexadecanoic acid) at a ratio of 1:1. The thickness of the nanofilm was reported as 120 nm. The pore size of the nanoporous film synthesized using a metallic corrosion technique was~20 nm. After characterization studies, nanoporous film was coated onto a nanowell-based QCM gold chip surface using the SAM technique. Then, the specific aptamer was attached to a QCM aptasensor through covalent bonding. The mechanism of the NH 2 -aptamer immobilization is displayed in. QCM gold electrode characterization was carried out through scanning electron microscopy (SEM). The linear concentration range was obtained from 2 −4 to 2 4 hemagglutination units (HAUs)/50 µL. The limit of detection was found to be 2 −4 HAU/50 µL for AIV H5N1. No signal was observed for non-target AIV subtypes, including H1N1, H2N2, H7N2, and H5N3. The authors reported that further development of this aptasensor could be applied to detect different viruses [bib_ref] A nanowell-based QCM aptasensor for rapid and sensitive detection of avian influenza..., Wang [/bib_ref].
omachines 2022, 13, x FOR PEER REVIEW 7 of gold electrode characterization was carried out through scanning electron microscop (SEM). The linear concentration range was obtained from 2 −4 to 2 4 hemagglutination un (HAUs)/50 μL. The limit of detection was found to be 2 −4 HAU/50 μL for AIV H5N1. N signal was observed for non-target AIV subtypes, including H1N1, H2N2, H7N2, an H5N3. The authors reported that further development of this aptasensor could be appli to detect different viruses [bib_ref] A nanowell-based QCM aptasensor for rapid and sensitive detection of avian influenza..., Wang [/bib_ref]. Another AIV H5N1 QCM aptasensor platform to enhance the signal produced f detection of the AIV H5N1 was reported by Brockman et al. First, streptavidin was coat onto the QCM electrode's gold surface after binding biotin-labeled aptamers. Afterward QCM aptasensor response was enhanced by adding aptamer-attached magnetic nanopa ticles. The magnetic nanoparticles' amplification of the aptasensor response was effecti at low AIV H5N1 concentrations. The LOD value for this aptasensor was calculated as HAU [bib_ref] QCM Aptasensor for Rapid and Specific Detection of Avian Influenza Virus, Brockman [/bib_ref].
In another study, a QCM aptasensor for label-free detection of HepBV virus was e tablished by Giamblanco et al. They designed a system for sensing HepBV DNA by im mobilizing a thiol-ssDNA aptamer on the surface of the QCM gold electrode. QCM ele trode gold surfaces thus functionalized with thiolated ssDNA were characterized usi Another AIV H5N1 QCM aptasensor platform to enhance the signal produced for detection of the AIV H5N1 was reported by Brockman et al. First, streptavidin was coated onto the QCM electrode's gold surface after binding biotin-labeled aptamers. Afterwards, QCM aptasensor response was enhanced by adding aptamer-attached magnetic nanoparticles. The magnetic nanoparticles' amplification of the aptasensor response was effective at low AIV H5N1 concentrations. The LOD value for this aptasensor was calculated as 1 HAU [bib_ref] QCM Aptasensor for Rapid and Specific Detection of Avian Influenza Virus, Brockman [/bib_ref].
In another study, a QCM aptasensor for label-free detection of HepBV virus was established by Giamblanco et al. They designed a system for sensing HepBV DNA by immobilizing a thiol-ssDNA aptamer on the surface of the QCM gold electrode. QCM electrode gold surfaces thus functionalized with thiolated ssDNA were characterized using atomic force microscope (AFM) measurement. The QCM aptasensor was able to detect fmol/cm 2 target HepBV virus with an ssDNA probe without using any amplification steps or labeling method. The authors were able to perform more sensitive determination by controlling the ssDNA density on the electrode surface. They reported that these results facilitated the basic use and portability of the developed POC biosensor device for label-free and quick detection of HepBV [bib_ref] Single-step label-free hepatitis B virus detection by a piezoelectric biosensor, Giamblanco [/bib_ref].
A biotinylated-DNA immobilized QCM aptasensor for detecting hepatitis C virus (HCV) in serum was developed by Skladal et al. The functionalization process of the QCM electrode surface included the immobilization of cysteamine and activation with glutaraldehyde followed by addition of either avidin or streptavidin; 10 MHz AT-cut gold electrode (diameter 5 mm) quartz crystals were used in this work. The authors reported results showing significantly higher immobilization efficiency with avidin as compared to streptavidin. The piezoelectric aptasensor was able to perform real-time monitoring of hybridization in 10 min. The biotinylated-DNA-avidin-immobilized aptasensor was reused 30 times. From the economic point of view, the reusability of QCM aptasensors is quite promising [bib_ref] Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C..., Skládal [/bib_ref].
## Bacteria
The quick, reliable, accurate, and highly sensitive detection of bacteria is a focus of diverse areas, particularly public health [bib_ref] Bacteria Detection: From Powerful SERS to Its Advanced Compatible Techniques, Zhou [/bib_ref]. Therefore, the progress of novel quick, specific, and sensitive biosensors for the determination of pathogens is of remarkable importance [bib_ref] Detection of pathogenic bacteria via nanomaterials-modified aptasensors, Sharifi [/bib_ref]. Salmonella typhimurium is a pathogen bacteria that causes outbreaks of diseases. S. typhimurium infection causes fever, diarrhea, abdominal cramps, and even death. It is important to develop a quick, selective, and sensitive system to detect S. typhimurium pathogen bacteria. Wang et al. designed a novel QCM aptasensor for label-free and real-time detection of S. typhimurium employing an AT-cut 7.995 MHz quartz crystal gold electrode. The LOD value was calculated as 10 3 CFU/mL of S. typhimurium within one hour. The preparation of the QCM aptasensor process is shown in. facilitated the basic use and portability of the developed POC biosensor device for labelfree and quick detection of HepBV [bib_ref] Single-step label-free hepatitis B virus detection by a piezoelectric biosensor, Giamblanco [/bib_ref]. A biotinylated-DNA immobilized QCM aptasensor for detecting hepatitis C virus (HCV) in serum was developed by Skladal et al. The functionalization process of the QCM electrode surface included the immobilization of cysteamine and activation with glutaraldehyde followed by addition of either avidin or streptavidin; 10 MHz AT-cut gold electrode (diameter 5 mm) quartz crystals were used in this work. The authors reported results showing significantly higher immobilization efficiency with avidin as compared to streptavidin. The piezoelectric aptasensor was able to perform real-time monitoring of hybridization in 10 min. The biotinylated-DNA-avidin-immobilized aptasensor was reused 30 times. From the economic point of view, the reusability of QCM aptasensors is quite promising [bib_ref] Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C..., Skládal [/bib_ref].
## Bacteria
The quick, reliable, accurate, and highly sensitive detection of bacteria is a focus of diverse areas, particularly public health [bib_ref] Bacteria Detection: From Powerful SERS to Its Advanced Compatible Techniques, Zhou [/bib_ref]. Therefore, the progress of novel quick, specific, and sensitive biosensors for the determination of pathogens is of remarkable importance [bib_ref] Detection of pathogenic bacteria via nanomaterials-modified aptasensors, Sharifi [/bib_ref]. Salmonella typhimurium is a pathogen bacteria that causes outbreaks of diseases. S. typhimurium infection causes fever, diarrhea, abdominal cramps, and even death. It is important to develop a quick, selective, and sensitive system to detect S. typhimurium pathogen bacteria. Wang et al. designed a novel QCM aptasensor for label-free and real-time detection of S. typhimurium employing an AT-cut 7.995 MHz quartz crystal gold electrode. The LOD value was calculated as 10 3 CFU/mL of S. typhimurium within one hour. The preparation of the QCM aptasensor process is shown in. Escherichia coli (E. coli) O157:H7 infection causes various symptoms including severe abdominal cramps, acute hemorrhagic diarrhea, and hemolytic uremic syndrome. The design of a highly sensitive and specific technique is critically important for controlling outbreaks and disease progression in infected individuals. Yu et al. developed a singlestranded DNA aptamer-attached QCM aptasensor for real-time detection of E. coli O157:H7. Whole cells of E. coli O157:H7 bacteria were employed using the SELEX process. The detection limit was 1.46 × 10 3 CFU/mL of E. coli O157:H7 within 1 h. The presented results show that the specific ssDNA aptamer chosen by means of whole-bacterium SELEX possesses high affinity [bib_ref] Whole-bacterium SELEX of DNA aptamers for rapid detection of E.coli O157:H7 using..., Yu [/bib_ref]. Escherichia coli (E. coli) O157:H7 infection causes various symptoms including severe abdominal cramps, acute hemorrhagic diarrhea, and hemolytic uremic syndrome. The design of a highly sensitive and specific technique is critically important for controlling outbreaks and disease progression in infected individuals. Yu et al. developed a singlestranded DNA aptamer-attached QCM aptasensor for real-time detection of E. coli O157:H7. Whole cells of E. coli O157:H7 bacteria were employed using the SELEX process. The detection limit was 1.46 × 10 3 CFU/mL of E. coli O157:H7 within 1 h. The presented results show that the specific ssDNA aptamer chosen by means of whole-bacterium SELEX possesses high affinity [bib_ref] Whole-bacterium SELEX of DNA aptamers for rapid detection of E.coli O157:H7 using..., Yu [/bib_ref].
## Proteins
The detection of proteins can provide valuable information for clinical diagnosis applications [bib_ref] Coffee ring aptasensor for rapid protein detection, Wen [/bib_ref]. Thrombin is a significant biomarker, and its rapid and selective detection is very important for diagnosis and prevention of related diseases [bib_ref] Role of thrombin in the pathogenesis of atherosclerosis, Jaberi [/bib_ref]. Xi et al. designed a target-triggered delivery of cargo molecules-based QCM aptasensor. Gold nanocages (AuNCs) were characterized using a transmission electron microscope (TEM). Empty nanocages were loaded with ssDNA molecules, capped with specific aptamers, then coated on a QCM chip gold surface for real-time detection of thrombin [bib_ref] Label-Free Quartz Crystal Microbalance Biosensor Based on Aptamer-Capped Gold Nanocages Loaded with..., Xi [/bib_ref] , showing a broad linear concentration dynamic range of 0.0086 nM−86 nM. The LOD values were calculated as 7.7 pM and 1.2 nM in PBS buffer and a human serum sample, respectively. The preparation of this QCM aptasensor design for the detection of thrombin is shown in.
## Proteins
The detection of proteins can provide valuable information for clinical diagnosis applications [bib_ref] Coffee ring aptasensor for rapid protein detection, Wen [/bib_ref]. Thrombin is a significant biomarker, and its rapid and selective detection is very important for diagnosis and prevention of related diseases [bib_ref] Role of thrombin in the pathogenesis of atherosclerosis, Jaberi [/bib_ref]. Xi et al. designed a target-triggered delivery of cargo molecules-based QCM aptasensor. Gold nanocages (AuNCs) were characterized using a transmission electron microscope (TEM). Empty nanocages were loaded with ssDNA molecules, capped with specific aptamers, then coated on a QCM chip gold surface for real-time detection of thrombin [bib_ref] Label-Free Quartz Crystal Microbalance Biosensor Based on Aptamer-Capped Gold Nanocages Loaded with..., Xi [/bib_ref] , showing a broad linear concentration dynamic range of 0.0086 nM−86 nM. The LOD values were calculated as 7.7 pM and 1.2 nM in PBS buffer and a human serum sample, respectively. The preparation of this QCM aptasensor design for the detection of thrombin is shown in. The electrode was covered by DNA aptamers of the electrochemical indicator methylene blue (MB), which was bonded to thrombin They reported that MB can be used to detect thrombin with high sensitivity and selectivity. The lower limit of the detection QCM method was 1 nM [bib_ref] Detection of aptamer-protein interactions using QCM and electrochemical indicator methods, Hianik [/bib_ref]. The electrode was covered by DNA aptamers of the electrochemical indicator methylene blue (MB), which was bonded to thrombin They reported that MB can be used to detect thrombin with high sensitivity and selectivity. The lower limit of the detection QCM method was 1 nM [bib_ref] Detection of aptamer-protein interactions using QCM and electrochemical indicator methods, Hianik [/bib_ref].
Iijima et al., developed a thrombin-binding DNA-aptamer attached QCM aptasensor. They used DNA-aptamer in their previously developed~30 nm bio-nanocapsules (ZZ-BNC). ZZ-BNC was modified by replacing the ZZ domain with a DNA-binding single-chain lambda Cro (scCro) domain to expand the versatility of ZZ-BNC. The nanocapsule-coated scCro-BNC-QCM aptasensor chip immobilized with thrombin-binding DNA aptamers showed~5.5-fold higher thrombin binding capacity and~6000-fold higher detection sensitivity compared to a QCM aptasensor chip directly coated with DNA aptamers. They reported that the number of bound thrombin molecules per DNA aptamer molecule in-creased~7.8-fold with scCro-BNC coating [bib_ref] Bio-nanocapsules for oriented immobilization of DNA aptamers on aptasensors, Iijima [/bib_ref].
Deng et al. developed a first-time combined QCM and surface-enhanced Raman spectroscopy (SERS) platform for detection of thrombin. The functionalization process of the QCM electrode included a 1,6-hexanedithiol (HDT) monolayer with gold nanoparticles (20 nm AuNPs) assembled on immobilized HDT with stable Au-S linkage. Then, thiolmodified aptamers were assembled on this electrode surface. AuNPs were used to amplify the frequency signal significantly. The limit of detection for thrombin was 0.1 µM. In the concentration range of 0.1 to 1.0 µM, a good linear correlation was obtained for the determination of thrombin. This presented combination could further develop the progress and application of QCM and SERS in protein analysis with aptamers.
Aptasensor platforms for analysis of HIV-1 Tat protein by immobilizing a specific RNA aptamer on a QCM electrode were reported by Tombelli et al. This QCM aptasensor was compared with a surface plasmon resonance (SPR)-based aptasensor. The biotin-avidin linking was immobilized onto the gold surface of the quartz crystal chip. Both devices displayed similar reusability, sensitivity, and specificity. The linear detection range of the QCM was from 0 to 1.25 ppm [bib_ref] Aptamer-based biosensors for the detection of HIV-1 Tat protein, Tombelli [/bib_ref].
Minunni et al. developed a specific RNA aptamer-immobilized QCM aptasensor for the trans-activator of transcription (Tat) protein of HIV-1. In this work, a specific RNA aptamer was utilized as a biological recognition element. The antibody was immobilized on a layer of carboxylated dextran previously deposited on the QCM gold chip surface. The linear range with the antibody was from 0 to 2.5 ppm, and the limit of detection was 0.25 ppm [bib_ref] Development of biosensors with aptamers as bio-recognition element: The case of HIV-1..., Minunni [/bib_ref].
Yao et al. developed a QCM aptasensor for fast sensing of Immunoglobulin E (IgE) in human serum samples. Aptamers were immobilized non-covalently using a monolayer of avidin on the QCM gold surface. They reported this sensor to be suitable for the detection of IgE within 15 min. It showed a linear detection range between 2.5 µg/L and 200 µg/L in buffer solution and human serum, respectively. This QCM aptasensor was suitably designed for label-free and selective detection of proteins, and represents an innovative device for future proteomics [bib_ref] Aptamer-based piezoelectric quartz crystal microbalance biosensor array for the quantification of IgE, Yao [/bib_ref].
## Cells
Recently, investigations of the interaction of whole cells with QCM sensors have been reported [bib_ref] Selective microorganism detection with cell surface imprinted polymers, Hayden [/bib_ref] [bib_ref] Whole cell imprinting based Escherichia coli sensors: A study for SPR and..., Yilmaz [/bib_ref]. Leukemia is one of the most common deadly cancers [bib_ref] Applications of electrochemical biosensor of aptamers-based (APTASENSOR) for the detection of leukemia..., Nur [/bib_ref]. It is caused by blood or bone marrow cancer [bib_ref] Cancer statistics, Acheson [/bib_ref]. A sensitive and accurate diagnosis is important for efficient treatment of this disease. The methods employed today for analysis of leukemia cells are polymerase chain reaction [bib_ref] Molecular detection and characterisation of circulating tumour cells and micrometastases in solid..., Ghossein [/bib_ref] , flow cytometry [bib_ref] Multiparameter flow cytometry in the diagnosis and management of acute leukemia, Peters [/bib_ref] , and fluorescence measurement [bib_ref] Coformulation of doxorubicin and curcumin in poly(d, l-lactide-co-glycolide) nanoparticles suppresses the development..., Misra [/bib_ref]. There is a need to produce simple and cost-effective technologies for rapid, label-free, and selective detection of leukemia cells. Shan et al. developed a new method for specific detection of leukemia cells. They first prepared aminophenylboronic acid-modified gold nanoparticles (APBA-AuNPs) which could bind to cell membranes. Then, these APBA-AuNPs were employed for labeling of cells. Signal amplification was achieved via silver enhancement. A good linear relationship was obtained between 2 × 10 3 -1 × 10 5 cells/mL. The limit of detection was calculated as 1160 cells/mL. This QCM aptasensor offers a quick, rapid, label-free, and cheap technology for sensitive detection of leukemia cells [bib_ref] An aptamer-based quartz crystal microbalance biosensor for sensitive and selective detection of..., Shan [/bib_ref]. The QCM detection process is shown in. In another study, a label-free QCM aptasensor for selective detection of the hepatocellular carcinoma cell line (HepG2) was reported by Kashefi-Kheyrabadi et al. A sandwich architecture was used on the electrode surface. The related HepG2 cells were captured by a TLS11a aptamer covalently attached to a gold surface. This QCM aptasensor showed a broad linear range between 1 × 10 2 and 1 × 10 6 cells/mL, and the limit of detection was 2 cells/mL. The authors reported that this method offers a simple, cheap, and stable technology for sensitive detection of liver cancer as well as other cancel cell [bib_ref] Ultrasensitive detection of human liver hepatocellular carcinoma cells using a label-free aptasensor, Kashefi-Kheyrabadi [/bib_ref].
## Conclusions and future perspectives
QCMs are highly reliable for sensing the mass of deposited target samples in both liquid and gas matrices. Moreover, they allow real-time monitoring and have relatively low manufacturing and processing costs. These properties make mass-sensitive devices In another study, a label-free QCM aptasensor for selective detection of the hepatocellular carcinoma cell line (HepG2) was reported by Kashefi-Kheyrabadi et al. A sandwich architecture was used on the electrode surface. The related HepG2 cells were captured by a TLS11a aptamer covalently attached to a gold surface. This QCM aptasensor showed a broad linear range between 1 × 10 2 and 1 × 10 6 cells/mL, and the limit of detection was 2 cells/mL. The authors reported that this method offers a simple, cheap, and stable technology for sensitive detection of liver cancer as well as other cancel cell [bib_ref] Ultrasensitive detection of human liver hepatocellular carcinoma cells using a label-free aptasensor, Kashefi-Kheyrabadi [/bib_ref].
## Conclusions and future perspectives
QCMs are highly reliable for sensing the mass of deposited target samples in both liquid and gas matrices. Moreover, they allow real-time monitoring and have relatively low manufacturing and processing costs. These properties make mass-sensitive devices feasible for numerous nanoscale applications such as detection of cells, viruses, antibody interactions, and DNA hybridizations. While QCM biosensors are mostly used owing to their low cost, they are limited to the operating temperature of quartz,~350 - C, although high-temperature piezoelectric sensors resisting up to 1250 - C have been reported for different applications [bib_ref] High-temperature piezoelectric sensing, Jiang [/bib_ref]. The limit of detection of a QCM biosensor is lower compared to a surface acoustic wave (SAW). However, QCMs can provide access to physical parameters of samples by measuring the dissipation factor or another equivalent electrical parameter, providing a more detailed analysis of the surface and interactions thereon that is not limited to measurement of the mass per unit area. The viscoelastic and conformational characteristics of a sample are monitored based on the dissipation parameter. The application of the QCM technique in biomedical applications can be very helpful.
Many different biosensors have been developed based on diverse transducers, including optical, electrochemical, and mass-sensitive varieties. Among these techniques, aptamer-based mass-sensitive biosensors have been comprehensively characterized owing to their high sensitivity, high stability, cost-effectiveness, and simplicity of fabrication. It is well known that early determination of diseases and epidemics is essential to ensuring efficient treatment, and aptamers are highly promising biomolecules in this critical area. The selection of aptamers for biorecognition of related viruses, cancer cells, and proteins has already been achieved. [fig_ref] Table 1: The advantages and major challenges of QCM biosensors prepared by various techniques [/fig_ref] summarizes different advantages and challenges of the QCM biosensor concept.
# Method and materials
Advantages Challenges Ref.
Aptamer label-free detection, specific recognition, online, rapid, highly sensitive analysis, simple to functionalize, non-aggregating, very stable in dehydrated form, more resistant to thermal degradation anchoring to the surface of QCM electrode, low reproducibility, costly [bib_ref] Electrochemical Microfluidic Paper-Based Aptasensor Platform Based on a Biotin-Streptavidin System for Label-Free..., Ming [/bib_ref] Antibody selective affinity to target molecules, sensitive assays, reproducible results, substantial decrease in bioactivity owing to the denaturation and random orientation, costly production [bib_ref] A novel electrochemical piezoelectric label free immunosensor for aflatoxin B1 detection in..., Chauhan [/bib_ref] Molecular imprinting polymer (MIP)
high selectivity to template molecule, long-term storage stability, potential re-usability, cheap creates wide cavities, template molecule may covalently bound to the polymer, difficult target removal [bib_ref] The rational development of molecularly imprinted polymer-based sensors for protein detection, Whitcombe [/bib_ref] Metal-organic frameworks (MOFs)
high sensitivity to target, low power consumption, easy modification
Large-scale manufacturing, improved selectivity, enhancing reproducibility, miniaturized manufacturing methods [bib_ref] Metal-organic frameworks for QCM-based gas sensors: A review, Wang [/bib_ref] When compared to antibodies, aptamers are an especially good match for the recognition of small molecules with high specificity and affinity. Therefore, the improvement of aptamer-based biosensors of macromolecules, even small molecules, could be an efficient way to expand the range of easily measurable analytes. Another potential advantage of aptamer sensors is that they can be stored at variable temperatures and are reusable for certain time periods. Aptamers are thus suitable as miniaturized and portable biosensors that can be kept for extended time periods.
Due to their potential applications, aptamer production has increased significantly over the past few years. Aptamers are convenient for use in biosensors as sensitive and selective recognition elements with a variety of transducer technologies that allow them to be highly sensitive. It is quite beneficial to unite aptamers with QCM as the transducer. Quartz crystal microbalance has emerged as one of the most popular biosensing devices over the past fifteen years. QCM devices are capable of fast, label-free, real-time, and on-site detection of analytes that are of great public health importance, including influenza and hepatitis B virus (HBV), among others, as well as bacteria and proteins. As such, their use in medical diagnostic applications has increased significantly. It is most important that the QCM is evenly distributed over the entire electrode surface when the target analyte is delivered to the recognition surface. The repeatability of measurements made by QCM in practical applications is limited largely by the unevenness of the sensitivity distribution.
In this review, we have examined QCM biosensors and indicated the applicability of establishing aptamer-attached QCM biosensors for quick, high affinity, sensitive, and label-free detection of biological analytes. We have presented and discussed a considerable amount of research on the use of aptamer-based quartz crystal microbalance technology. We have divided these studies into various sections according to analyte and technique, summarizing aptamer-based QCM platform information in [fig_ref] Table 1: The advantages and major challenges of QCM biosensors prepared by various techniques [/fig_ref]. By developing more aptamers, new aptasensors can be designed which can play a significant role in the development of future diagnostic methods. The design of aptamers with QCM devices has been successful for both quantitative and qualitative medical applications. Aptamers are considered smart receptors for specific binding of target molecules, including viruses, cells, proteins, bacteria, and biomarkers. Several methods have been advanced to transform target molecule−aptamer binding combinations into physically detecTABLE Signals. The progress of QCM biosensing based on aptamers holds great prospects for the development of new medical applications and analytical platforms. Advanced research, particularly that dedicated to the precision, accuracy, and robustness of the reviewed techniques, is needed in this area.
[fig] Figure 1: Process of SELEX technology. Reprinted with permission from Ref. [31] 2021, Liu. [/fig]
[fig] Figure 2: Working principle of a biosensor. [/fig]
[fig] Figure 3: A quartz crystal with gold electrodes: (a) typical AT-cut piezoelectric crystal coated with two gold electrodes, one on each side; reprinted with permission from Ref.[75] 2015, Bragazzi. Modified gold electrode (b) and bare gold surface (c) of AT-cut quartz with keyhole electrode. Reprinted with permission from Ref.[105] 2010, Biemmi. [/fig]
[fig] Figure 4: Preparation of aptamer hydrogel-coated QCM aptasensor and QCM sensorgram: ( created on the sensor in equilibrium with distilled water; (2) NHS/EDC activation; (3) poly mide) co-aptamer polymer hydrogel immobilization; (4) washing to obtain the baseline. Re with permission from Ref.[114] 2013, Wang. [/fig]
[fig] Figure 5: (a) Bifunctional dithiol immobilization process onto QCM gold surface; (b) SEM image nanowell-based QCM electrode: (I) the bare QCM electrode and (II) the nanoporous gold film mo ified QCM electrode; (c) design process of NH2-aptamer immobilization and QCM aptasensor se sorgram graph: (1) the SAM generated on the sensor in equilibrium with distilled water; NHS/EDC activation; (3) NH2-aptamer immobilization; and (4) washing to obtain the baseline. R printed with permission from Ref. [115] 2017, Wang. [/fig]
[fig] Figure 6: (a) SELEX process of DNA aptamers; (b) preparation of the QCM aptasensor. Reprinted with permission from Ref.[106] 2017, Wang. [/fig]
[fig] Figure 7: (A) Illustration of process of loading cargo molecules into AuNCs; (B) preparation of aptasensor for detection of thrombin; and (C) thrombin-triggered release of cargo molecules. Reprinted with permission from Ref. [126] Xi. Hianik et al. developed a novel QCM aptasensor device for the detection of thrombin. [/fig]
[fig] Figure 8: (a) Design of QCM aptasensor for detection of leukemia cells, reprinted with permission from Ref. [140] 2014, Shan. (b) Capture of HepG2 Cells on aptamer immobilized QCM gold surface electrode, reprinted permission from Ref. [141] 2014, Kashefi-Kheyrabadi. [/fig]
[table] Table 1: The advantages and major challenges of QCM biosensors prepared by various techniques. [/table]
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A Rare Case of Radius Head Epiphyseal Aneurysmal Bone Cyst with Predisposing Factor as Trauma Tuberculosis of Elbow apart from Genetic
Introduction: Aneurysmal bone cysts (ABCs) are benign aggressive bone lesions arising predominantly in the second decade of population in long bones at metaphyseal region which can cause local pain, swelling, and pathologic fracture. With two types, primary and secondary. diagnosis is made with various imaging modalities, although biopsy is important for diagnosis. Currently, the standard treatment is curettage or excision. Along with adjuvant therapy, chemical physical radiotherapy pharmacological to decrease recurrence was more in previous decades. Case Report: A 40-year-old male presented with the complaint of pain swelling over the right elbow but no fever for the past10days with a history of trauma 15 days back. In the past, history of tuberculosis of the elbow treated 10 years back on antituberculosis therapy, antitubercular drugs, and surgery was done and recovered with satisfactory result. On investigation X-ray -pathological fracture of radial head with eccentric ballooned expanded radiolucency margin well circumscribed lesion bone cyst. Surgery aim was to excise diseased part and get good functional recovery. Wide excision of tumor was done via anterolateral approach with injection of phenol at excised tumor site Tumor bone soft tissues was send for biopsy after surgery .Post-operative recovery was satisfactory with good reasonable range of movement of elbow was achieved Conclusion: A rare case of primary ABC at epiphyseal region rarest site less common bone involved radius has been reported with no reconstruction done due to it extensive extent with local predisposing factor apart from genetic. ABCs are aggressive benign lesions with high rates of recurrence challenging treatment, but it is curable when approached with multimodality treatment surgical along with radiotherapy pharmacological chemical physical.AbstractCase Report
# Introduction
Dr. Jaffe and Lichenstein described first aneurysmal bone cysts (ABCs) in pelvic and spine lesions in 1942.Non-neoplastic expansile lytic lesion consisting of blood filled spaces separated by connective tissue septa containing bone or osteoid and osteoclast giant cells also known as multilocular hematic cyst or giant cell reparative granuloma accounting1.4 % of all tumors is locally aggressive benign cyst lesion. Aetiology is unknown but develop due to vascular disturbance increase vascular pressure in venous network of bone tumor that result in dilatation of small vessel haemorrhage which causes erosion and resoprtion of bone matrix lead to uncommon expansile osteolytic lesion of bone consisting of a proliferation of vascular tissue that forms a lining around blood filled cystic lesion genetically, ABCs harbor translocation of TRE17/USP6, leading to its transcriptional up regulation. TRE17 encodes a ubiquitin-specific protease and a TBC domain that mediates binding to the Arf6 GTPase. However, TRE17 over expression contribute to tumor pathogenesis. ABCs are commonly seen in childhood and young adulthood with 90% of lesions which are found before age 30 s decades of life. ABC is more common in female with a male:female sex ratio of 1:1.16.ABCs are more common in metaphysis of long bones 67% (femur tibia fibula upper extremity) pelvis 9% Spine 15% posterior element. Also seen in clavicle, foot, and fingers.ABCs are mostly solitary and are currently thought to arise either as a primary neoplasm 70% genetic as cause or secondary lesions 30% arising secondary to Osteoblastomas, Chondroblastoma's, or Giant cell tumors(GCT) Osteosarcoma chondromyxoid fibroma, nonossifying fibromas, or fibrous dysplasia. These secondary ABCs account for nearly 30% of all ABCs, and they are not considered a neoplasm because no known translocation or genetic aberrancy is present. Differential diagnosis of ABC are unicameral bone cyst GCT osteoblastoma telangiectatic osteosarcoma chondromyxoid fibroma. Clinically, patients present with pain due to the destruction of bone and pathological fracture # swelling at the involved extremity. In the spine, backache and neurological deficit may present and clinically present as (a) inactive,(b) active, and (c) aggressive types.Radiograph radiolucent lesion with expanded cortex arising in medullary canal of metaphysis aneurysmal expanded appearance of cortex is contained by periosteum and thin shell of bone marked cortical thinning and erosion with periosteal elevation. This lesion rarely penetrates the articular surface or growth plate; computed tomography (CT) scan look for fluidfluid level(blood/serum CT scan shows a cystic lesion with intraosseous and extraosseous extend of lesion). Cortical thinning and multiple septal like structures were noted and not appreciated on the radiograph. Bone scan shows intense uptake in the margin of the lesion, with normal background or decreased uptake in its center. Magnetic resonance images (MRI)bright on T2 and fat suppression and intermediate or low signal on T1 double density fluid level, and septation are also suggestive of ABC expansile lytic eccentric septated lesion containing character i st ic f luid-f luid level.
Histopathology pathology lesion is formed by a thin shell of bone enclosing cystic blood-filled space with friable mass. Microbiology -spongy bone/marrow replaced by pools of blood enclosed in fibrous-osseous sacs which are filled fibroblast, multinucleated osteoclast type gaint cells and reactive woven bone rimmed by osteoblasts 30% cases have bone basophilic called blue bone. Treatment gold standard is open surgery, resulting in good local control excision or curettage w ith local adjuvant treatment w ith some complication rate. Non-invasive methods such as embolization. Radiotherapy has also been used, but a major concern is the risk for secondary malignancies. More recently, the use of sclerotherapy has proved an easy and safe method which is associated with good local control and few side effects. Recently, medical treatments, denosumab, block the osteolytic pathway, which has given very promising result. Curettage and bone grafting with adjuvant therapy to decrease recurrence are the main stay of treatment. Curettage can be aggressive, and excision can be marginal or wide with or without bone grafting is preferable treatment. Curettage is associated with an acceptable rate of local control.Adjuvant treatment is chemical-physical radiotherapy pharmacological to decrease recurrence phenol, cryosurgery, arterial embolization, argon beam coagulation, polymethylmethacrylate (PMMA), liquid nitrogen, sclerotherapy, and high-speed burrmedical treatment -denosumab bisphosphonate doxycycline. Overall recurrence 5-35% in young average time before recurrence was 18.7 months. En bloc excision, or complete resection, is surgical procedure with high morbidity with the lowest rates of recurrence which is subperiosteal resection of lesion. Cement following curettage, bone graft reconstruction is commonly done to promote osseous healing of the resultant cavity. PMMA cement in pediatric benign bone lesions provides immediate stabilization for the resultant cavity, and it exothermic effect helps in reducing recurrence after cementJournal of Orthopaedic Case Reports Volume 9 Issue 1 Jan-Feb 2019 Page 23-27 | | | | Chhawra S et al : (a-d)Pre-operative and post-operative X-rays. : (a-c) Intraoperative pictures. hardens. Highspeed burr is done to augment curettage by mechanical disruption of the lesion. Phenol carbolic acid helps by sterilizing or washing the lesion, removing remaining neoplastic cells following curettage. Selective arterial embolization N 2-butylcyanoacrylate can be used as an adjunct to surgery, but it has also been employed as a primary treatment in ABC lesions that are difficult to access (i.e., pelvis and sacrum) or are at considerable risk for hemorrhage. Sclerotherapy acts by damaging the endothelium of vessels, triggering the coagulation cascade, and resulting in thrombosis. By inducing sclerosis of the ABC vascular network, local control of the lesion can be achieved. (a) Polidocanalinjection s c l e r o r a n t , ( b ) d o x y c y l i n e i n j e c t i o n , a n d (c)Ethiblocareradiopaque alcoholic solutions that cause local fibrogenic and thrombogenic effects on contact with ABCs which havegoodresult. Cryosurgery is the use of liquid or aerosolized nitrogen to generate freezing temperatures that have a cytotoxic effect on the ABC lesion following curettage. Radiotherapy consists of external beam radiation to induce cellular death as an adjuvant therapy in cases of recurrence, and in inoperable ABC lesions, it is emerging treatment for lesion that is not amenable to other treatment but adverse side effects. Radionuclide ablation involves the intralesional injection of radioisotopes, which emit ionizing radiation that ablate adjacent tissue. Argon beam coagulation produces a unipolar electrical current through tissue to induce desiccation and coagulation. Directing argon beam therapy at an ABC lesion following curettage has been shown to reduce recurrence rates. Pharmacological treatment is very effective in tumor.
In bone cell receptor-activator of nuclear kappa B ligand (RANKL) signalling pathway is mediator in bone homeostasis by promoting osteoclast activation, bone resorption and remodelling. In various benign and malignant bone neoplasms, the role of RANKL expression is there.ABCs have higher than normal levels of RANKL expression. RANKL signaling is inhibited by denosumab in which a human monoclonal antibody is used in osteoporosis, GCT of bone, in metastasis and recently has being started in ABC with promising result. Dox yc ycl ine i s an ant i b iot ic inhi b it ion of matr i x metalloproteinase angiogenesis and anti-neoplastic properties play a role in inhibiting ABC expansion within bone.
Bisphosphonates are pyrophosphate analogs that inhibit osteoclast-mediated bone resorption with antineoplastic characteristics possibly by inducing apoptosis, inhibiting tumor cell adhesion and invasion by inhibiting angiogenesis so help in pain relief. So both drugs doxycycline Bisphosphonates used in ABC.
## Case report
A 40-year-oldmale presented with the complaint of pain swelling over the right elbow but no fever for the past 10 days with a history of trauma 15 days back. In the past, a history of tuberculosis (TB) of elbow treated 10 years back on antituberculosis therapy, antituberculardrugs, and surgery synovectomy debridement of elbow joint through lateral approach later patient recovered well with satisfactory result. On physical examination, there was tenderness over lateral aspect of the elbow with restricted movement of elbow and wasting of forearm muscle. Swelling over elbow with no sign of inflammation or infection skin was normal. On investigation Xray -pathological fracture of Radial head with eccentric ballooned expanded Radiolucency margin well circumscribed lesion Bone cyst . MRI -mildly expansile lesion at proximally epiphysis-metaphysis adjacent to diaphyseal location of radius extending upto sub articular location abutting articular cartilage. Surgery was planned; the aim was to excise diseased part and get good functional recovery wide excision of tumor through anterolateral approach with injection of phenol . Tumor bone soft tissues send for biopsy including Radial head which was involved up to articular cartilages,Radial head reconstruction was not done because ABC extended from metaphysic -diaphysis junction to epiphysis cartilage of radial head. Post-operative recovery was satisfactory with good reasonable range of movement of the elbow.
# Discussion
This case is reported with rarity in percentage and rarity in the location of site and bone. The epiphyseal ABC is rare to present with location over proximal radius head which is only 3%. The ABC common site presentation is long bones humerus femur tibia with location over metaphyseal region here in this case epiphyseal location rare to see. In this case apart from genetic cause recent trauma and 10 years back, the treated case of TB elbow may be predisposing factor which is something unusual different. The treatment of ABC is surgery curettage -excision, reconstruction and adjuvant procedure to prevent recurrence such as liquid nitrogen, phenol or PMMA injection at the site of tumor arterial embolization. Recent denosumab injection therapy had decreased recurrence of tumor. In this case, wide excision done with phenol injection was given, but no reconstruction was done due to epiphyseal radial head involvement and its extension to metaphyseal region reconstruction not done to prevent recurrence. Post-operative patient gained a reasonable range of movement with stability. Recurrence is 10-60% which is observed more in young children if surgery is done alone curettage with no adjuvant treatment. Hence, now, treatment protocol is surgical excision with various adjuvant treatments to prevent recurrence with minimum complication and morbidity.
# Conclusion
A rare case of primary ABC at epiphyseal region rarest site less common bone radius is involved has been reported. Surgery has being done to excise tumor without reconstruction of radius head because of it extensive extent with local predisposing factor apart genetic. ABCs are aggressive benign lesions with high rates of recurrence challenging treatment, but it is curable when approached with multimodality treatment surgical along with chemical-physical radiotherapy pharmacological. The standard treatment remains curettage or excision with grafting to fill the bone void, with different adjuvant or alternative treatment methods to reduce recurrence. ABCs in anatomic locations where surgery would cause significant morbidity are most often treated with embolization or radiotherapy, and recently, medical management with denosumab has given a promising result.
Journal of Orthopaedic Case Reports Volume 9 Issue 1 Jan-Feb 2019 Page 23-27 | | | |
## Clinical message
ABC is rare tumor challenging in its presentation in bone with it site percentage and approach of treatment. The mainstay of treatment is surgical excision or curettage with adjuvant treatment chemical-physical radiotherapy pharmacological treatment to prevent recurrence with regular follow-up which gives the best curable result with less complication and morbidity. |
Adjusting ferritin concentrations for inflammation: Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project
Background: The accurate estimation of iron deficiency is important in planning and implementing interventions. Ferritin is recommended as the primary measure of iron status, but interpretability is challenging in settings with infection and inflammation. Objective: We assessed the relation between ferritin concentrations and inflammation and malaria in preschool children (PSC) (age range: 6-59 mo) and women of reproductive age (WRA) (age range: 15-49 y) and investigated adjustment algorithms to account for these effects. Design: Cross-sectional data from 15 surveys for PSC (n = 27,865) and 8 surveys for WRA (24,844), from the Biomarkers Reflecting the Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed individually and combined with the use of a meta-analysis. Several approaches were explored to estimate depleted iron stores (ferritin concentration ,12 mg/L in PSC and ,15 mg/L in WRA) in inflammation and malaria settings as follows: 1) increase ferritin-concentration cutoff to ,30 mg/L; 2) exclude individuals with C-reactive protein (CRP) concentrations .5 mg/L or a-1-acid glycoprotein (AGP) concentrations .1 g/L; 3) apply arithmetic correction factors; and 4) use a regression correction approach. Results: Depleted iron-store estimates incrementally increased as CRP and AGP deciles decreased (4% compared with 30%, and 6% compared with 29% from highest compared with lowest CRP deciles for pooled PSC and WRA, respectively, with similar results for AGP). Depending on the approach used to adjust for inflammation (CRP plus AGP), the estimated prevalence of depleted iron stores increased by 7-25 and 2-8 absolute median percentage points for PSC and WRA, respectively, compared with unadjusted values. Adjustment for malaria in addition to CRP and AGP did not substantially change the estimated prevalence of depleted iron stores. Conclusions: Our results lend support for the use of internal regression correction to estimate the prevalence of depleted iron stores in regions with inflammation. This approach appears to mathematically reflect the linear relation of ferritin concentrations with acute-phase proteins. More research is warranted to validate the proposed approaches, but this study contributes to the evidence base to guide decisions about how and when to adjust ferritin for inflammation.Am J Clin Nutr 2017;106(Suppl):359S-71S.
# Introduction
Iron deficiency remains an important public-health problem primarily in women and children [bib_ref] Iron homeostasis, Andrews [/bib_ref] [bib_ref] Iron deficiency: global prevalence and consequences, Stoltzfus [/bib_ref]. Iron deficiency has been estimated to affect 1.62 billion people worldwide, although these figures are approximations because of limited available estimates of iron deficiency globally [bib_ref] Worldwide prevalence of anaemia, WHO Vitamin and Mineral Nutrition Information System, Mclean [/bib_ref]. The accurate measurement of iron deficiency is important in the planning and implementation of interventions and for comparisons between countries or geographic areas.
The WHO currently recommends low ferritin concentrations (termed depleted iron stores) as the primary measure of population-level iron deficiency because it reflects body iron stores, has a long history of use with well-established laboratory methods and cutoffs, is easier to measure than more-invasive methods (e.g., bone marrow iron), and is responsive to interventions. However, in populations with high levels of inflammation, ferritin concentrations not only reflect nutritional iron but are also impacted by the acute-phase response (APR) [bib_ref] Assessing micronutrient status in the presence of inflammation, Tomkins [/bib_ref]. The APR is an immunologic process that causes certain acutephase proteins (APPs) in the body to rise or fall in response to microbial invasion, tissue injury, immunologic reactions, and inflammatory processes [bib_ref] Interpreting indicators of iron status during an acute phase response-lessons from malaria..., Ca [/bib_ref]. In addition to its role as a marker of iron stores, ferritin is a positive APP that becomes elevated during an APR. In an inflammatory state, ferritin synthesis appears to be upregulated by cytokines independent of iron homeostasis [bib_ref] Acute-phase proteins and other systemic responses to inflammation, Gabay [/bib_ref]. Ferritin concentrations may also increase in the presence of malaria infection potentially because of impaired iron incorporation or malaria-associated inflammation [bib_ref] Serum levels of erythropoietin in acute Plasmodium falciparum malaria, Burgmann [/bib_ref] [bib_ref] Increased serum hepcidin and alterations in blood iron parameters associated with asymptomatic..., De Mast [/bib_ref] [bib_ref] Assessment of urinary concentrations of hepcidin provides novel insight into disturbances in..., De Mast [/bib_ref]. Thus, not accounting for changes in the control mechanisms of iron during states of inflammation or malaria infection may translate to a significant underestimate of depleted iron stores [bib_ref] Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in..., Thurnham [/bib_ref].
Interest has emerged in methods to account for inflammation and malaria infection in the interpretation of iron biomarkers [bib_ref] Inflammation and Nutritional Science for Programs/Policies and Interpretation of Research Evidence (INSPIRE), Raiten [/bib_ref] [bib_ref] Plasma ferritin and soluble transferrin receptor concentrations and body iron stores identify..., Engle-Stone [/bib_ref] [bib_ref] Effects of inflammation and Plasmodium falciparum infection on soluble transferrin receptor and..., Righetti [/bib_ref] [bib_ref] The use of adjustment factors to address the impact of inflammation on..., Thurnham [/bib_ref]. Because it is not possible to determine the level of inflammation in apparently healthy people with the use of clinical signs or symptoms, the WHO has recommended measuring other APPs, namely C-reactive protein (CRP) and a-1-acid glycoprotein (AGP), concurrently with ferritin to confirm the presence of an APR. The WHO has suggested measuring both of these biomarkers (6) because they reflect different stages of the APR [bib_ref] Inflammation and Nutritional Science for Programs/Policies and Interpretation of Research Evidence (INSPIRE), Raiten [/bib_ref].
Several approaches have been proposed about how to use CRP and AGP to account for inflammation and infections such as malaria to increase the sensitivity of detecting depleted iron stores at the population level [bib_ref] Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in..., Thurnham [/bib_ref] [bib_ref] Plasma ferritin and soluble transferrin receptor concentrations and body iron stores identify..., Engle-Stone [/bib_ref] [bib_ref] Effects of inflammation and Plasmodium falciparum infection on soluble transferrin receptor and..., Righetti [/bib_ref]. These approaches include 1) increasing the ferritin-concentration cutoff to ,30 mg/L for the entire sample or only in individuals with elevated inflammation (practitioners have interpreted the WHO recommendations on this approach both ways); 2) excluding individuals with elevated inflammation; 3) applying arithmetic correction factors (CFs); and 4) using a regression correction (RC) approach. The first 2 approaches are currently recommended by the WHO, but there is a need to re-evaluate other approaches and reach a consensus on how to improve the estimates of the prevalence of depleted iron stores in populations with a high prevalence of inflammation.
The accurate assessment of iron deficiency is imperative to identify populations who are most in need of iron interventions (or not in need of additional iron) and to better understand the contributions of iron to other conditions including anemia. Therefore, in this article, the following 4 questions are explored: 1) Is there a need to measure biomarkers of inflammation when measuring ferritin? 2) Is there a need to measure .1 inflammation biomarker (CRP and AGP) to adjust ferritin? 3) Is an additional adjustment needed to correct ferritin concentrations in the presence of malaria infection? and 4) How do the different adjustment approaches for correcting for inflammation and for calculating the estimated prevalence of depleted iron stores compare with each other? We used data from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project (www.BRINDA-nutrition.org) [bib_ref] Overview of the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA)..., Suchdev [/bib_ref]. The BRINDA protocol was reviewed by the institutional review boards of the NIH and was deemed to be non-human subjects research. The methods for identifying data sets, inclusion and exclusion criteria, and data management for the BRINDA project have been described in detail elsewhere [bib_ref] Overview of the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA)..., Suchdev [/bib_ref]. The surveys were nationally or regionally representative, and the inclusion criteria were surveys that were 1) conducted after 2004, 2) had target groups including preschool children (PSC), nonpregnant women of reproductive age (WRA), or both and 3) measured $1 marker of iron (ferritin or soluble transferrin receptor) or vitamin A status (retinol binding protein or retinol) and $1 marker of inflammation (AGP or CRP). Surveys included in this analysis were those with measures of ferritin and inflammation (CRP or AGP). In all surveys in which WRA data were collected, PSC data were also collected as part of the same survey. Of the 16 PSC and 10 WRA BRINDA data sets, data from 15 PSC and 9 WRA surveys were applicable for analysis for this article. Malaria was measured in 5 PSC and 3 WRA data sets. Both CRP and AGP were measured in 8 of 15 PSC data sets (n = 13 surveys with CRP; n = 10 surveys with AGP) and 4 of 9 WRA data sets (n = 9 surveys with CRP; n = 4 surveys with AGP).
# Laboratory analysis
Venous or capillary blood was collected from each respondent, and plasma or serum was stored at 2208C until analysis; one survey used dried blood spots. Ferritin, AGP, and CRP concentrations were assessed with the use of a sandwich ELISA at the VitMin Laboratory in 8 data sets (Bangladesh, Cameroon, Cote d'Ivoire, Laos, Liberia, Kenya 2007 and 2010, and the Philippines) [bib_ref] Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive..., Erhardt [/bib_ref]. Ferritin was measured with the use of an immunoassay in 7 data sets (Colombia, Georgia, Mexico 2006 and 2012, Nicaragua, Pakistan, and the United States). CRP was measured with the use of an immunoassay in one data set (United States), turbidimetry in 2 data sets (Colombia, Georgia), and nephelometry in 2 data sets (Mexico 2006 and 2012). AGP was measuring with the use of an immunoassay in Pakistan and turbidimetry in Nicaragua.
A comparison between the ELISA assay at the VitMin Laboratory and commercially available immunoassay assays has yielded high specificity and sensitivity and low intra-assay and interassay variability [bib_ref] Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive..., Erhardt [/bib_ref]. The CDC laboratory also regularly conducts round-robin analyses to ensure comparability between 360S the immunoassay and ELISA assay (D Whitehead, CDC, International Micronutrient Malnutrition Prevention and Control program, personal communication, 2016). In the few cases when turbidimetry and nephelometry were used, heterogeneity may have been of concern because of the lack of literature comparing these methods with the ELISA assay and immunoassay.
Current malaria infection was assessed with the use of microscopy in Kenya and Côte d'Ivoire, and current or recent malaria infection was assessed with the use of the Paracheck Pf rapid-diagnostic test (Orchid Biomedical System) in Liberia and histidine-rich protein 2 (Cellabs Pty Ltd.) in Cameroon. A standardizing malaria diagnostic was not used because a sensitivity analysis showed a minimal impact [bib_ref] Standardizing plasmodium falciparum infection prevalence measured via microscopy versus rapid diagnostic test, Mappin [/bib_ref]. The methods for identifying data sets, inclusion and exclusion criteria, and data management for the BRINDA project have been described in the methodologic overview in this supplement, which is an open access publication.
## Case definitions
Depleted iron stores were defined as concentrations of ferritin ,12 mg/L in PSC and ,15 mg/L in WRA. The WHO-defined concentration cutoff in the presence of inflammation of ,30 mg/L was also applied in PSC and WRA (although not currently part of the WHO recommendations for WRA); the higher cutoff was applied to the entire sample as well as only in individuals with subclinical inflammation. Malaria infection was defined as either positive or negative. Inflammation was defined as a CRP concentration .5 mg/L, AGP concentration .1 g/L, or both [bib_ref] Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in..., Thurnham [/bib_ref]. A household socioeconomic status (SES) asset score was defined by survey investigators who applied a principal component analysis within each survey to household characteristics and item ownership. The poorest wealth quintile was compared with the higher quintiles. In Georgia and the United States, where these data were unavailable, household employment was used in Georgia, and a poverty-index ratio of family income to family size for the comparison of lowest to higher levels was used in the United States. Maternal education was defined as any school compared with no school.
# Statistical analysis
Descriptive statistics were calculated with the use of STATA 12.0 software (StataCorp) and cross-checked with SAS 9.4 software (SAS Institute). Correlations between ferritin, CRP, and AGP concentrations and malaria infection were calculated with the use of Kendall's t coefficient with the use of the SOMERSD package. The Taylor linearization method was used to obtain unbiased estimates that incorporated the sampling weight, strata, and cluster (as applicable) when analyzing individual surveys. To combine data, individual survey analyses, accounting for the complex survey design, were performed with the use of the survey package in R 3.2.2 software (R Core Team) [bib_ref] Analysis of complex survey samples, Lumley [/bib_ref]. Individual survey estimates were combined with the use of a meta-analysis approach with the use of the metafor package in R 3.2.2 software [bib_ref] Conducting meta-analyses in R with the metafor package, Viechtbauer [/bib_ref] [bib_ref] Meta-analysis in clinical trials, Dersimonian [/bib_ref]. The heterogeneity of estimates across the surveys was assessed with the use of Cochran's heterogeneity test [bib_ref] Conducting meta-analyses in R with the metafor package, Viechtbauer [/bib_ref] [bib_ref] Meta-analysis in clinical trials, Dersimonian [/bib_ref].
The prevalence of depleted iron stores (ferritin concentration ,12 or ,15 mg/L in WRA) was estimated without any adjustments to ferritin and was referred to as the unadjusted estimate. The following 4 approaches were used to adjust ferritin for inflammation and malaria: a higher ferritin-concentration cutoff of ,30 mg/L, exclusion, CF, and RC.
## Higher ferritin cutoff approach
The higher ferritin-cutoff adjustment approach uses a higher ferritin-concentration cutoff of ,30 mg/L. We tried 2 different approaches when the higher ferritin cutoff was used. We applied the cutoff to the entire sample as well as to the subset of individuals with elevated CRP and AGP (as defined by a CRP concentration .5 mg/L, AGP concentration .1 g/L, or both).
## Exclusion approach
The exclusion approach uses the inflammation, malaria-biomarker information, or both to exclude individuals with elevated inflammation (as defined by a CRP concentration .5 mg/L, AGP concentration .1 g/L, or both) or individuals with malaria infection from the analysis; this resulted in a smaller sample size. We excluded these individuals and calculated the estimated prevalence of depleted iron stores in the remaining individuals.
## Cf approach
The CF approach, as proposed by , uses a arithmetic CF. We calculated the CF by grouping inflammation into the following 4 groups: 1) reference (both CRP concentration #5 mg/L and AGP concentration #1 g/L); 2) incubation (CRP concentration .5 mg/L and AGP concentration #1 g/L); 3) early convalescence (both CRP concentration .5 mg/L and AGP concentration .1 g/L); and 4) late convalescence (CRP concentration #5 mg/L and AGP concentration .1 g/L). We also calculated the CF by grouping inflammation or malaria into 2 groups in which CRP, AGP, or malaria were used independently. CFs were defined as the ratio of geometric means of the reference group (nonelevated CRP, AGP, or malaria negative depending on the grouping used) to those of the respective inflammation group or malaria group. CFs were calculated based on internal survey-specific data [termed the internal correction factor (ICF)], a previous meta-analysis with the use of distinct data from the BRINDA data set [termed the Thurnham correction factor (TCF)] [bib_ref] Inflammation and Nutritional Science for Programs/Policies and Interpretation of Research Evidence (INSPIRE), Raiten [/bib_ref] , and the metaanalyzed BRINDA data set [termed the BRINDA correction factor (BCF)]. The CF approach was performed for CRP alone (termed ICF-CRP, TCF-CRP, BCF-CRP), AGP alone (termed ICF-AGP, TCF-AGP, BCF-AGP), and both CRP and AGP (termed ICF-CRP1AGP, TCF-CRP1AGP, BCF-CRP1AGP). CFs were further calculated for malaria with the use of the ICF approach only.
## Rc approach
The RC approach uses linear regression to adjust ferritin concentrations by the CRP and AGP concentrations on a continuous scale and malaria infection as a dichotomous variable as defined in the BRINDA methods article. Briefly, the adjusted ADJUSTING FERRITIN FOR INFLAMMATION 361S ferritin equation was calculated by subtracting the influence of CRP, AGP, and malaria as follows:
[formula] Ferritin adjusted ¼ ferritin unadjusted 2 b 1 À CRP obs 2 CRP ref Á 2 b 2 À AGP obs 2 AGP ref Á 2 b 3 malariað1Þ [/formula]
Depending on the available data, CRP, AGP, or malaria can be included in the model. b 1 is the CRP regression coefficient, b 2 is the AGP regression coefficient, b 3 is the malaria regression coefficient, obs is the observed value, and ref is the external reference value generated to define low inflammation (the maximum values of the lowest CRP or AGP decile with the use of combined BRINDA data with nonlogged reference values as follows: CRP concentration in PSC: 0.10 mg/L; CRP concentration in WRA: 0.16 mg/L; AGP concentration in PSC: 0.59 g/L; AGP concentration in WRA: 0.54 g/L). CRP, AGP, and ferritin were all ln transformed; CRP and AGP were continuous variables, and malaria was a dichotomous variable. The correction was only applied to individuals with ln CRP greater than ln CRP ref or ln AGP greater than ln AGP ref to avoid overadjustments. The first step in the RC approach was to ln transform ferritin, CRP, and AGP concentrations to approximate normality on the basis of regression diagnostics. CRP data contained values of zero, and thus, the survey-specific lowest value was used to replace zeros before applying the ln transformation. Second, linear regression coefficients for CRP, AGP, malaria, or a combination were estimated (bivariate and multivariate) with ferritin as the outcome. A test of multicollinearity between ln CRP and ln AGP or between malaria and ln CRP or ln AGP was assessed on the basis of a test of tolerance (.0.1) and a variance inflation factor (,5) to determine whether it was appropriate to include all variables in the model. Third, an ln-CRP reference value was subtracted from the ln-CRP value, and an ln-AGP reference value was subtracted from ln-AGP concentrations in the regression equation.
The RC approach is presented based on internal surveyspecific data [termed the internal regression correction (IRC)] and the meta-analyzed BRINDA data [termed BRINDA regression correction (BRC)]. An illustrative example of how to adjust ferritin concentrations for inflammation with the use of the IRC approach on the basis of real data from Liberia in PSC is provided in Supplemental [fig_ref] FIGURE 1: Estimated prevalence [% [/fig_ref]. The BRC approach entailed the replacement of CRP and AGP b coefficients in the adjusted ferritin Equation 1 with the meta-analysis b coefficients. The same external reference values were used when applying both the IRC and BRC approaches. Each of these RCs was performed for CRP alone (termed IRC-CRP, BRC-CRP), AGP alone (termed IRC-AGP, BRC-AGP), or both CRP and AGP (termed IRC-CRP1AGP, BRC-CRP1AGP). The IRC was also performed for malaria alone, malaria plus CRP alone, malaria plus AGP alone, and malaria plus both CRP and AGP. The BRC approach was not applied when including malaria in the model because of the limited number of data sets that measured malaria. We also tested whether potential confounders significantly influenced the relation between ferritin and inflammation. We added sex, age (months in PSC and years in WRA), maternal education, and SES to the model, and the CRP and AGP slopes that were adjusted for these confounders were extracted and used in the adjusted ferritin equation.
## Comparison of adjustments
Unadjusted and adjusted prevalence estimates of depleted iron stores were compared with the use of McNemar's chi-square statistics; statistical significance was defined as P , 0.05 before applying the Bonferroni corrections to correct for multiple comparisons (P = 0.05 O k, where k equals the number of comparisons). To examine whether malaria infection remained associated with estimated depleted iron stores after adjustment for CRP and AGP, we stratified the adjusted estimated prevalence of depleted iron stores (ferritin concentrations adjusted with the use of the IRC approach) by malaria status and compared the prevalence of individuals with and without malaria with the use of the adjusted Wald's test to determine the effect of malaria in addition to elevated APPs. It was decided a priori that adjustments for CRP and AGP together and for CRP, AGP, and malaria combined would be explored irrespective of the significance between the malaria and nonmalaria estimated prevalences of depleted iron stores.
# Results
## Participant characteristics
Our study sample was restricted to participants with no missing values for ferritin, CRP, AGP, or malaria (in surveys that measured malaria); this resulted in a total loss of 6.4% (1901 of the 29,766) of the observations that met the BRINDA inclusion criteria in PSC and of 3.4% (887 of 25,731) of the observations that met the BRINDA inclusion criteria in WRA. Participants who were excluded because of missing ferritin, CRP, AGP, or malaria data did not differ from those who were included with regard to sex, age, or SES (data not shown). The PSC mean age range was 8.3-41.5 mo, and there was considerable age-range variability notably for Bangladesh (6-11 mo), Liberia, Kenya 2007 and 2010 (6-35 mo), and the Philippines (6-23 mo), whereas the age range in the remaining 10 surveys was from either 6-59 mo (n = 5) or 12-59 mo (n = 5) [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. In contrast, WRA age ranged from 15 to 49 y in all surveys with a mean age range of 27.2-34 y [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. The prevalence of inflammation varied across surveys in PSC (CRP concentration .5 mg/L: 6.0-40.4%; AGP concentration .1 g/L: 21.2-64.5%) and WRA (CRP concentration .5 mg/L: 7.9-29.5%, AGP concentration .1 g/L: 7.2-26.9%) [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. The prevalence of malaria varied by 13 percentage points (pps) in both PSC (19.7-32.5%) and WRA (5.0-17.9%) [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref].
## Relation between ferritin, inflammation, and malaria
The rank correlations between the 2 markers of the APR, CRP and AGP ranged from 0.48 to 0.63 in PSC and from 0.26 to 0.41 in WRA and were significant (P , 0.001) in all surveys (Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. There was a moderately positive association between ferritin with CRP and AGP, and the strength of the relations were similar for both APPs and across PSC surveys (CRP range: 20.02 in Georgia to 0.44 in Kenya 2010; AGP range: 0.04 in Pakistan to 0.44 in Kenya 2010) except between ferritin and CRP in Georgia and between ferritin and AGP in Pakistan. The relation between ferritin with CRP and AGP was weaker in WRA than in PSC. The rank correlations between malaria (defined as positive or negative) and ferritin ranged from 0.14 to 0.25 in PSC and from .20.01 to 0.07 in WRA (Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. The rank correlations between malaria and CRP or AGP ranged from 0.12 to 0.27 in PSC and from 0.04 to 0.10 in WRA.
The relation between the estimated prevalence of depleted iron stores and inflammation deciles visually appeared to follow a linear pattern in PSC [fig_ref] FIGURE 1: Estimated prevalence [% [/fig_ref] and WRA [fig_ref] FIGURE 2: Estimated prevalence [% [/fig_ref] with the estimated prevalence in depleted iron stores incrementally increasing as the concentrations of CRP and AGP decreased. The relation between the estimated prevalence of depleted iron stores and inflammation declines did not vary according to whether subjects were above or below CRP and AGP cutoffs. In the lowest CRP decile group, the proportion of estimated depleted iron stores (ferritin concentration ,12 mg/L) was substantially higher (29.6%) than in the highest CRP decile group (4.2%) in PSC; similarly, the estimated prevalence of depleted iron stores was markedly higher in the lowest AGP decile (31.0%) than in the highest AGP decile (8.3%). The prevalences between highest and lowest decile of inflammation for WRA followed the same pattern as for PSC; the estimated prevalence of depleted iron stores (ferritin concentration ,15 mg/L) was 6.1% and 29.0% for the highest and lowest CRP decile groups, respectively, and 7.9% and 26.0% for the highest and lowest AGP decile groups, respectively.
## Cfs for ferritin, inflammation, and malaria
The geometric mean of ferritin was lowest in individuals with no inflammation in PSC and WRA compared with in individuals with inflammation with and without malaria (as a dichotomous variable) [fig_ref] TABLE 2: Ferritin in preschool children and women of reproductive age according to inflammation... [/fig_ref]. Individuals who were stratified by malaria status (as a dichotomous variable) and by the 4-inflammation groups had the highest ferritin concentrations in the group with malaria and elevated CRP and AGP in PSC but not in WRA [fig_ref] TABLE 2: Ferritin in preschool children and women of reproductive age according to inflammation... [/fig_ref]. The CFs were ,1 across all group combinations (CRP, AGP, and malaria) and surveys for both PSC and WRA. The TCFs that were derived from a previous meta-analysis in which all population groups were combined (incubation: 0.77; early convalescence: 0.53; late convalescence: 0.75), were similar to the BCFs although we showed the BCFs in PSC (incubation: 0.68; early convalescence: 0.38; late convalescence: 0.65) were lower than in WRA (incubation: 0.66; early convalescence: 0.52; late convalescence: 0.82).
## Rc slopes for ferritin, inflammation, and malaria
Bivariate linear regression with ln ferritin as the outcome resulted in an unstandardized ln-CRP slope that ranged from ,20.01 (Georgia) to 0.31 (Côte d'Ivoire) and an ln-AGP slope from 0.20 (Philippines) to 1.84 (Kenya 2010) in PSC (Supplemental [fig_ref] TABLE 2: Ferritin in preschool children and women of reproductive age according to inflammation... [/fig_ref]. In WRA, the unstandardized ln-CRP slope ranged from 0.06 (Colombia) to 0.19 (Mexico 2012) for CRP and from 0.45 (Laos) to 0.71 (Cameroon and Liberia) for AGP (Supplemental. There was no multicollinearity between ln CRP and ln AGP or between ln CRP, ln AGP, and malaria in the PSC and WRA models across surveys. A multivariate analysis of ln ferritin with both ln CRP and ln AGP in the model generally dampened the ln-CRP slope and ln-AGP slope in PSC and WRA (Supplemental as did the inclusion of malaria (data not shown).
Heterogeneity was shown between the slopes that were metaanalyzed to obtain the external slope for the BRC approach. The test of residual heterogeneity QE (df = 21) = 645 and QE (df = 9) = 30 for PSC and WRA, respectively, when both CRP and AGP were in the model. The highest degree of heterogeneity was shown when creating a bivariate meta-analyzed AGP slope in PSC. Although all surveys were retained in the combined analyses, the removal of Pakistan from the model reduced the heterogeneity from QE (df = 18) = 1779 to QE (df = 16) = 593, although it was still significant (P , 0.001). Similarly, if Georgia was excluded from the WRA data when creating a bivariate meta-analyzed CRP slope, the heterogeneity changed from QE (df = 16) = 2358 to QE (df = 14) = 376. Neither Georgia nor Pakistan was included when creating the multivariate meta-analyzed CRP and AGP slopes because both surveys only included one marker of inflammation.
## Unadjusted prevalence of depleted iron stores
There was substantial variation in the prevalence of unadjusted depleted iron stores (ferritin concentration ,12 mg/L in PSC and ,15 mg/L in WRA) across surveys ( 1 Reference is defined as a CRP concentration #5 mg/L and AGP concentration #1 g/L; incubation is defined as a CRP concentration .5 mg/L and AGP concentration #1 g/L; early convalescence is defined as a CRP concentration .5 mg/L and AGP concentration .1 g/L; and late convalescence is defined as an AGP concentration .1 g/L and CRP concentration #5 mg/L. AGP, a-1-acid glycoprotein; BRINDA, Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia; CRP, C-reactive protein.Surveys had to have a marker for CRP, AGP, and malaria to be included and were as follows: Cameroon, Côte d'Ivoire, Kenya 2007, Kenya 2010, and Liberia (preschool children); and Cameroon, Côte d'Ivoire, and Liberia (women of reproductive age). [bib_ref] Iron homeostasis, Andrews [/bib_ref] Geometric mean; 95% CI in parentheses (all such values).
## 364s
in the surveys that measured both CRP and AGP (PSC range: 8.0-38.9%; WRA range: 12.9-22.7%). In the surveys in which data were available for both PSC and WRA, there was considerable variation in terms of which target group (PSC or WRA) had a higher prevalence of depleted iron stores.
Adjusted estimated prevalence of depleted iron stores with the exclusion of subjects with inflammation to adjust ferritin for inflammation
The exclusion of PSC with elevated CRP or AGP resulted in a substantial sample-size loss of 26-68% in PSC and 14-34% in WRA. In the surveys in which CRP was measured, the greatest absolute change in the estimated prevalence of depleted iron stores of subjects with CRP concentrations #5 mg/L compared with that of the full population was in Kenya 2010 (an estimated prevalence of 27.4% compared with 19.2%, respectively) for PSC and in Mexico 2012 (30.4% compared with 27.9%, respectively) for WRA. In surveys in which AGP was measured, the greatest absolute change in the estimated prevalence of depleted iron stores of subjects with AGP concentrations #1 g/L compared with that of the full population was in Kenya 2010 (34.1% compared with 19.2%, respectively) in PSC and in Côte d'Ivoire (an estimated prevalence of 16.2% compared with 13.6%, respectively) in WRA. The greatest absolute change in the estimated prevalence of depleted iron stores in subjects with CRP concentrations #5 mg/L and AGP concentrations #1 g/Lcompared with that in the full population was an estimated prevalence of 35.2% compared with 19.2%, respectively (Kenya 2010), in PSC and an estimated prevalence of 16.6% compared with 13.6%, respectively (Côte d'Ivoire), in WRA.
Adjusted estimated prevalence of depleted iron stores with the use of CRP or AGP to adjust ferritin for inflammation by the CF and RC adjustment approach
The estimated prevalence of depleted iron stores in PSC incrementally increased compared with the unadjusted prevalence when CRP only, AGP only, and both CRP and AGP were used to adjust ferritin concentrations in general across all surveys and across adjustment methods (Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. For example, with the use of the ICF approach, the estimated prevalence of depleted iron stores in surveys that measured CRP and AGP resulted in an absolute median increase of 4.6 pps (range: 1.0-7.8 pps) when the ICF-CRP and of 5.9 pps (range: . The estimated prevalence of depleted iron stores with the use of the IRC approach compared with the unadjusted prevalence in surveys that measured CRP and AGP resulted in a higher absolute median increase when the IRC-CRP approach was used (19.7 pps; range: 4.7-26.6 pps) than when the IRC-AGP approach was used (15.9 pps; range: 7.3-31.8 pps); however, in most individual surveys, the absolute increase was greater when the IRC-AGP approach was used than when the IRC-CRP approach was used in PSC [fig_ref] FIGURE 3: Estimated prevalence [% [/fig_ref] , Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. Similar to PSC, the estimated prevalence of depleted iron stores in WRA increased when correcting for CRP, AGP, or both (Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. However, the estimated prevalence of depleted iron stores in WRA in surveys that measured CRP and AGP was much lower than in PSC and showed an absolute median increase of 1.4 pps (range: 1.0-1.7 pps) with the use of the ICF-CRP approach and of 0.95 pps (range: 0.7-2.3 pps) with the use of the ICF-AGP approach compared with unadjusted values (Supplemental . The estimated prevalence of depleted iron stores in WRA in surveys that measured CRP and AGP showed an absolute median increase of 6.0 pps (range: 4.0-9.5 pps) with the use of the IRC-CRP approach and of 6.1 pps (range: 2.9-6.9 pps) with the use of the IRC-AGP approach compared with unadjusted values (Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref].
## Comparison of estimated prevalence of depleted iron stores with the use of different approaches to adjust ferritin for inflammation
The application of adjustments to ferritin concentrations, irrespective of the method, increased the estimated prevalence of depleted iron stores compared with unadjusted estimates; generally, there was a greater pp difference in PSC than in WRA [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. The use of a higher ferritin-concentration cutoff of ,30 mg/L for all PSC (regardless of inflammation status) resulted in large absolute increase in estimates of depleted iron stores upwards of 35 pps compared with unadjusted values (Supplemental . All other adjustments that required the use of inflammation data and comparisons between the different approaches described in this section are limited to adjustments for both CRP and AGP. Adjustments with the use of only CRP or AGP are shown in Supplemental Tables With the use of a higher cutoff (ferritin concentration ,30 mg/L) in PSC with elevated inflammation (CRP concentration .5 mg/L or AGP concentration .1 g/L) resulted in an absolute median increase of 12.2 pps (range: 8.0-22.4 pps) in the estimated prevalence of depleted iron stores compared with unadjusted values [fig_ref] FIGURE 4: Estimated prevalence [% [/fig_ref] , Supplemental and a much lower increase in WRA (absolute median: 3.0 pps; range: 2.1-5.8 pps) [fig_ref] FIGURE 5: Estimated prevalence [% [/fig_ref] , Supplemental . Prevalences that were generated with the use of the higher cutoff in individuals with elevated inflammation minus the prevalences that were generated with the use of the ICF-CRP+AGP approach resulted in a lower estimated prevalence of depleted iron stores (absolute median difference: 25.6 pps; range: 27.1 to 2.7 pps) and, with the use of the IRC-CRP+AGP approach, resulted in a higher estimated prevalence of depleted iron stores (absolute median difference: 11.7 pps; range: 21.6 to 22.7 pps) in PSC. When nonmalaria and malaria countries were examined separately, the IRC-CRP+AGP was similar (absolute median difference: 20.8 pps; range: 21.6 to 1 pps) to the higher cutoff that was applied to individuals with elevated inflammation in nonmalaria countries, but not in the malaria countries (absolute median difference: 16.7 pps; range: 11.4-22.7 pps) in PSC. The difference between the higher cutoff in individuals with elevated inflammation resulted in a
## 366s
lower difference (absolute median difference: = 21.4 pps; range: 20.9 to 23.2 pps) and a higher difference (absolute median difference: 3.7 pps; range: 2.1-6.5 pps) than was shown with the use of the ICF-CRP+AGP and IRC-CRP+AGP approaches, respectively, in WRA.
When the ICF-CRP+AGP, BCF-CRP+AGP, and TCF-CRP+AGP adjustment approaches were applied to ferritin concentrations, the estimated prevalence of depleted iron stores increased by an absolute median difference of 6.5 pps (range: 2.6-16.6 pps), 7.3 pps (range: 4.3-14.6 pps), and 4.0 pps (range: 1.7-9.7 pps), respectively, . In contrast, the increase in the estimated prevalence in WRA was much lower [ICF-CRP+AGP absolute median difference: 1.6 pps (range: 1.2-2.6 pps); BCF-CRP+AGP absolute median difference: 1.6 pps (range: 1.3-2.9 pps); and TCF-CRP+AGP absolute median difference: 1.5 pps (range: 0.8-2.7 pps)] compared with unadjusted values [fig_ref] FIGURE 5: Estimated prevalence [% [/fig_ref] , Supplemental Tables 7-9). The difference between the TCF-CRP+AGP and the BCF-CRP+AGP adjusted estimates of depleted iron stores was consistently lower by a small amount (absolute median difference: 23 pps; range: 22.6 to 4.9 pps), although in 2 surveys (Laos and Liberia), the difference between TCF and BCF was close to 5 pps in PSC (Supplemental . The difference between the TCF-CRP+AGP and BCF-CRP+AGP approaches was negligible in WRA with a difference of ,1 pp (Supplemental .
The use of the IRC-CRP+AGP approach to adjust ferritin concentrations in PSC increased the estimated prevalence of depleted iron stores by an absolute median of 24.7 pps (range: Comparison of estimated prevalence of depleted iron stores with the use of different approaches to adjust ferritin for inflammation and malaria Adjustment for malaria alone with the use of an ICF approach increased the estimated prevalence of depleted iron stores by an absolute median of 5.1 pps (range: 2.6-6.3 pps) in PSC and of 0.5 pps (range: 0-1.2 pps) in WRA (Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. After the adjustment of ferritin concentrations with the use of the IRC-CRP+AGP approach, the estimated prevalence of depleted iron stores was significantly different (P , 0.0001) in subjects with and without malaria in 4 of 5 surveys that included malaria in PSC and in 1 of 3 surveys that included malaria in WRA [fig_ref] FIGURE 6: Estimated prevalence [% [/fig_ref] , Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref]. Adjustment of CRP and AGP alone (IRC-CRP+AGP) resulted in similar point estimates of depleted iron stores to those when adjusting for CRP and AGP plus malaria with the use of the IRC approach [absolute median differences: 20.9 pp (range: 23.9 to 0.7 pps) in PSC and 0 pps (range: 20.3 to 0.3 pps) in WRA)] [fig_ref] FIGURE 6: Estimated prevalence [% [/fig_ref] , Supplemental [fig_ref] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive... [/fig_ref].
Estimated prevalence of depleted iron stores with the use of the IRC approach to adjust ferritin for inflammation taking into account confounders There was little difference between the prevalence of depleted iron stores adjusting for ferritin with the use of the IRC approach when controlling for SES, maternal education, age, and sex (maternal education data were unavailable in Bangladesh, Liberia, Georgia, Mexico, and the United States for PSC and in all surveys for WRA, and SES data were unavailable in Bangladesh and Nicaragua). The absolute median difference of adjusted depleted iron stores controlling for confounders compared with not controlling for confounders was 0.40 pps (range: 21.2 to 3.8 pps) for IRC-CRP, 0.45 pps (range: 21.4 to 2.8 pps) for IRC-AGP, and 0.05 pps (range: 21.9 to 2.5 pps) for IRC-CRP+AGP in PSC (data not shown). The absolute median difference of depleted iron stores compared with not controlling for these factors was 20.90 pps (range: 23.7 to 0.2 pps) for IRC-CRP, 0.05 pps (range: 20.2 to 0.3 pps) for IRC-AGP, and 20.35 pps (range: 21.6 to 0 pps) for IRC-CRP+AGP in WRA (data not shown).
# Discussion
The results of this multicountry analysis showed that inflammation and malaria infection affected ferritin concentrations such that individuals with inflammation or malaria infection had a higher ferritin concentration and were less likely to be classified as having depleted iron stores than were subjects without inflammation or malaria.
In this study, the first question we sought to answer was whether it is necessary to measure biomarkers of inflammation to estimate the prevalence of depleted iron stores. We showed a consistent positive correlation between ferritin with CRP and AGP concentrations, and the strength of the association was similar for both biomarkers; ferritin concentrations were highest when CRP and AGP were elevated (23) as have been shown by others [bib_ref] Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in..., Thurnham [/bib_ref]. This finding lends support to the hypothesis that the magnitude of change in ferritin concentrations differs depending on the stage in the inflammatory process [bib_ref] The use of adjustment factors to address the impact of inflammation on..., Thurnham [/bib_ref]. Perhaps, more importantly, we showed that ferritin concentrations were altered well below the CRP and AGP cutoffs. This result indicates that clinically defined cutoffs for CRP and AGP may have less relevance when attempting to capture the influences of CRP and AGP on ferritin concentrations. These results corroborate findings by Duncan et al. [bib_ref] Quantitative data on the magnitude of the systemic inflammatory response and its..., Duncan [/bib_ref] , whereby low concentrations of CRP effected the concentrations of micronutrient biomarkers.
Our second question was whether both CRP and AGP are required to interpret ferritin concentrations. CRP tends to rise quickly in response to injury or infection and has a half-life of w2 d, whereas AGP rises slower but remains elevated with a half-life of $5 d [bib_ref] The use of adjustment factors to address the impact of inflammation on..., Thurnham [/bib_ref]. Not surprisingly, as has been shown elsewhere [bib_ref] The acute phase response affected traditional measures of micronutrient status in rural..., Bresnahan [/bib_ref] , adjustments with the use of both CRP and AGP increased the estimated prevalence of depleted iron stores compared with the use of either CRP or AGP alone, although AGP frequently provided similar estimates to those when adjusting for CRP and AGP. Nevertheless, CRP is more routinely measured and should continue to be measured along with AGP until we better understand the relation between ferritin, CRP, and AGP.
In answer to our third question, malaria infection was independently associated with ferritin after controlling for CRP and AGP. This result could reflect impaired iron incorporation in 368S response to malaria infection by the suppression of erythropoietin [bib_ref] Serum levels of erythropoietin in acute Plasmodium falciparum malaria, Burgmann [/bib_ref] [bib_ref] Increased serum hepcidin and alterations in blood iron parameters associated with asymptomatic..., De Mast [/bib_ref] [bib_ref] Assessment of urinary concentrations of hepcidin provides novel insight into disturbances in..., De Mast [/bib_ref]. Alternatively, or in addition, it may be the effect of residual confounding of malaria-associated inflammation that are not fully captured by CRP or AGP [bib_ref] Influence of malaria on markers of iron status in children: implications for..., Das [/bib_ref]. However, malaria adjustments alone resulted in lower adjustments to CRP and AGP, which were consistent with past studies [bib_ref] Effects of inflammation and Plasmodium falciparum infection on soluble transferrin receptor and..., Righetti [/bib_ref]. When CRP and AGP are measured, there appears to be limited utility in measuring malaria status to adjust ferritin concentrations.
Our fourth question was to compare adjustment approaches to estimate the prevalence of depleted iron stores in populations with inflammation. To compare approaches, we considered the following 3 criteria: 1) precision, 2) variability reflecting relation between nutrient biomarkers and severity of inflammation, and 3) feasibility to implement across countries. The application of different adjustment approaches resulted in a high degree of variability in the estimated prevalence of depleted iron stores. The exclusion approach resulted in a loss of precision (averaging a 50% sample loss when excluding subjects with elevated CRP and AGP) and may have introduced bias. The use of a fixed ferritin-concentration cutoff of ,30 mg/L (in subjects with elevated CRP or AGP) was relatively equivalent to the IRC approach in nonmalaria settings, whereas in malaria settings, the IRC approach resulted in a greater estimated prevalence of depleted iron stores. The fixed higher cutoff is a crude but easy adjustment approach, and its sensitivity and specificity should be further assessed because it was derived from a single study in pregnant women [bib_ref] Iron status in pregnant women: which measurements are valid?, Van Den Broek [/bib_ref] [bib_ref] Etiology of anemia in pregnancy in south Malawi, Van Den Broek [/bib_ref]. Depleted iron stores were defined as a ferritin concentration ,12 mg/L in preschool children and a ferritin concentration ,15 mg/L in women of reproductive age. Only surveys that measured malaria are presented. Bars without a common lowercase letter within a given survey differ, P , 0.05 (adjusted by using Bonferroni correction). -CRP+AGP, adjusting for C-reactive protein and a-1-acid glycoprotein; -CRP+AGP+malaria, adjusting for C-reactive protein, a-1-acid glycoprotein, and malaria.
## Adjusting ferritin for inflammation
## 369s
The ICF approach compared with the TCF or BCF approach has the advantage of using the inflammation profile that is specific to the data being adjusted. However, the precision of the ICF approach is lower because the estimated prevalence of depleted iron stores is based on the sample size of the reference group. Nevertheless, only in circumstances when the sample size is not sufficient to calculate a reliable ICF, an external CF should be considered. In our analysis, the BCFs were lower in children than in women. We suggest the use of the age-specific BCF instead of the TCF that was derived from a previous meta-analysis, which combined pregnant and nonpregnant women, men, children, and HIV-positive adults [bib_ref] Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in..., Thurnham [/bib_ref].
We showed that the IRC approach had promise because it better reflected the relation of ferritin concentrations with inflammation than the categorical approaches. Unlike in our study, in which we showed larger changes in the estimated prevalence of depleted iron stores after making IRC adjustments, other authors showed limited differences between a regression and categorical approach [bib_ref] Plasma ferritin and soluble transferrin receptor concentrations and body iron stores identify..., Engle-Stone [/bib_ref]. These disparate results were likely due to previous studies having applied adjustments only to individuals above the cutoffs for CRP and AGP [bib_ref] Plasma ferritin and soluble transferrin receptor concentrations and body iron stores identify..., Engle-Stone [/bib_ref]. We also showed that there was a high degree of variability in the strength of the linear relation (e.g., slopes) between ferritin and CRP and AGP across surveys, and thus, internal slopes (i.e., ICF approach) should be used instead of an external slope (i.e., BRC approach). The variation in slopes across surveys necessitates further investigations to identify potential modifiers of the ferritin-CRP-AGP relation in particular the examination of differential effects by the type of infection or inflammatory event.
We showed a stronger relation between inflammatory and ferritin biomarkers in children than in women, which translated into a greater increase in the estimated prevalence of depleted iron stores in children when adjusted for inflammation. This result may be indicative of differences in the response of ferritin to an inflammatory event across the life cycle. Although this hypothesis needs to be further explored, there has been supporting evidence of age-specific differences when it comes to iron metabolism [bib_ref] Iron requirements, absorption and metabolism in infancy and childhood, Domellö F [/bib_ref] , malaria [bib_ref] Anemia, interleukin-10, tumor necrosis factor alpha, and erythropoietin levels in children with..., Ageely [/bib_ref] , and pharmacokinetics [bib_ref] Factors and mechanisms for pharmacokinetic differences between pediatric population and adults, Fernandez [/bib_ref].
The principal limitations of this study are the selection of data sets on the basis of convenience (i.e., the availability of data from BRINDA partners), the cross-sectional nature of the data, and the absence of a gold-standard measure of iron status. With the use of the BRINDA database, we were unable to determine whether CRP and AGP incompletely capture the relation between ferritin and inflammation or, alternatively, whether the adjustment approaches overadjust ferritin concentrations on the basis of a third unknown confounder. A comparison of adjustment approaches against a gold standard (e.g., bone marrow iron) and the use of longitudinal data would further contribute to the evidence.
An exploration of the potential utility of other iron biomarkers that are less affected by inflammation and infections would be worthwhile. One such biomarker, soluble transferrin receptor, was also shown to be influenced by inflammation and infections both individually and as part of the body iron equation with the use of a similar adjustment approach [bib_ref] Adjusting soluble transferrin receptor concentrations for inflammation: Biomarkers Reflecting Inflammation and Nutritional..., Rohner [/bib_ref] [bib_ref] Adjusting total body iron for inflammation: Biomarkers Reflecting Inflammation and Nutritional Determinants..., Mei [/bib_ref]. In addition, ferritin may also be elevated in the presence of genetic disorders [bib_ref] Genetic hemoglobin disorders, infection, and deficiencies of iron and vitamin A determine..., George [/bib_ref] and obesity [bib_ref] Inflammation profile in overweight/obese adolescents in Europe: an analysis in relation to..., Ferrari [/bib_ref] , and adjustments for these factors should be further studied.
In conclusion, the accurate assessment of iron status is critical to guide policy and program decisions. This study is meant as a step forward in identifying approaches to adjust ferritin concentrations in populations with high levels of inflammation. Our findings lend support for the use of the IRC approach. The use of this type of approach is not new to the field and has been widely adopted to adjust hemoglobin concentrations for altitude [bib_ref] Haemoglobin adjustments to define anaemia, Sullivan [/bib_ref]. However, the use of the IRC approach often results in a substantial increase in the estimated prevalence of depleted iron stores, which, in some cases, has been by a magnitude of 2-to 3-fold. Therefore, it will be important to describe the IRC approach to decision-makers or other audiences without an epidemiologic background to facilitate its use. The development of an automatic macro would also increase the usability of the IRC approach. This study contributes to the evidence on how to adjust ferritin concentrations for inflammation.
[fig] FIGURE 1: Estimated prevalence [% (95% CI)] of depleted iron stores in preschool children by CRP (A) and AGP (B) deciles: the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. The analysis was restricted to countries (Bangladesh, Cameroon, Côte d'Ivoire, Kenya 2007, Kenya 2010, Laos, Liberia, and Philippines) that measured both CRP and AGP for comparability between CRP and AGP relations with depleted iron stores. Sample size: n = 8458. Depleted iron stores were defined as a ferritin concentration ,12 mg/L. Bold vertical lines indicate the commonly used cutoffs for CRP and AGP. AGP, a-1-acid glycoprotein; CRP, C-reactive protein. [/fig]
[fig] FIGURE 2: Estimated prevalence [% (95% CI)] of depleted iron stores in women of reproductive age by CRP (A) and AGP (B) deciles: the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. The analysis was restricted to countries (Cameroon, Côte d'Ivoire, Laos, and Liberia) that measured both CRP and AGP for comparability between CRP and AGP relations with depleted iron stores. Sample size: n = 4352. Depleted iron stores were defined as a ferritin concentration ,15 mg/L. Bold vertical lines indicate the commonly used cutoffs for CRP and AGP. AGP, a-1-acid glycoprotein; CRP, C-reactive protein. [/fig]
[fig] FIGURE 3: Estimated prevalence [% (95% CI)] of depleted iron stores on the basis of internal regression correction adjusting for CRP, AGP, or both in preschool children: Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. Depleted iron stores were defined as a ferritin concentration ,12 mg/L. Internal regression correction reference values were as follows: C-reactive protein = 22.26 ln(mg/L) [QE (df = 10) = 439.9, P , 0.0001]; and a-1-acid glycoprotein = 20.52 ln(g/L)] [QE (df = 10) = 584.6, P , 0.0001]. Bars without a common lowercase letter within a given survey differ, P , 0.05 (adjusted by using Bonferroni correction). AGP, a-1-acid glycoprotein; CRP, C-reactive protein. [/fig]
[fig] FIGURE 4: Estimated prevalence [% (95% CI)] of depleted iron stores with the use of different inflammation-adjustment approaches in preschool children: BRINDA project. Depleted iron stores were defined as a ferritin concentration ,12 mg/L except in the higher-cutoff approach in which depleted iron stores were defined as a ferritin concentration ,30 mg/L. Only surveys that measured both AGP and CRP are presented. Bars without a common lowercase letter within a given survey differ, P , 0.05 (adjusted by using Bonferroni correction). BRINDA correction factors were as follows-incubation phase: 0.68 (95% CI: 0.60, 0.79); early convalescence phase: 0.38 (95% CI: 0.30, 0.49); and late convalescence phase: 0.65 (95% CI: 0.58, 0.74) [QE (df = 28) = 515.9, P , 0.0001]. Internal regression correction and BRINDA regression correction reference values were as follows-CRP: 22.26 ln(mg/L) [QE (df = 10) = 439.9, P , 0.0001]; and AGP: 20.52 ln(g/L) [QE (df = 10) = 584.6, P , 0.0001]. BRINDA regression correction coefficients were as follows-ln CRP = 0.12 and ln AGP = 0.71 [QE (df = 21) = 644.8, P , 0.0001]. AGP, a-1-acid glycoprotein; BRINDA, Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia; CRP, C-reactive protein; -CRP+AGP, adjusting for C-reactive protein and a-1-acid glycoprotein. [/fig]
[fig] FIGURE 5: Estimated prevalence [% (95% CI)] of depleted iron stores with the use of different inflammation-adjustment approaches in women of reproductive age: BRINDA project. Depleted iron stores were defined as a ferritin concentration ,15 mg/L except in the higher-cutoff approach in which depleted iron stores were defined as a ferritin concentration ,30 mg/L. Only surveys that measured both AGP and CRP are presented. Bars without a common lowercase letter within a given survey differ, P , 0.05 (adjusted by using Bonferroni correction). BRINDA correction factors were as follows-incubation phase: 0.66 (95% CI: 0.53, 0.83); early convalescence phase: 0.52 (95% CI: 0.44, 0.61); and late convalescence phase: 0.82 (95% CI: 0.68, 0.99) [QE (df = 12) = 38.7, P , 0.0001]. Internal regression correction and BRINDA regression correction reference values were as follows-CRP: 21.83 ln(mg/L) [QE (df = 6) = 1142.4, P , 0.001]; and AGP-0.63 ln(g/L) [QE (df = 4) = 83.8, P , 0.0001]. BRINDA regression correction coefficients were as follows-ln CRP = 0.11; and ln-AGP: 0.34 [QE (df = 9) = 29.6, P , 0.001]. AGP, a-1-acid glycoprotein; BRINDA, Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia; CRP, C-reactive protein; -CRP+AGP, adjusting for C-reactive protein and a-1-acid glycoprotein. compared with unadjusted values in PSC (Figure 4, Supplemental [/fig]
[fig] FIGURE 6: Estimated prevalence [% (95% CI)] of depleted iron stores with the use of different inflammation-and malaria-adjustment approaches in preschool children (A) and women of reproductive age (B): Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. [/fig]
[table] TABLE 1: Age, inflammation, and malaria infection in preschool children and women of reproductive age: the BRINDA project 1 [/table]
[table] Table 3: The prevalence range was wider in the surveys that measured either CRP or AGP (PSC range: 0.3-46.9%; WRA range: 1.4-27.9%) (Table 3) than [/table]
[table] TABLE 2: Ferritin in preschool children and women of reproductive age according to inflammation stage and malaria status: the BRINDA project1 [/table]
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Combating COVID-19 with integrated traditional Chinese and Western medicine in China
, an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. China has achieved rapid containment of this highly infectious disease following the principles of early detection, early quarantine and early treatment with integrated traditional Chinese and Western medicine. The inclusion of traditional Chinese medicine (TCM) in the Chinese protocol is based on its successful historic experience in fighting against pestilence. Current findings have shown that the Chinese medicine can reduce the incidence of severe or critical events, improve clinical recovery and help alleviate symptoms such as cough or fever. To date there are over 133 ongoing registered clinical studies on TCM/integrated traditional Chinese and Western medicine. The three Chinese patent medicines (Lianhua
# Introduction
Coronavirus disease 2019 (COVID- [bib_ref] Utilization of the PICO framework to improve searching PubMed for clinical questions, Schardt [/bib_ref] is an emerging infectious disease caused by SARS coronavirus 2 (SARS-CoV-2). People with COVID-19 have had a wide range of symptoms from mild symptoms such as fever, cough and fatigue, to severe illness such as acute respiratory distress syndrome (ARDS) likely precipitated by a cytokine storm [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China, Huang [/bib_ref] [bib_ref] Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia..., Chen [/bib_ref]. The World Health Organization (WHO) declared the COVID-19 outbreak a public health emergency of international concern (PHEIC) on 30 January 2020 and a pandemic on 11 March 2020. Since the outbreak, China has taken unprecedented public health interventions to contain the spread of the virus within China 5 , and has achieved rapid containment of COVID-19 infection following the principles of early detection, early quarantine and early treatment.
According to WHO, there are no vaccines nor specific antiviral treatments for COVID-19. China's treatment protocol using integrated traditional Chinese and Western medicine attracted increasing attention of the international community. Ever since the COVID-19 outbreak, China's National Health Commission has issued seven versions of Diagnosis and Treatment Protocol for COVID-19 6e10 . Traditional Chinese medicine treatment was included in the third version of integrative treatment protocol officially released on January 22, 2020. A body of evidence from clinical practice and research has shown that integrated traditional Chinese and Western medicine played an important role for China's successful battle with COVID-19 [bib_ref] Traditional Chinese and Western medicines jointly beat COVID-19 pandemic, Qing [/bib_ref].
## Chinese protocol for treatment of covid-19
After the outbreak of COVID-19, the National Health Commission of China issued the diagnosis and treatment protocol, and recommended antiviral drug candidates. The National Administration of traditional Chinese medicine recommended traditional Chinese medicine based on pattern identification from the TCM perspective. So far, the protocol has been updated seven times.
The protocol classifies confirmed cases into mild, moderate, severe and critical, and recommends the use of antiviral drugs that might work to combat COVID-19, for example, chloroquine phosphate (500 mg bid for adults), arbidol (200 mg tid for adults), and combination of ribavirin with interferon or lopinavir/ritonavir. There is no specific antiviral medicine yet to prevent or treat this condition. These recommendations [fig_ref] Table 1: Development of treatment protocol for COVID-19 [/fig_ref] are based on the experience in treating the infection of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).
Right after the COVID-19 outbreak, traditional Chinese medicine doctors rushed to the frontline of the epidemic to work with doctors of modern medicine to control the disease and collect clinical data from the TCM perspective. This firsthand information is essential to pattern identification, treatment recommendations, and national prevention and treatment protocol. Their research paper was published online on January 29, 2020 [bib_ref] TCM clinical features and pattern identification for novel coronavirus pneumonia, Wang [/bib_ref].
As we learn more about COVID-19, the protocol of traditional Chinese medicine treatment is continuously updating.
In Trial Version 3, there were no disease stages; just four patterns and four recommended formulas for clinical reference.
In Trial Version 4, there were two periods: medical observation (for suspected cases) and clinical treatment (for confirmed cases). The clinical treatment was further classified into four stages (early/mild, middle/moderate, severe, and convalescence). The protocol recommended five types of Chinese patent medicine for patients during medical observation. These include Huoxiang Zhengqi Wan (Agastache Qi-Correcting Pills), Lianhua Qingwen Zhiji (Forsythiae and Honeysuckle Flower Pestilence-Clearing Preparations), Shufeng Jiedu Keli (Wind-Expelling and Toxin-Removing Granules), and Fangfeng Tongsheng Zhiji (Saposhnikoviae Heat-Clearing Preparations). Besides individualized traditional Chinese medicine decoction, the protocol also recommended four Chinese medicine injections for patients during clinical treatment, including Xiyanping Injection, Xuebijing Injection, Shenfu Injection and Shengmai Injection.
In Trial Version 6 and 7, the protocol recommended a specific formula for COVID-19dQingfei Paidu Tang (Lung-Cleansing and Toxins-Removing Decoction), along with modified formulas for different stages (mild, moderate, severe, critical, and convalescence). In addition, Fangfeng Tongsheng Zhiji was removed from the recommended lists of Chinese patent medicine and four Chinese medicine injections were added (
## The successful experience of traditional chinese medicine in containing pestilence
The understanding of pestilence has been documented throughout the history of traditional Chinese medicine (TCM). These theories have evolved over time and played a significant role in epidemic control. Some examples of great creativity became the inspiration of modern medical breakthroughs, such as variolation for the eradication of smallpox, and the discovery of artemisinin for malaria. From 243 before common era (BCE) to 1949, China suffered at least 500 devastating epidemic diseases. Unlike the Antonine Plaque (165e180 BCE) and Spanish flu pandemic in 1918 that killed millions of lives, epidemic disease in the history of China had been contained soon after the outbreaks. Over time, these successful experiences contributed to an established system to infectious diseases intervention using herbal medicine, nondrug therapies, and numerous single or compound formulas or techniques, such as the well-known Artemisiae Annuae for malaria and the use of variolation for smallpox. In 2003, TCM and traditional medical physicians made a major contribution to combating SARS in China.
Traditional Chinese medicine shares similar understandings of infectious diseases with modern medicine. It believes that the emergence, outbreak and threat of infectious diseases are closely associated with three interactive factorsdepidemic pathogens (pathogenic microorganism), living environment and host (human being, .
Most fundamental theories of traditional Chinese medicine are founded on the Huang Di Nei Jing (Yellow Emperor's Inner Classic), an ancient Chinese medical text written in the 2nd century BCE. This text mentioned the idea of "prevention before disease occurrence", and the strategies of "prevention of transmission after occurrence" and "prevention of recurrence after recovery". The measures to "prevent before disease occurrence" include staying away from the source of infection, cutting off the route of transmission, minimizing the environmental impact on susceptible population, and building the body's self-defending ability against pathogens. After disease occurrence, active measures need to be taken to prevent mild conditions from developing into severe or critical ones. It is also important to take precautions to prevent recurrence upon recovery.
In traditional Chinese medicine, the essential principles for disease prevention and treatment are to "reinforce healthy (antipathogenic) qi and remove pathogenic qi" and "treat diseases according to three categories of etiological factors". To reinforce healthy qi requires regulating the body functions, achieving homeostasis, and maximizing the body's self-defending ability. In addition to Chinese medicine decoction or patent medicine, nondrug intervention methods include acupuncture, tuina (Chinese 3-3-2020 Antiviral therapy: atomization inhalation of a-interferon, lopinavir/ritonavir tablets, ribavirin (intravenous injection), chloroquine phosphate, and arbidol (using three or more antiviral drugs at the same time is not recommend). Immunotherapy: use tocilizumab for patients with elevated level of IL-6 (contraindicated for patients with active infection such as tuberculosis). Others: intravenous injection of g-globulin can be used for severe and critically ill children.
Same as version 6 therapeutic massage), qigong, mental adjustment and nutrition. To treat disease by three categories of three etiological factors requires the physician to conduct individualized precision intervention according to different timing, geographic region and population groups. The Shang Han Lun (Treatise on Cold Damage) written in the 2nd century is another classic text of traditional Chinese medicine, which preliminarily established the system of Chinese herbal medicine. This text recorded a total of 113 compound formulas and usage for infectious diseases. Like precision medicine, these formulas are modified according to individual conditions. As most of these formulas are directed at regulating the body functions, they have also been widely used for noncommunicable diseases in China and Japan. They have survived the passage of time and are still regarded as having great relevance in today's environment. The recommended formulas [fig_ref] Figure 2: A brief history timeline [/fig_ref] in the Chinese protocol for the diagnosis and treatment of COVID-19 have all been proven effective in the long history of combating infectious diseases. The safety and efficacy of some formulas have been proven by evidence-based clinical trials, for example, Ma Xing Shi Gan Tang (Ephedra, Apricot Kernel, Gypsum and Licorice Decoction)dYin Qiao San (Lonicera and Forsythia Powder) for H1N1 15 .
## Current status of clinical studies on integrated traditional chinese and western medicine for covid-19
Unlike evidence-based modern medicine, traditional Chinese medicine is an empirical medicine developed on accumulated clinical observations gathered over centuries of practice. It not only deals with the etiological factor to eradicate the pathogenic microbial, but also supports the body's immune function to help fight the disease and ameliorate its consequences. In ancient times, when facing infectious disease, specific pathogens could not be A diagrammatic illustration: essential TCM theory on pestilence prevention and treatment. This includes prevention and treatment strategies. Prevention strategy: to stop pathogens from attacking the host, change the existing environment of pathogens, and make the host stay away from pathogens. Treatment strategy: to reinforce healthy qi to boost the host's ability to defend against diseases, and to remove pathogenic factors, i.e., to fight off pathogens.
identified; however, doctors could observe the patient's sign and symptoms, assess the condition and develop strategies and formulas for the different stages of the illness. So far there are over 133 ongoing registered clinical studies on TCM/integrated traditional Chinese and Western medicine. The results of 16 published papers on this topic (not including case reports) and a systematic review on 8 clinical trials (804 cases) have suggested that integrated traditional Chinese and Western medicine can reduce the incidence of severe or critical events, improve clinical recovery and help alleviate symptoms such as cough or fever [bib_ref] Clinical effects of integrated traditional Chinese and Western medicine on coronavirus disease-2019:..., Wu [/bib_ref]. Two papers [bib_ref] Clinical observation on Jinhua qinggan granules for COVID-19, Duan [/bib_ref] [bib_ref] Clinical practice and use of Qingfei Paidu decoction, Zhang [/bib_ref] reported the detailed information of its adverse reactions during the treatment for COVID-19.
## Overview of registered clinical studies
A search was performed on clinical trial registries of privately and publicly funded clinical trials worldwide. We selected Chinese clinical trial registries (www.chictr.org.cn/) and U.S. (https:// clinicaltrials.gov/). Our search strategy was designed to identify all the clinical trials that were registered for COVID-19. The following search terms were used for our search to capture relevant studies: "2019 novel coronavirus", "2019-nCoV", "COVID-19", "SARS-CoV-2", and "novel coronavirus pneumonia (in Chinese)". The eligibility criteria were developed using the Patient Intervention Comparison Outcomes Study type (PICOS) framework [bib_ref] Utilization of the PICO framework to improve searching PubMed for clinical questions, Schardt [/bib_ref].
Up until March 25, 2020, 642 registered clinical trials on COVID-19-related were retrieved from Chinese Clinical Trial Registry (ChinCTR) and ClinicalTrials.org, among which 514 were registered in ChinCTR, and 133 were about traditional Chinese medicine/integrated traditional Chinese and Western medicine. The research subjects of the 133 trials include confirmed cases (mild, moderate, and convalescent), suspected cases and those who had been in close contact with a confirmed case. The planned sample size was 52,744 cases. The smallest sample size was 16 cases (case-control study, Guangzhou). The largest sample size was 20,000 cases (Health Service Research, Chengdu). 103 of the 133 trials were related to interventions (including 13 nondrug therapies such as exercise and acupuncture), among which 77 were randomized controlled clinical trials (74.76%) and only 6 were double-blind design. Conventional Western medicine was used as control (symptomatic treatment or standard treatment). These trials measured 416 outcomes, including 220 primary outcomes such as time to defervescence, routine blood test, pulmonary functions, chest CT scan, St. George's Respiratory Questionnaire (SGRQ), TCM patterns, length of hospital stay and the viral negative-transforming time. [fig_ref] Table 3: Registered TCM clinical studies on COVID-19 [/fig_ref]. found that the ethical approval during an epidemic outbreak was more quickly; however, the review standards were not lowered.
# Ethical review process
## Role of current research findings in clinical practice
According to a retrospective study on 1305 COVID-19 cases treated in Wuhan University Tongren Hospital, patients who took Chinese medicine preparations had a smaller death risk than those who did not (0.273, P < 0.05); severe/critically ill patients had 74.364 times increased death risk than patients with moderate conditions (P < 0.05); and patients with pre-existing medical conditions had 29.420 times increased death risk (P < 0.05) [bib_ref] Analysis of influencing factors of death in patients with COVID-19, Luo [/bib_ref]. In addition, we searched MEDLINE, EMBASE, WANFANG DATA and CNKI, manually removed papers irrelevant to traditional Chinese medicine or clinical trials, and retrieved 16 clinical trial papers on traditional Chinese medicine/integrated traditional Chinese and Western medicine for COVID-19 27À41 , involving a total of 1529 cases (shown in [fig_ref] Table 4: Characteristics of clinical trials [/fig_ref]. Preliminary study findings have shown that integrated traditional Chinese and Western medicine had a shorter time to defervescence than standard Western medicine treatment alone.
A multicenter, prospective, and randomized controlled trial (n Z 284) showed Lianhuaqingwen Capsule, a repurposed marketed Chinese herb product, could be considered to ameliorate clinical symptoms of COVID-19. Compared with control group, the recovery rate (91.5% vs. 82.4%, P Z 0.022), the rate of improvement in chest computed tomographic manifestations (83.8% vs. 64.1%, P < 0.001) and clinical cure (78.9% vs. 66.2%, P Z 0.017) were significantly higher, the median time to symptom recovery (median: 7 vs. 10 days, P < 0.001), time to recovery of fever (2 vs. 3 days), fatigue (3 vs. 6 days) and coughing (7 vs. 10 days) were significantly shorter in treatment group [bib_ref] Efficacy and safety of Lianhuaqingwen Capsules, a repurposed Chinese herb, in patients..., Hu [/bib_ref]. Another retrospective analysis on 308 cases [bib_ref] Retrospective analysis on 308 cases of COVID-19 and clinical application program of..., Fang [/bib_ref] have suggested that, after receiving traditional Chinese medicine treatment, all mild and moderate COVID-19 cases recovered and none became severe/critical.
In terms of adverse reactions, Jinhua Qinggan Granules caused diarrhea in 32.93% (27/82) of the patients 17 ; Zhang et al. [bib_ref] Clinical practice and use of Qingfei Paidu decoction, Zhang [/bib_ref] reported Qingfei Paidu Decoction caused excessive sweating in 2 cases, gastric pain in 20 causes, nausea and vomiting in 8 cases, diarrhea in 16 cases, elevated serum transaminase in 11 cases, palpations in 4 cases, elevated blood pressure in 2 cases, elevated blood sugar in 3 cases, insomnia in 7 cases, and skin allergy in 1 case. The author advised modification for individual patients, for example, Qingfei Paidu Decoction is not indicated for patients with a red tongue with scanty coating (due to stomach yin deficiency). It is also worth noting some adverse reactions are associated with the combination of Western medication, for example, the serum transaminase can be restored normal after discontinuation of arbidol alone or discontinuation of both arbidol and Qingfei Paidu Decoction.
## Limitations of clinical studies
On March 18, Drifa et al.published a brief review of antiviral drugs evaluated in registered clinical trials for COVID-19 in medRxiv. Their study underlines the need to register as much details as possible on clinical trials registries during outbreaks in order to inform the development of future trials. And they believe reporting as much details as possible is key to have consistent clinical trials and to enhance the reproducibility of the results, especially as studies are more often associated with a low number of planned inclusions and composite or weak outcomes that can limit the efficacy assessment of the treatments. Our study data found similar limitations in registered TCM clinical trials.
As shown in [fig_ref] Table 4: Characteristics of clinical trials [/fig_ref] , except for three randomized controlled trials, the rest of the published clinical studies are observational, whose overall evidence is relatively low. Due to insufficient studies and a large heterogeneity in outcome measures, it is too soon to conduct a systematic review. Higher levels of evidence are further needed to justify the efficacy of traditional Chinese medicine treatment for COVID-19. It is worth noting that it is challenging to conduct large-scale randomized controlled trials (RCT) during an ongoing pandemic, especially the ethical issue of RCTS with a double-blind design. However, it is a unique setting to prioritize the patients' safety, deliver precision treatment and, at the same time, conduct pilot studies in clinical research.
## Pharmacological studies
Chinese medicine formulas recommended in the Chinese protocol are composed of multiple ingredients. The roles of these ingredients are categorized into monarch, minister, assistant and guide according to their roles. This categorization is based on the two characteristics of traditional Chinese medicine: a holistic concept and treatment based on pattern identification. The sequences of R&D strategy in Chinese medicine are safety and efficacy in patients (clinical experience), then safety and efficacy in animals, and finally clinical trials. The "clinical experienceclinical trial" model works for epidemic control and saving lives.
Application of TCM in the treatment of the patients with COVID-19 is largely inspired by the treatment of SARS in the late of 2002 [bib_ref] Traditional Chinese medicine in the treatment of patients infected with 2019-new coronavirus..., Yang [/bib_ref]. Different from Western medicine, TCM exerted broadspectrum antiviral and immune regulatory effects to treat COVID- Chest-soothing and qi-regulating manipulation, tuina (2 studies). [bib_ref] Utilization of the PICO framework to improve searching PubMed for clinical questions, Schardt [/bib_ref]. For example, Lianhua Qingwen Capsules exerted its anticoronavirus activity by inhibiting the SARS-CoV-2 replication and reducing the cytokine release from host cells, thus supporting the clinical application for COVID-19 [bib_ref] Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2), Li [/bib_ref]. In addition, Liu Shen
Capsule significantly inhibited SARS-CoV-2 replication in Vero E6 cells, greatly reduced the production of pro-inflammatory cytokines (TNF-a, IL-6, IL-1b, IL-8, CCL-2/MCP-1 and CXCL-10/ IP-10), and regulated the activity of NF-kB/MAPK signaling [bib_ref] Progress in the study of traditional Chinese medicine for the treatment of..., Gao [/bib_ref]. In the past decade, scientists have confirmed multiple components in TCM with immune regulatory and antiviral activity. Due to the similarity of SARS-CoV-2 and other RNA viruses, these naturally occurring compounds from TCM in previous studies may have the capacity to inhibit SARS-CoV-2 [bib_ref] Traditional Chinese medicine in the treatment of patients infected with 2019-new coronavirus..., Yang [/bib_ref]. Yang et al. [bib_ref] Traditional Chinese medicine in the treatment of patients infected with 2019-new coronavirus..., Yang [/bib_ref]
## Host-directed regulation
COVID-19 caused by SARS-CoV-2 is mainly associated with rapid virus replication, massive inflammatory cell infiltration and elevated pro-inflammatory cytokine responses [bib_ref] Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2), Li [/bib_ref] [bib_ref] Study of the lymphocyte change between COVID-19 and non-COVID-19 pneumonia cases suggesting..., Zheng [/bib_ref]. TCM works not only to inhibit the virus, but to block the infection, regulate the immune response, cut off the inflammatory storm, and promote the repair of the body 50 , such as Lianhua Qingwen Capsules, which suppressed the increased cytokine (TNF-a, IL-6, CCL-2/ MCP-1, and CXCL-10/IP-10) release in a dose-dependent manner, when host cells were infected with HCoV-229E and SARS-CoV-2 [bib_ref] Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2), Li [/bib_ref]. Since viral pneumonia involves infection, inflammation, immune response, blood coagulation, tissue injury, and genetic polymorphism, Ye et al.constructed a component-targetpathway to analyze the components in Lianhua Qingwen Capsules and pathways to boost immunity, such as T cells, B-cell receptor signaling, natural killer (NK) cell-mediated cytotoxicity, and anti-inflammatory Fc epsilon RI, ErbB, and MAPK signaling pathways. Of ten inflammatory and immune signals, formononetin, rutin, emodin 8-O-b-D-glucoside, hyperoside, loganic acid, and salidroside are more important than the other components. Consequently, Lianhua Qingwen Capsules have antiviral effect as well as anti-inflammatory and immune mechanism of action. Network pharmacological study 52 has suggested that the compound formula Qingfei Paidu Decoction contains 948 chemical components, which act via modulation of 790 potential target proteins. The proteineprotein interactions may form a molecular network. As modulations of these targets may regulate immunityand cytokine-related pathways, inhibit the activation of cytokines, and thus mediate overactive immune response and reduce inflammation, multiple active components of Chinese medicine have been found to protect organ damage from virus infection, virus replication and secondary inflammatory factors. Liu et al. [bib_ref] Study on chemical components of Qingfei Paidu decoction and tissue distribution in..., Liu [/bib_ref] studied major chemical components in Qingfei Paidu Decoction and tissue distribution in mice using UHPLCeQ-Orbitrap HRMS technology. They identified 39 chemical components, among which 9 can be rapidly absorbed and distributed over multiple tissues. Except for baicalin (exhibited a peak value at 2 or 4 h), the serum and tissue concentrations of the other eight components in exhibited a peak value within 0.5 h. At 0.5 h, the exposed components in the lung tissues (in sequence) are ephedrine, prunasin, pseudoephedrine, amygdalin, hesperidin, irisflorentin, baicalin, hyperin, and liquiritin; and the concentrations of ephedrine, prunasin, pseudoephedrine and baicalin remained high at 2 and 4 h. These findings provided pharmacokinetic information for its further efficacy study and clinical use. Previous studies have suggested that ephedrine, pseudoephedrine and amygdalin have notable anti-inflammatory [bib_ref] Pseudoephedrine/ephedrine shows potent anti-inflammatory activity against TNFa-mediated acute liver failure induced by..., Wu [/bib_ref] and immunomodulatory actions [bib_ref] Amygdalin analogues inhibit IFN-g signalling and reduce the inflammatory response in human..., Paoletti [/bib_ref] , and therefore, are main components in therapeutic drugs for respiratory disorders. Zhang et al. [bib_ref] Protective effect of amygdalin on LPSinduced acute lung injury by inhibiting NF-kB..., Zhang [/bib_ref] found that amygdalin could protect acute lipopolysaccharide (LPS)-induced lung damage through inhibiting nuclear factor kappa-B (NF-kB) and LRR and PYD domains-containing protein 3 (NLRP3) signaling pathways.
Network pharmacology and molecular docking have also been used in potential targets and action mechanism of recommended formulas in the Diagnosis and Treatment Protocol for COVID-19 57e71 . In traditional Chinese medicine treatment protocol for COVID-19, Xuanfei Paidu Keli
## Anti-coronavirus effects
Some Chinese medicine directly exhibit broad-spectrum antiviral effects, some were found to possess their anti-coronaviruses effects via potential targets such as SARS-CoV-2 3CL protease, RNA dependent RNA polymerase (RdRp), papain-like protease (PL pro ), helicase, and spike glycoprotein (Spike). Ye et al.studied the molecular mechanism of Lianhua Qingwen Capsules for COVID-19 via molecular docking. By docking and scoring of 21 compounds and SARS-CoV-2 3CL protease, they found that the scores of rutin, forsythoside B and hyperoside are higher than lopinavir; and hyperoside might be the most potential SARS-CoV-2 3CL protease inhibitor. Wu et al. [bib_ref] Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by..., Wu [/bib_ref] systematically analyzed all the proteins encoded by SARS-CoV-2 genes, selected a total of 21 targets, including two human targets (ACE2 and TMPRSS2) and screened a ZINC drug database (2924 compounds) and natural products database of their own (containing 1066 chemicals), and a database of 78 anti-viral drugs using target-based computer virtue screening. They found that some natural flavanoids, licoflavonol from Glycyrrhiza uralensis, cosmosiin from Scutellaria baicalensis, neohesperidin from Citrus aurantium, mangostin from Garcinia mangostana, kouitchenside D from Swertia kouitchensis, excoecariatoxin from Excoecaria agallocha, phyllaemblicin G7 from Phyllanthus emblica, and piceatannol from Vitis vinifera, exhibited high binding affinity. The only compound that could target the binding interface between Spike and ACE2 was hesperidin. This study provided new lead compounds, targets and research strategy for further in vivo and in vitro anti-SARS-CoV-2 research. Chinese medicinal plant Jin Yin Hua (Flos Lonicerae Japonicae) can remarkably inhibit influenza virus 72 , EV71 73 , and Dengue virus type 2 [bib_ref] Honeysuckle aqueous extract and induced let-7a suppress dengue virus type 2 replication..., Lee [/bib_ref]. Its active antiviral components include chlorogenic acid [bib_ref] Antiviral activity of chlorogenic acid against influenza A (H1N1/H3N2) virus and its..., Ding [/bib_ref] , flavonoids and triterpene saponins [bib_ref] Systematic review of herbal tea (a traditional Chinese treatment method) in the..., Li [/bib_ref]. It is a major component of Yinhuang Injection, Shuanghuanglian Injection, Yinhuang Tablets, Vitamin C Honeysuckle Pills and Yinhuang Tablets, which are commonly used for fever and respiratory tract infections 77 . Huang Qin (Radix Scutellariae) can inhibit respiratory syncytial virus, influenza virus and Coxsackie virus 78e80 . Its chemical components include flavonoids, flavonols (baicalin, baicalein, wogonoside, wogonin, dihydrobaicalin, etc.), terpenoids, volatile oil, trace elements and polysaccharide 81 . Rao and his team 82 screened baicalein, a SARS-CoV 3CL protease inhibitor, and assumed that flavonoids have the similar activity as they share the similar structure. Enzyme inhibitory assays have found that scutellarein, quercetagetin, myricetin and robinetin can inhibit SARS-CoV 3CL protease. During the study on active anti-SARS-CoV compounds, we screened a natural product quercetin-3-bgalactoside, a potential SARS-CoV 3CL protease inhibitor, from MDL-ACD database. Its inhibition mechanism has been confirmed by molecular simulation, point mutation experiment, FRET method and SPR technology [bib_ref] Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL (pro):..., Chen [/bib_ref] [bib_ref] The Chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of..., Ding [/bib_ref]. What's more, it can significantly inhibit the SARS-CoV-2 replication, affect virus morphology and exert anti-inflammatory activity in vitro. These findings indicate that it protects against the virus attack and thus, can be used as a novel strategy for controlling the COVID-19 [bib_ref] Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2), Li [/bib_ref] In addition, Lianqiao Baidu Pian (Forsythia Toxin-Resolving Tablets), Xiongju Shangqing Wan (Chuanxiong and Chrysanthemum Pills) and Qingwen Baidu Pian (Epidemic-Clearing Toxin-Resolving Tablets) have better anticoronavirus effects. Compound Qinlan (Scutellariae and Isatidis) Oral Liquid and Reyanning Mixture have shown a good anti-HCoV-229E efficacy, probably by inhibiting viral replication within the lung, improving gastrointestinal function, boosting the immune function and reducing the expression of inflammatory cytokines within pulmonary cells [bib_ref] Therapeutic effect of compound Qinlan Oral Liquid on treating combining disease with..., Bao [/bib_ref] [bib_ref] Application of Reyanning Mixture in evaluating combining disease with syndrome of human..., Bao [/bib_ref]. These Chinese medicines and their components provided reference for anti-SARS-CoV-2 drug screening and clinical treatment as well as the R&D of anti-coronavirus drugs.
# Discussion
To date, there is no specific therapeutics and vaccines for COVID-19, a new emerging infectious disease. During the fight against the outbreak of COVID-19 in China, traditional Chinese medicine showed some significant benefit in alleviating symptoms and preventing disease deterioration. It must be pointed out that the updated versions of traditional Chinese medicine treatment in the protocol are based more on essential theories and clinical experience rather than high-quality clinical evidences. Most published TCM clinical studies are observational, but the epidemic context is not an ideal setting to conduct large-scale randomized controlled trials. Historically, traditional Chinese medicine has been proven effective in combating epidemics of infectious disease. Despite its often-unknown mechanism of action and low level of evidence, the TCM concepts and methods deserve attention for the management of sudden unexpected outbreaks of infectious diseases, especially when there is no specific medicine to help cure the illness.
The COVID-19 is a stark reminder of the ongoing challenge presented by emerging and re-emerging infectious pathogens. The outbreak of this infection was not the first threat and will not be the last. We need to be better prepared to cope with these infection outbreaks especially when there are no specific medicine/approaches for management. According to traditional Chinese medicine, SARS-CoV-2 is the external cause of COVID-19; however, its contagion and severity are also associated with climate, geographic regions and body functions. As a result, the TCM treatment strategy for infectious disease is to reinforce the anti-pathogenic qi and remove pathogenic factors. This is similar to the concept of combining antiviral therapy with HDT. The clinical value of TCM is necessary to be evaluated on base of the TCM theory-based method.
In addition, TCM doctors, with their experience and understanding on infectious diseases, can often notice special manifestations during an early stage that may not be considered by conventional medicine. For example, the traditional Chinese medicine treatment in the Trial Version 3 protocol clearly stated that COVID-19 cases do not necessarily develop a fever 6 , whereas conventional medicine lists fever as one initial symptom necessary to identify patients. Modern medicine also confirmed the predictive analytics of TCM for this infectious disease, such as "dampness tends to affect the spleen and stomach" (indicating the nature of virus and its effects on gastrointestinal tract), and "over time, pathogens may also affect the heart" in severe stage. All these analyses are consistent to the autopsy report on the first patient who died of COVID-19 [bib_ref] Pathological findings of COVID-19 associated with acute respiratory distress syndrome, Xu [/bib_ref] : the diffuse alveolar damage and pulmonary hyaline membrane formation as in ARDS, pathologic pulmonary changes as in SARS and MERS, mild liver fatty degeneration and abnormal hepatic lobules and portal vein by liver biopsy (indicating liver damage might be induced by virus or drugs), and presence of a small amount of inflammatory monocyte infiltration without substantial damage (indicating the heart tissue is also affected by the virus). Another example is the association with gastrointestinal symptoms: some COVID-19 cases experienced diarrhea during an early stage, which has later been confirmed by stool samples (nucleic acid positive in fecal specimens or virus isolation (indicating viral multiplication in the gastrointestinal tract upon infection of COVID-19) [bib_ref] Nucleic acid detection of fecal samples from confirmed cases of COVID-19, Wu [/bib_ref]. Timely and effective communication between TCM and Western medical doctors is helpful in managing infectious diseases like COVID-19. Furthermore, TCM concepts may be beneficial to the identification of herbal active components useful as antiviral drugs. TCM can thus help to improve the clinical diagnosis and treatment of infectious diseases.
Despite the large number of registered clinical trials, there are still many limitations. We believe that it is necessary to avoid duplicate studies and conduct appropriate clinical trials to satisfy the TCM features and epidemic control. A rational design of clinical trials for COVID-19 should put the patients' interests first. An emergency censorship policy should be set up during major public health events to mobilize and integrate resources to ensure reliable research findings [bib_ref] Insights into current clinical trials of new Chinese medicine in corona virus..., Yuan [/bib_ref]. There are great uncertainties in the clinical research of TCM in emerging infectious diseases, and it is very difficult to fully carry out randomized double blind controlled multicenter clinical trial and provide high-quality evidence-based medical evidence for COVID-19 treated by TCM.
We also believe that the future advancement in TCM lies in getting high-quality evidence using modern scientific approaches. To explore the scientific value in TCM therapies, it is important to discover specific active herbal ingredients like artemisinin, but it is equally important to highlight the synergistic effect of TCM via multidisciplinary approaches. A Chinese medicine formula, no matter it contains a single ingredient or multiple ingredients, is a complex system involving multiple ingredients, targets and pathways. Despite its advantage in overall regulation, it is inevitably challenging to study its effective components and assess its clinical safety and efficacy using the method to study a single chemical substance, either in laboratories or a clinical setting. However, the particular TCM value cannot be overlooked simply because it is hard to be assessed scientifically.
The ongoing studies of TCM on mechanism of action for COVID-19 represent the future research orientations in Chinese medicine. These include the conservation and sustainable use of Chinese medicine resource and studies on their active components and mechanism of action, standardization and quality control techniques, new compound Chinese medicine, efficacy evaluation and clinical studies, and modern industrial technologies. The essential theories and clinical experiences are worthy of further studies using modern concepts and approaches. Today, with the help of big data technology, systems biology, integrative pharmacology, network pharmacology, and progress in information & computing science, chemistry and the life sciences, we can now develop multidisciplinary and multi-tiered screening and evaluation to better understand the effective components, action mechanism and pattern of prescriptions against COVID-19. From the ongoing global fight against COVID-19, the integration of traditional Chinese and modern medicine may be may offer valuable experience since it can contribute to an all-around progress in human medicine.
[fig] Figure 2: A brief history timeline: traditional Chinese medicine for pestilence prevention and treatment of infectious diseases. [/fig]
[table] Table 2: including Reduning Injection, Tanreqing Injection, Xingnaojing Injection (major ingredients: moschus, borneol, Gardeniae Fructus, etc.) and Shenmai Injection (ingredients: red ginseng and ophiopogon tuber). In fighting with COVID-19, three Chinese patent medicines and three herbal formulas have been screened for the effective COVID-19 treatment. The three Chinese patent medicines, i.e., Lianhua Qingwen Keli/Jiaonang (Forsythiae and Honeysuckle Flower Pestilence-Clearing Granules/Capsules), Jinhua Qinggan Keli (Honeysuckle Flower Cold-Relieving Granules) and Xuebijing (Stasis-Resolving & Toxin-Removing) Injection, were approved by the National Medical Products Administration to list COVID-19 as an additional indication. Among the three herbal formulas, i.e., Qingfei Paidu Fang (Lung-Cleansing & Toxin-Removing Formula), Huashi Baidu Fang (Dampness-Transforming & Toxin-Removing Formula) and Xuanfei Baidu Fang (Lung-Dispersing and Toxin-Removing Formula), the first two were approved by the National Medical Products Administration to perform clinical trials for the COVID-19 treatment. [/table]
[table] Table 1: Development of treatment protocol for COVID-19. [/table]
[table] Table 3: Registered TCM clinical studies on COVID-19. Tanreqing Injection/Capsules, Babao Elixir, Reduning Injection, Xiyanping Injection, Shenfu Injection, Jingyin Granules, Xuebijing Injection, Kesouting Syrup, Keqing Capsule, Jinyinhua Oral Liquid, Xiangxue Kangbingdu Oral Liquid, Lianhua Qingwen Capsules/Granules, Shuanghuanglian Oral Liquid, Shenqi Fuzheng Oral Liquid, Jinye Baidu Granules, Liushen Pills/Capsules, Fufang Yuxingcao Mixture, Zedoray Turmeric Oil Injection, Qingqiao Kangbingdu Granules, Fufang Yinchai Granules, Shufeng Jiedu Capsules, [/table]
[table] Table 4: Characteristics of clinical trials (16 published clinical trial papers on traditional Chinese medicine/integrated traditional Chinese and Western medicine for COVID-19). ②improvement of main symptoms (cough, fatigue, a poor appetite and diarrhea); ③inflammatory factors; ④blood test results; ⑤ improvement by CT (scan inflammatory absorption shown in chest X-ray); ⑥oxygenation index; ⑦death rate; ⑧ clinical recovery; ⑨ numbers and ratios from moderate to severe or fatal; ⑩ negative of novel coronavirus nucleic acid; ⑪ numbers and ratios of adverse reactions; ⑫ Hamilton Anxiety Scale. [/table]
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Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC′ and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1's affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC′ and FG loops. The structure of the apo triplemutant PD-1 shows that the CC′ loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.PD-1 | PD-L2 | immune checkpoint | drug discovery
eliminating the need for refrigeration during transportation and storage, in contrast to mAbs. Despite substantial efforts, there are no well-characterized small-molecule ligands for PD-1.
PD-1 has two known endogenous ligands, PD-L1 and PD-L2. These ligands both bind the same surface on PD-1, and current anti-PD-1 mAb drugs block binding of both ligands. The available evidence indicates that the primary effect of anti-PD-1 mAb drugs in cancer immunotherapy is mediated through interference with the PD-1/PD-L1 checkpoint pathway.
The ligand-binding surface of human PD-1 is generally flat, lacking pockets considered suitable for binding small molecules. However, upon binding to PD-L1, a modest cavity forms on the ligand-binding surface of PD-1. A similar cavity forms in murine PD-1 upon binding of PD-L1. Importantly, when murine PD-1 binds a different ligand, PD-L2, this cavity extendsto a volume comparable to that occupied by established small-molecule inhibitors. Unfortunately, this murine structure is insufficient to provide a structural model for Significance Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has transformed the treatment of cancer. Small-molecule PD-1 drugs have the potential to offer increased efficacy, safety, and global access. Despite substantial efforts, such small-molecule drugs have been out of reach. We identify a prominent pocket on the ligand-binding surface of human PD-1 that appears to be an attractive small-molecule drug target. The pocket forms when PD-1 is bound to one of its ligands, PD-L2. Our high-resolution crystal structure of the human PD-1/ PD-L2 complex facilitates virtual drug-screening efforts and opens additional avenues for the design and discovery of small-molecule PD-1 inhibitors. Our work provides a strategy that may enable discovery of small-molecule inhibitors of other "undruggable" protein-protein interactions.
Author contributions: S.T. and P.S.K. designed research; S.T. performed research; S.T. contributed new reagents/analytic tools; S.T. and P.S.K. analyzed data; and S.T. and P.S.K. wrote the paper. the analogous pocket in the human PD-1/PD-L2 complex, as the human and murine PD-1 proteins share sequence identities of only 63%.
Although the murine PD-1/PD-L2 structure was determined over a decade ago, the structure of the human complex has not been reported. Our previous attempts to obtain diffractionquality crystals of the human PD-1/PD-L2 complex were unsuccessful. Analyses of earlier structural studiesrevealed that formation of cavities on the ligand-binding surface of PD-1 is accompanied by changes in the structures of the CC′ and FG loops. We therefore hypothesized that substitutions in these loops could have an allosteric effect on the conformations of PD-1 in the pocket region and alter its affinity for PD-L2. Using deep mutational scanningand yeast surface display, we selected for CC′ and FG loop variants of human PD-1 with enhanced PD-L2 binding. We identified a triplemutant PD-1 that binds PD-L2 with nanomolar affinity and is amenable to crystallization, both alone and as a complex. The resulting X-ray crystal structures confirm that a prominent pocket forms in human PD-1 upon binding of PD-L2 and support the notion of allostery between the pocket and the CC′ and FG loops. The pocket identified here in human PD-1 can serve as a template for virtual drug discoveryand opens up additional avenues for the discovery of small-molecule PD-1 inhibitors.
# Results
Engineering Human PD-1 Loop Variants with Enhanced PD-L2 Affinity.
Substantial efforts by us and othersto crystallize the human PD-1/PD-L2 complex were previously unsuccessful. Earlier studiesindicated that the PD-1 ligand-binding interface consists of a hydrophobic core, the CC′ loop, and the FG loop, and that formation of a complex with ligands results in loop movement and pocket formation in the hydrophobic core. We hypothesized that mutations in these two loops of PD-1 were coupled to pocket formation and could alter PD-1's affinity for PD-L2. Consistent with this hypothesis, we found that polyglycine mutants of these loops in human PD-1 substantially decreased affinities for PD-L2 (SI Appendix,A and B).
Since we were particularly interested in the structure of the PD-1 pocket when bound to PD-L2, we maintained residues in the hydrophobic core and performed directed evolution exclusively in the CC′ loop (residues 70-78) and the FG loop (residues 127-133) of human PD-1. We used deep mutational scanningto construct loop-variant libraries with trinucleotides encoding each of 20 residues at each position. We next used yeast surface display (27) (SI Appendix,with a recombinant human PD-L2-human Fc fusion protein as the selection agent. After two rounds of selection using magnetic-activated cell sorting and fluorescence-activated cell sorting, we isolated human PD-1 loop-variant clones with single-residue substitutions. Substitutions at two residues were identified in the CC′ loop (N74G and T76P) and at one residue in the FG loop (A132V, A132L). In contrast, when we used the same yeast library and selected with PD-L1-Fc, we only isolated the A132 substitutions as high-affinity variants (SI Appendix,C-E), suggesting that the N74G and T76P variants are PD-L2-binding specific. We chose PD-1 T76P as a template to generate a second PD-1 loop variant library and selected for further enhancement of PD-L2 binding. As a result, we obtained a PD-1 triple mutant (SI Appendix,F and G) that contains all three substitutions identified from the first library: N74G, T76P, and A132V.
## Pd-1 loop variants showed increased binding affinity and
Association Kinetics for PD-L2 and PD-L1. To validate the detected enhancement in affinity, we recombinantly expressed and purified human PD-1 and the loop variants, as well as the human PD-L2 and PD-L1 ectodomain proteins. Using bio-layer interferometry, we compared the binding of PD-L2 to wild-type (WT) PD-1 and to the variantsand SI Appendix,. WT PD-1 binds PD-L2 with a K D of 500 nM; the variants all exhibit increased PD-L2 affinity, with K D values of 170 nM for N74G, 12 nM for T76P, and 69 nM for A132V. Remarkably, the PD-1 triple mutant has a K D of 2.6 nM for PD-L2, constituting a ∼200-fold increase in affinity. The triple mutant also shows substantially higher affinity for PD-L1. The A132V mutant has higher affinity for PD-L1, consistent with previous reports, but the N74G and T76P single mutants have minor effectsF and G and SI Appendix,B and C). Thus, this human PD-1 triple mutant exhibits a potent enhancement of binding affinity for both PD-L1 and PD-L2.
Bio-layer interferometry of ligand binding also enabled us to determine association constants (k on ). Compared to WT PD-1, all loop variants showed increased k on for binding PD-L2. The PD-1 triple mutant underwent a 3-fold increase of k on for PD-L2, and a 14-fold increase for PD-L1. These results suggest that these amino acid substitutions in the loops stabilize the ligand-bound state among the conformational ensembles of apo-PD-1 (ref. 32; see, however, ref..
X-Ray Crystal Structure of the Human PD-1/PD-L2 Complex. We then attempted to crystalize the human PD-1/PD-L2 complex using the PD-1 triple mutant. Site-directed mutagenesis was used to remove all N-linked glycosylation sites in each protein in an effort to aid crystallization (SI Appendix, . Coexpression of the PD-1 triple mutant and the immunoglobulin variable (IgV) domain of PD-L2 yielded a stable and 1:1 stoichiometric complex (SI Appendix,. We successfully obtained crystals of the human PD-1 N74G T76P A132V /PD-L2 IgV complex and determined an X-ray cocrystal structure at 2.0 Å resolutionand SI Appendix,. The crystal contains one PD-1/ PD-L2 complex per asymmetric unit, with space group P 2 1 2 1 2 1. This structure reveals that the human PD-1/PD-L2 complex adopts an overall architecture similar to that previously determined for the murine PD-1/PD-L2 complex (21) with a C α root-mean-square deviation (rmsd) of 3.8 Å. To our knowledge, no human PD-L2 structures have been previously described.
The human PD-1/PD-L2 interface is formed by the front β-sheets of both IgV domains, burying 1,840 Å2 (14% of the total) of the solvent-accessible surface area. In the interface, notable interacting residues include the three highly conserved aromatics W110 L2 , Y112 L2 , and Y114 L2 from βG of the PD-L2 IgV domain. The side chains of these residues point into the center of the PD-1 ligand-binding surface (SI Appendix,. To validate whether the PD-1/PD-L2 interface of the PD-1 triple mutant complex resembles the WT PD-1/PD-L2 interactions, we performed site-directed mutagenesis on several PD-1 and PD-L2 interfacial residues using the natively glycosylated WT proteins. Bio-layer interferometry revealed reduced binding of PD-1 interface mutants to PD-L2, and PD-L2 interface mutants to PD-1 (SI Appendix,, consistent with our cocrystal structure. The high-affinity loop substitutions of PD-1 localize to the interface. Among them, T76P and A132V make additional contacts to PD-L2 that likely contribute to the increase in affinity (SI Appendix,E-H).
X-Ray Crystal Structures of Human Apo-PD-1 Loop Variants. To assist analyses of the conformational changes in PD-1 associated with PD-L2 binding, we crystallized two human apo-PD-1 loop variants (SI Appendix, and determined X-ray crystal structures at 1.2 Å and 1.4 Å resolution for PD-1 N74G T76P A132V (SI Appendix, A and C) and PD-1 T76P A132V (SI Appendix, , respectively. Crystals of both variants contain a single PD-1 molecule per asymmetric unit, with space group P 3 2 2 1. Both PD-1 variants were well defined by the electron density maps, with the notable exception of the CC′ loop discussed further below (SI Appendix, . Superimposing the apo and PD-L2-bound PD-1 N74G T76P A132V structures resulted in a C α rmsd of 1.6 Å.
The C′D loop of PD-1 (residues 83-92) is a major part of the pembrolizumab epitope. This loop is not resolved in earlier structures of human PD-1 in the absence of pembrolizumabbut is clear in both of our apo-PD-1 structures. Our results indicate that the conformation of the loop changes substantially upon antibody binding (SI Appendix, .
## Formation of a prominent pocket in human pd-1 upon binding pd-l2
with an Architecture Distinct from the Murine Pocket. Our crystal structures of the human PD-1/PD-L2 complex and apo-PD-1 variants allowed us to examine formation of the human PD-1 pocket in the PD-1/PD-L2 interface. Although the human apo-PD-1 variant has a flat ligand-binding interface, there are rearrangements in this interface upon binding PD-L2. These rearrangements involve residues in βC (F63, V64, N66, Y68), βF (L122, G124, I126), βG (I134, E136), and the C′D loop (E84) to form a deep and extended pocket. Each of these residues in PD-1 is within 4.4 Å of a PD-L2 residue (SI Appendix,. This pocket accommodates PD-L2 side chains including the aromatic residues W110 L2 and Y112 L2.
Comparison of the PD-1 pockets in the human and murine PD-1/PD-L2 complexes revealed striking differences in pocket geometries. The human pocket adopts an open, funnel-shaped architecture. Compared to the murine pocketand SI Appendix, , the human pocket has a wider entrance and a narrower exit. The distinct pocket geometries arise from at least two factors. First, human PD-1 employs a different subset of interfacial residues to form the pocket than the murine version. Human PD-1 lacks an ordered βC′′ strand and, thus, the open pocket is formed by rearranging residues F63, V64, and E84. In contrast, the murine pocket is closed, with side chains of A81 and S83 forming a boundary (SI Appendix, . Second, several sequence variations exist among the residues that form the pocket. For example, V64 and Y68 in human PD-1 are substituted with M64 and N68 in murine PD-1, respectively. To quantitatively evaluate the pocket dimensions, we measured pocket volumes using POCASA 1.1. The human and murine pockets have volumes of 170 Å 3 and 220 Å 3 , respectively. Notably, these pockets are comparable in size to other protein cavities with established small-molecule inhibitors (160-800 Å 3 ).
We compared our human PD-1/PD-L2 structure (SI Appendix, with the previously determined human PD-1/PD-L1 structure . Superimposing the two structures resulted in a C α rmsd of 1.5 Å for PD-1 residues. Binding PD-L1 triggers formation of a much smaller cavity in human PD-1, with a volume of 80 Å 3 (SI Appendix, . PD-L1 lacks a large aromatic side chain corresponding to W110 L2 , so the PD-1 rearrangement only involves accommodation of a small subset of the interfacial residues, including the side chain of Y123 L1 , which corresponds to PD-L2 residue Y112 L2 (SI Appendix, . These results indicate that the core of the human PD-1 interface has remarkable structural plasticity, with the ability to form pockets with varied dimensions to interact with different PD-1 ligands.
## The cc′ loop in triple-mutant pd-1 adopts a ligand-bound
Conformation in the Absence of Ligand. We also detected conformational changes in the CC′ and FG loops when human PD-1 binds PD-L2. Earlier studies reported that the CC′ loop undergoes a substantial conformational change when human PD-1 binds PD-L1. This CC′ loop conformational change is even larger in the human PD-1/PD-L2 structure reported here. Strikingly, in the absence of ligands, the CC′ loop conformations of the PD-1 triple and double mutants resemble those of the ligand-bound conformations (SI Appendix, . For example, a 4.8 Å shift occurs between the C α of T76 and P76 in the PD-1 triple mutant of apo-PD-1. When the PD-1 triple mutant binds PD-L2, the side chain of P76 maintains the same conformation as the unbound protein. An increased population of the ligand-bound conformations of the mutant apo-PD-1 proteins is consistent with increased association constants (k on ) of the PD-1 variants.
In contrast, the conformations of the FG loop are the same in all three apo-PD-1 structures: one with an A132L substitution in the FG loopand the triple and double mutants described here (SI Appendix, . Upon binding PD-L1, there are no substantial conformational changes in the FG loop. There is, however, a drastic shift in the FG loop conformation upon binding PD-L2.
Structural Plasticity of the Human PD-1 Ligand-Binding Interface. To further investigate how the observed loop changes are associated with pocket formation, we superimposed the apo and PD-L2-bound structures of our human triple-mutant PD-1. Upon binding PD-L2, a large conformational change occurs in the PD-1 ligand-binding interface. A three-residue shortening of βC occurs (SI Appendix, , and βC and βF move apart to create a deep cleft. The rearrangements in the pocket propagate to the edge of the FG loop, resulting in a remarkable 8.2 Å lateral shift.
We note that the overall change is less dramatic in murine PD-1 (SI Appendix, . The closed architecture of the murine pocket does not require flipping of residues E84 and F63, as seen in human PD-1, and there is no secondary structure change in βC in murine PD-1 (SI Appendix, . Taken together, our results provide a structural basis for systematic rearrangements at the human PD-1 ligand-binding interface that couple pocket formation and changes in the loops of PD-1 when it binds PD-L2.
# Discussion
A prominent pocket forms in human PD-1 upon binding PD-L2. This pocket has a volume of 170 Å 3 , comparable to pockets that bind small-molecule drugs. The structure of this pocket is quite distinct from the corresponding pocket in murine PD-1 bound to PD-L2.
We speculate that this pocket represents an attractive drug target. How would a pocket-binding drug bind to a flat protein surface? We conceptualize an ensemble of PD-1 conformations (SI Appendix, in which the predominant species of apo-PD-1 has a flat ligand-binding surface (K i < 1). A pocket-binding drug will stabilize the PD-1 conformation containing the pocket (K iii ). Drug binding via an induced-fit mechanism (K iv > 1) can also occur.
The human PD-1/PD-L2 structure reported here will facilitate virtual drug screening to identify potential lead compounds (e.g., ref. 28). Specifically, we envision a small molecule binding to PD-1 contacting all or many of the residues that form the pocket, particularly F63, V64, N66, Y68, E84, L122, G124, I126, I134, and E136 in a conformation similar to that formed in the complex with PD-L2. In addition, the structures of the indole and phenol rings and neighboring side chains of PD-L2 when bound to the pocketare potentially useful starting points for the design of fragment-based screening scaffolds.
Since the PD-1 pocket is not populated substantially in the absence of PD-L2, it is not straightforward to use traditional drug-screening methods to identify small molecules that bind the pocket. Nonetheless, we speculate that conformational changes in the CC′ and FG loops and formation of pockets in the ligandbinding interface of PD-1 are thermodynamically coupledand that this coupling can be used to enable drug-discovery efforts. We envision that PD-1 exists in an ensemble of conformations in the absence of ligands, populating predominantly structures that contain a flat ligand-binding face (i.e., K 1 < 1). PD-1 molecules with a preformed pocket have a higher affinity for PD-L2 (K 3 > K 2 ). Thermodynamics dictates that K 1 K 3 = K 2 K 4 , so K 4 > K 1 . In this model, the PD-1 loop variants studied here increase K 1 and lead to a higher proportion of apo-PD-1 in the PD-L2-bound conformation.
The higher association constants (k on ) for binding ligands by the mutant PD-1, as compared to WT PD-1and SI Appendix,, support this model. Such kinetic properties are consistent with an increased fraction, relative to WT PD-1, of unliganded mutant PD-1 molecules that are in a ligand-bound conformation (ref.; see, however, ref.. In addition, the CC′ loop shifts toward the PD-L2-bound conformation in the apo-PD-1 triple and double mutants (SI Appendix, .
(While there are only minimal changes in the pocket in both apo-PD-1 mutants [], the pocket residues and a neighboring FG loop have substantial crystal contacts in the lattice [SI Appendix, ] that likely interfere with conformational changes.)
Coupling between the pocket and the loops would stabilize the pocket in the absence of a ligand, for example if the loops were held in their PD-L2-bound conformations with antibodies or aptamers. Alternatively, or in addition, new mutations (e.g., amino acid replacements, insertions, and/or deletions) could be selected for or designed to induce conformational changes in the loops. This coupling could therefore enable more traditional approaches to small-molecule drug discovery, such as highthroughput screeningand/or the discovery of allosteric regulators of PD-1 activity. More generally, our work has implications for enhancing discovery of small-molecule inhibitors of other "undruggable" protein-protein interactions.
# Materials and methods
Additional information is provided in SI Appendix, SI Materials and Methods.
Protein Expression and Crystallization. The human apo-PD-1 N74G T76P A132V and human apo-PD-1 T76P A132V proteins (SI Appendix, were overexpressed in and refolded from the inclusion bodies of Escherichia coli BL21(DE3) (Invitrogen). The human apo-PD-1 N74G T76P A132V protein was crystallized in 100 mM NaCl, 100 mM Tris:HCl pH 8.0, and 27% (wt/vol) PEG-MME 5000. The human apo-PD-1 T76P A132V protein was crystallized in 100 mM NaCl, 100 mM Tris:HCl pH 8.0, and 36% (wt/vol) PEG 3350. The human PD-1 N74G T76P A132V and human PD-L2 IgV protein complex (SI Appendix, was produced using the human Expi293F cell line (Gibco). The complex was crystallized in 200 mM magnesium acetate and 10% (wt/vol) PEG 8000. of apo-PD-1 N74G T76P A132V and PD-L2-bound PD-1 N74G T76P A132V , are indicated with sticks. The arrow highlights a 4.8-Å C α shift for residue 76 (T-to-P) from apo-PD-1 A132L to apo-PD-1 N74G T76P A132V . (B) L132 of apo-PD-1 A132L , as well as V132 of apo-PD-1 N74G T76P A132V and PD-L2-bound PD-1 N74G T76P A132V , are indicated with sticks. The arrow highlights a 3.7-Å C α shift for V132 from apo-PD-1 N74G T76P A132V to PD-L2-bound PD-1 N74G T76P A132V . (C) Pocket formation is associated with the loop change. Overlay of ribbon diagrams of human apo-PD-1 N74G T76P A132V (pale yellow) and PD-L2-bound PD-1 N74G T76P A132V (dark green). A subset of pocket residues that undergo main-chain rearrangements (arrows) are indicated with sticks. Distances of C α shifts from the unbound to the PD-L2-bound states are indicated. The FG loop shift of 8.2 Å for human PD-1 was measured using the C α of P130. (D) A thermodynamic cycle for PD-1 binding PD-L2. For clarity, only two of the states in the conformational ensemble of apo-PD-1 (Upper) are depicted. In one of these states (Left), the ligand-binding interface is flat. In the second state (Right), a pocket is formed and the loops have moved. Mutations or external agents (e.g., antibodies) could stabilize the loops in the PD-L2 bound conformation (i.e., increase K 1 ), thereby increasing the population of apo-PD-1 molecules in the bound conformation. Equilibrium constants for each step in the thermodynamic cycle are indicated (see Discussion). |
Qualitative exploration of melanoma awareness in black people in the USA
## Version 1 -author response
Reviewer: 1 Dr. Amelia Smit, The University of Sydney Comments to the Author: This is an interesting and important contribution to the literature on prevention and early detection of melanoma among Black people in the United States. The findings support the need for more tailored (or precision) public health strategies that better target population subgroups and better account for diversity in the community.
We thank Dr Smit for her time reviewing our article and her helpful feedback.
I have some minor comments: -recruitment details are needed in the abstract.
We have added these, thank you.
-In the key strengths, I would re-word the first dot point and remove the word 'extent' (as this sounds more quantitative) to: illuminates that the health information regarding melanoma provided to Black People in the US is insufficient.
We have made this edit, thank you.
-On page 4: details about the length of the interviews should be moved to Results (and include the mean, standard deviation in addition to the range).
Thank you, we have moved this to the results and added mean and standard deviation.
Reference to triangulation should be further explained because typically I have read this in relation to combining qualitative and quantitative data. When was data saturation achieved?
We are referring to investigator triangulation; we have added the following reference to help clarify this: Data saturation was achieved within 16 interviews; we have added this and also added the following explanation:
We interviewed a total of 26 participants to confirm data saturation applied across a varied sample in terms of geographic location, age and level of education completed.
-On page 6, where will the community-facing materials be made available to the community?
Thank you, we have added the following:
Our dissemination plans include the development of a community-facing animated video to describe our key messages to the Black community, which we plan to make freely available for use by dermatologists and primary care providers across the US. We will also make the video publicly available on our team website https://pcrt.stanford.edu/.
-In the Results, Themes 2-5 are well synthesised and explained. Theme 1 could be better presented. As it stands, the long table of quotes doesn't really tell the reader how the researchers have interpreted the results.
We have made our reason for presenting table 1 more explicit by re-phrasing the following:
Participants' responses to the question: 'What does the word melanoma mean to you?', which are presented in , exemplify the lack of understanding of the term melanoma across our study population. We hope you agree it is very striking and important for the reader to have the opportunity to see all the responses to this question, as it exemplifies the considerable lack of awareness that we think will be surprising to many readers.
Reviewer: 2 Dr. Caroline Watts, University of Sydney Comments to the Author: This is a qualitative analysis exploring skin cancer knowledge and beliefs in Black people in the US who have lower melanoma survival rates compared to White people diagnosed with melanoma due to later stage at diagnosis. Implementation strategies that could change awareness or behavior related to skin cancer risk are therefore required to improve melanoma related mortality in Black people. The community support and involvement in this project is noted. I enjoyed reading your paper and have some comments which I hope you will find helpful.
Thank you very much to Dr Watts for her time reviewing our article, and we have indeed found her comments very helpful.
As an area of little research, I think there should be clarification in the background about the type of melanomas being referenced with regard to Black people. While late diagnosis is an issue wherever melanomas are located on the body, it is not clear until page 6 line 5 in the Results when participants discuss awareness of the possible sites of melanoma, that there is discussion around acral melanomas. I think useful background information for the reader who may not be familiar with melanoma should include some information about melanoma and the differences of acral melanoma compared to other sub-types. That acral melanoma are relatively uncommon, but more common in people with darker skin color compared to lighter skin color should be noted.
Thank you, we have added the following to the introduction to prepare readers earlier on for the more in-depth discussion of this we have of this later on:
Current melanoma public health messaging also frequently fails to accurately depict where melanoma is most likely to occur in Black patients: although the majority of melanoma at a population level occurs on sun-exposed sites, the most common subtype of melanoma in Black people is acral lentiginous melanoma, which occurs on the palms, soles, and nail units. 7 I thought the themes you identified were very useful and informative but thought the manuscript quite quote heavy in relation to the limited analysis of the quotes. My suggestion to the authors is look at how these quotes may be further categorized and summarized rather than listing quotes under the themes which are repeated. Based on the categorization of these beliefs and behaviors can the authors suggest some potential implementation strategies for skin cancer public health campaigns such as to inform strategies such as self-skin examination and/or to shift perceptions around skin cancer risk in Black people.
Thank you for this useful feedback. We have a strong preference for including many quotes in our write-up to ensure the results remain close to the data and convey the powerful participant messages from our interviews. Our team is cautious about lengthening the article much further by doing a secondary analysis to merge themes. We agree with your suggestion to include more discussion of potential implementation strategies, and we have added the following paragraph to the end of the discussion to expand upon our key recommendations for public health messaging in this field:
Going forward, we call for provision of health information regarding melanoma for Black people which is specifically tailored to reflect the nuance and specifics about the presentation of melanoma in Black patients. This should not over estimate risk or promote fear, but should be clear, factual and informative. Specific content designed for Black people should: emphasize the specific body locations that melanoma most commonly presents in Black people, including the palms, soles and nails; describe how melanoma typically presents in Black people, for example a new brown streak under a nail which is not from an accident or bruise, or a changing or growing dark spot on the palm or sole that persists; explain the need to consult with their primary care provider if a patient notices a concerning lesion; and include visual representation of diverse skin types to increase message salience for people with skin of colour.
The reader's understanding could be improved through a broader discussion of factors that were facilitators or barriers to skin cancer awareness and through identifying the determinants; patient factors, professional factors, social factors or economic factors influencing perceptions and behavior related to skin cancer awareness. Could the authors add something around these factors.
Thank you for this important suggestion. We have added the following to the beginning of the discussion to help address this:
Lack of awareness of the potential to develop melanoma skin cancer is a crucial barrier to any interventions promoting early detection of melanoma: the value-expectancy theory the health belief model (HBM) posits that engaging in a particular public health behaviour is determined by perceived susceptibility to the health threat and perceived costs versus benefits of the recommended behaviour. Thus, awareness of risk is a key first step in empowering individuals to seek care for potentially concerning lesions. We agree a more in-depth exploration of these important questions is needed and we hope you agree with the call for further research we have now added to the end of our paper:
Further research is needed to evaluate the effectiveness of tailored public health messaging aiming to promote melanoma awareness among the Black community, with an exploration of barriers and facilitators to improving skin cancer awareness.
Re limitations-could the authors clarify if the educational level is representative of the Black population in the regions sampled?
Thank you for highlighting this important potential limitation; we have added the following to the limitations section of our manuscript:
Further, our population were highly educated: 77% of our participants were educated to college degree level or higher (compared to estimated 25% in Black population in the US). 22 However, we did not identify an influence of educational level on melanoma awareness, with melanoma awareness consistently low throughout our sample.
It was not clear where people got their health information. Could the authors elaborate on this please. Thank you for highlighting this. We found that many participants did not feel that they had received any health information regarding melanoma; we have addressed this under theme 4, 'Skin cancer awareness messages do not apply to or include Black people' with the following example: "Others felt |
Identifying Homogeneous Patterns of Injury in Paediatric Trauma Patients to Improve Risk-Adjusted Models of Mortality and Functional Outcomes
Injury is a leading cause of morbidity and mortality in the paediatric population and exhibits complex injury patterns. This study aimed to identify homogeneous groups of paediatric major trauma patients based on their profile of injury for use in mortality and functional outcomes risk-adjusted models. Data were extracted from the population-based Victorian State Trauma Registry for patients aged 0-15 years, injured 2006-2016. Four Latent Class Analysis (LCA) models with/without covariates of age/sex tested up to six possible latent classes. Five risk-adjusted models of in-hospital mortality and 6-month functional outcomes incorporated a combination of Injury Severity Score (ISS), New ISS (NISS), and LCA classes. LCA models replicated the best log-likelihood and entropy > 0.8 for all models (N = 1281). Four latent injury classes were identified: isolated head; isolated abdominal organ; multi-trauma injuries, and other injuries. The best models, in terms of goodness of fit statistics and model diagnostics, included the LCA classes and NISS. The identification of isolated head, isolated abdominal, multi-trauma and other injuries as key latent paediatric injury classes highlights areas for emphasis in planning prevention initiatives and paediatric trauma system development. Future risk-adjusted paediatric injury models that include these injury classes with the NISS when evaluating mortality and functional outcomes is recommended.
# Introduction
Injuries in children make a considerable contribution to disease burden globally, being a leading cause of mortality for children over one year of age and causing varying levels of disability affecting their development into adulthood. a 10-year review of the injury outcomes of children in Australia found that injury was the leading cause of death in children aged 1 to 16 years. The effects of rising globalisation, urbanisation, motorisation and environmental changes all impact on the risks and nature of childhood injuries around the world. However, regardless of age or country, traumatic injuries can be debilitating, affect a variety of regions of the body, cause future physical and/or mental impairment, and can be fatal. A single injury event in a child can result in highly variable patterns of injury and severity across age groups. Furthermore, typical types of traumatic injuries are not static through childhood and adolescence, with the cause, pattern and severity of injury varying with age. For example, falls and burns are common in children aged 1 to 5 years as they explore their environment in the context of rapid growth and development , whereas the advent of risk-taking behaviour in the ascendency to adolescence introduces mechanisms of injury related to activities such as the use of bicycles and off-road vehicles .
Many studies globally have focused on specific injury subsets (e.g., brain injury , spinal injuries, skeletal fractures, thoracic trauma or used single summary scores of severity such as the Injury Severity Score (ISS) or New ISS (NISS) . These approaches eliminate the ability to characterise patterns of injuries. Gaining a better understanding of the types and patterns of injuries sustained and how multiple injuries cluster together may allow for the improved modelling of injury mortality and functional outcomes.
Latent Class Analysis (LCA) measures one or more unobserved or latent classes inferred from a set of observed categorical variables . This technique has been found to be superior to traditional cluster analysis, as LCA is a model-based approach where the selection of the number of classes is based on a set of statistical indices. This approach has been widely used to create homogenous groups of individuals based on typologies in psychology, educational research and the social sciences.
Current risk-adjusted mortality models for paediatric trauma have predominantly used the ISS or NISS to control for the severity of the injury rather than pattern of injury. There is a need to focus on the individual paediatric trauma patientand incorporate homogenous injury patient-centric groupings from LCA in these models. This would enable patterns of injuries to be taken into consideration. Therefore, this study aimed to identify homogeneous groups of paediatric major trauma patients for use in risk-adjusted models of mortality and functional outcome.
# Materials and methods
## Sample
This study used data from the Victorian State Trauma Registry (VSTR). The VSTR is a population-based registry containing pre-hospital, acute care and long-term outcomes data for all major trauma patients in Victoria, Australia. The Victorian State Trauma System defines 'paediatric' as aged 0 to 15 years, triaging the majority of paediatric major trauma patients to a single, designated paediatric Major Trauma Service (MTS) for definitive care. Patients aged 0 to 15 years at the time of injury, injured between 2006 and 2016 (inclusive), with an ISS greater than 12were included in this study. The ISS ranges from 1 (least severe) to 75 (most severe) and an ISS>12 has been adopted to identify major trauma patients. The VSTR has Human Research Ethics Committee approval from the Department of Health and Human Services (DHHS) for all 138 trauma-receiving hospitals in Victoria, and the Monash University Human Research Ethics Committee (MUHREC) (CF13/3040-2001000165).
## Measures
Year of injury, demographics, injury event details, injury diagnoses, injury severity, and other relevant factors were extracted from the VSTR. Demographic data included sex (male, female) and age in years at the time of injury, categorised into four groups: <1 year, 1-5 years, 6-10 years, and 11-15 years. The Australia Bureau of Statistics (ABS) Socio-Economic Indexes for Areas (SEIFA) were accessed, with the Accessibility/Remoteness Index of Australia (ARIA) (0 = regional, 1 = major city) and the quintiles for the Index of Relative Socioeconomic Advantage and Disadvantage (IRSAD) (low score = greater disadvantage, high score = greater advantage) were included as indicative measures of socioeconomic status and geographic remoteness. a binary variable was created to indicate if the patient was definitively managed at an MTS (0 = No, 1 = Yes).
Injury diagnosis codes were assigned by trained coders using the Abbreviated Injury Scale (AIS) (2005 version; 2008 update). The AIS is an internationally recognised tool for ranking injury severity, and classifies individual injury severity on a six-point scale (1 = minor injury to 6 = maximal (currently untreatable) injury). Sixteen dichotomous injury groups were identified (0 = no injury present; 1 = 1 or more injuries within group) based on the AIS body region and severity (Appendix A). The Injury Severity Score (ISS), New Injury Severity Score (NISS) and number of injuries per patient (i.e., across the sixteen injury groups) were calculated. The ISS is calculated as the sum of squares of the highest AIS code in each of six body regions (head/neck, face, chest, abdominal/pelvic contents, extremities/pelvic girdle, external). If an injury is assigned an AIS of currently untreatable injury, the ISS score is automatically assigned the highest ISS score of 75. The NISS is calculated from the sum of squares of the three highest AIS scores, irrespective of the body region affected.
Mechanism of injury was collapsed into 13 groups: motor vehicle occupant, motorcycle, cyclist, pedestrian, horse-related, low fall from a standing height or <1 m, high fall from ≥1 m, submersion/drowning, other threat to breathing, fire/scalds/contact burn, cutting, piercing object, struck by or collision with person/object, and "other" cause.
Binary measures of in-hospital mortality (1 = died, 0 = alive) and the Kings Outcome Score for Closed Head Injury (KOSCHI) scale(1 = died in hospital/disability, 0 = good/intact recovery), administered at 6 months by telephone, were used for the final regression models.
## Analyses
Patients were clustered by hospital to ensure standard errors allowed for intragroup correlation. Individual hospital clusters of ≥10 patients were retained, with four clusters ranging in size from 17 to 1199 patients. Low volume hospitals (i.e., <10 patients) were classified into two clusters: metropolitan (n = 22) and regional (n = 29).
The main analyses consisted of six key steps, with steps 1 to 4 related to LCA and steps 5 to 6 related to the final regression models.
Four exploratory LCA Models were generated (M1, M2, M3, M4). To review the sensitivity of the final selection of the latent injury classes, two models excluded patients who sustained asphyxia or burn injuries (Ml and M3), and two models included patients with any asphyxia or burn injuries (M2 and M4). LCA M1 and M2 were initially analysed to establish the classes, then expanded to run the M3 and M4 multinomial logistic regression of the categorical latent variables on the covariates of age group and sex. Probabilities across models were compared to establish if these demographics influenced the latent injury class probabilities and latent classes (i.e., measurement known invariance explaining differences in class probabilities), or improved estimation. Consultation with a paediatric trauma surgeon supported the final latent classes chosen.
The LCA took account of hospital clusteringand latent classes one to six were tested for each model to ensure an adequate number of classes were evaluated. Complex mixture modelling used maximum likelihood estimation with robust standard errors. Initial stage optimisations were set, and number of random sets of starting values for the final stage optimisation was set to one quarter of the initial starting values to ensure model estimation converged on the global maximum likelihood. The maximum number of iterations in optimization was set to 20.
Patients were clustered by hospital to ensure standard errors allowed for intragroup correlation. Individual hospital clusters of ≥10 patients were retained, with four clusters ranging in size from 17 to 1199 patients. Low volume hospitals (i.e., <10 patients) were classified into two clusters: metropolitan (n = 22) and regional (n = 29).
The main analyses consisted of six key steps, with steps 1 to 4 related to LCA and steps 5 to 6 related to the final regression models. Goodness of fit statistics were compared to establish the optimal number of classes: Akaike Information Criterion (AIC), Bayesian Information Criterion (BIC), Vuong-Lo-Mendell-Rubin Likelihood Ratio Test (LMR-LR) and adjusted LMR LR (ALMR-LR) test, and Entropy. Smaller values of AIC/BIC and higher values for entropy indicated better model fit. The LMR-LR and ALMR-LR compared model fit improvement between models with k classes and (k − 1) classes. a p-value < 0.05 indicated rejecting the (k − 1) class model in favour of at least the current k class model. The LMR-LR and AMR-LR was applied to further classes to ensure a significant result for k + 1 classes (p-value > 0.05). The relative entropy criterion from MPlus was used for assessing the quality of class membership classification: 0.80 was considered high, 0.60 medium and 0.4 as low entropy.
Average latent class probabilities represented the proportion of the population expected to belong to a latent class. Estimated posterior probabilities ranged from 0 to 1, where higher posterior probabilities for injuries may indicate the label for that latent class.
Patients were classified into distinctive homogeneous groups, or latent classes, based on their posterior membership probabilities, given the model and the patient's data. Relative proportions of latent classes established adequate proportions per class. Detailed descriptive analysis ensured each latent class was distinctly different, theoretically and substantively meaningful, and interpretable. Differences between the latent classes were explored using chi-square tests with Pearson adjusted residuals (AR) > |2| considered significant, and Kruskal-Wallis equality-of-populations rank tests with Dunn's testwhere appropriate.
Five multivariable logistic regression models were run to investigate the impact of the inclusion of the final latent injury classes on the risk-adjusted models for in-hospital mortality and 6-month functional outcome (KOSCHI), controlling for potential demographic and injury confounders.
# Latent class analysis
Fit information from the LCA were generally consistent across models. All models replicated the best log-likelihood. Based on a combination of the fit criterion and evaluation of the posterior probabilities, three latent classes were selected from M1 and M3; four latent classes were selected from M2 and M4. The largest decline in both AIC and BIC was between latent classes one to three for M1 and M3, and one to four for M2 and M4. The BIC was lowest at latent classes three for all models; however, the four latent injury class models were selected for M2 and M4 as the BIC was virtually the same (i.e., <0.01% difference) but the entropy was higher and the classes made theoretical sense. The LMR-LR and ALMR-LR did not reach a significant result for the models, but the entropy values for all Models were above 0.8.presents the prevalence of each latent injury class and the predicted probability that patients assigned to an injury class would have certain injuries for each model. Exploratory analysis of the ARs for key characteristics across the final latent classes chosen found a number of consistent differences across the latent classes for the four models. Patients associated with the isolated head injury latent class were more likely to be in the 0-5 years age group, discharged for rehabilitation, and had falls (low/high) or were struck by or collided with a person/object as their mechanism for injury. Patients associated with the multi-trauma latent class were more likely to be in the 11-15 years age group, discharged to rehabilitation and had been a motor vehicle occupant or pedestrian or riding a motorcycle as their mechanism for injury. Those associated with the isolated abdominal latent class were more likely to be male, in the 6-15 years age group, discharged directly to home and been a motorcyclist, cyclist or struck by or collision with person/object as their mechanism for injury. Exploratory analysis of the ARs for key characteristics across the final latent classes chosen found a number of consistent differences across the latent classes for the four models. Patients associated with the isolated head injury latent class were more likely to be in the 0-5 years age group, discharged for rehabilitation, and had falls (low/high) or were struck by or collided with a person/object as their mechanism for injury. Patients associated with the multi-trauma latent class were more likely to be in the 11-15 years age group, discharged to rehabilitation and had been a motor vehicle occupant or pedestrian or riding a motorcycle as their mechanism for injury. Those associated with the isolated abdominal latent class were more likely to be male, in the 6-15 years age group, discharged directly to home and been a motorcyclist, cyclist or struck by or collision with person/object as their mechanism for injury.
## Logistic regression
## Mortality models
The four latent injury classes from M4 were used for the mortality regression models. Patients with asphyxia injuries were separated out from the other latent class due to their threat to life differing from those patients with burns and the other injuries in this latent class. The results from the five logistic regression models for mortality indicated the best performing mortality model contained both the NISS and the modified latent classes, yielding the lowest BIC, a high sensitivity, specificity, overall correctly classified percentage and highest R-squared values and AUC, Appendix B). In addition, the Hosmer-Lemeshow statistic indicated the model was a good fit, without specification error issues.
## Functional outcome models
As with the mortality modelling, the results from the five logistic regression models for the functional outcome models indicated the best performing functional outcomes model contained both the NISS and modified latent classes, yielding the lowest BIC, a high sensitivity, specificity, overall correctly classified percentage and the highest R-squared values for three of the four measures, Appendix B). There was only a marginal difference in the AUC between models (4) and (5), but the Hosmer-Lemeshow statistic indicated model (5) was superior, without specification error issues.
# Discussion
Irrespective of country or age, adequately characterising the number and complex pattern of injuries in major trauma patients can be challenging. This study used a novel methodological approach to classify key injury patterns across the whole paediatric major trauma population from Victorian registry data in Australia. For the first time, we are providing a quantified approach to succinctly describe injury patterns to the whole body, rather than relying on descriptions of injuries to individual body regions, and thereby providing data to directly inform injury prevention strategies. The use of exploratory LCA to identify three or four clusters provides an opportunity to target primary injury prevention strategies to prevent these specific injuries, and to focus strategies for optimizing the care of seriously injured paediatric trauma patients.
The identification of key injury patterns into the three main latent injury classes of isolated head injuries, isolated abdominal organ injuries and multi-trauma injuries was found to improve the model fit for both the mortality and 6-month functional outcome models. When considering childhood injury and the paediatric-specific trauma systems developed to prevent and care for such injury, the identification of 'at risk' injury populations, mechanisms and classes is of central importance. However, without accurate risk-adjustment, the design, application and evaluation of injury prevention strategies and trauma care quality improvements may be undermined by misleading epidemiological analyses. Therefore, we have proposed more accurate, paediatric-specific risk-adjustment modelling, to overcome these potential limitations, and so to promote more effective interventions for childhood injury, be that in prevention or trauma care delivery.
The findings highlight the role of isolated head and isolated abdominal injuries as common injury patterns in paediatric major trauma, elevating these for priority in the prevention and management of such injuries in childhood. Traumatic head injuries in children are a leading cause of death and a common cause of disability, often occurring in the very young. The abdominal organ latent injury class-predominantly an isolated injury-highlights the importance and relevance of abdominal trauma in childhood injury. Children have relatively larger abdominal solid organs (e.g., spleen, liver and kidneys) compared to adults, which protrude below a more compliant (and so less protective) rib cage. These and other age-specific anatomical differences make children particularly vulnerable to abdominal organ injuries, such as those caused by bicycle handlebars.
The complex multi-trauma latent class is another important injury group in children, with approximately one in five patients in our data sustaining more than three injury types. These children exemplify the quality and complexity of injured children, for whom a systematic and paediatric-specific approach to trauma management has been associated with improved delivery and outcomes of care . Not surprisingly, the multi-trauma latent class was significantly associated with higher energy mechanisms of injury including motor vehicle occupants, pedestrian collisions and motorcycle collisions.
Previous studies of paediatric trauma have commonly used composite scores to describe the severity of injuries, e.g., ISS , while other studies have used the most severe injuryor the presence of specific (non-mutually exclusive) injury types. This study identified patterns of injury in paediatric trauma to allow for a more informed and likely impactful modelling of injury in future paediatric trauma research. More parsimonious paediatric trauma statistical modelling was enabled by controlling for either three or four key latent injury classes, rather than each individual injury. The inclusion of injury classes in a model with NISS was superior in terms of all key fit statistics to the other four models. NISS has been shown to outperform the ISS for mortality in more severely injured adults, and this study showed that the NISS outperforms the ISS for mortality in more severely injured children.
A strength of this research is the ability of our LCA modelling to contextualise the mortality or functional outcome expectations of an individual injured child in terms of homogenous injury groups. This overcomes an important and recognised limitation of previous injury research, in which the complexity of patterns of injuries sustained has limited inclusion in risk-adjusted statistical models. This research has focussed on the investigation of major trauma injury typologies in children, resulting in the simplification of the complexity of patterns of injuries. The sample size of the cohort used was considerably more than the sample size of 500 as recommended by Finch and Bronkfor LCA. a further strength is the improvement in the fit of the statistical models by the adjustment of key injury classes.
A key limitation of this study is its focus on major trauma, given patterns of childhood injury are likely to differ in less-severely injured cohorts. This acknowledged bias notwithstanding, a mature trauma system aiming to reduce death and disability due to injury will similarly focus on patients with major trauma as the cohort of primary concern. Furthermore, the modelling presented in this study may have limited generalizability in non-Australian populations with dissimilar paediatric injury profiles, e.g., far higher rates of firearm trauma in the United Statesor penetrating injury in the United Kingdom. The initial classification of the sixteen injury groups used in the LCA relied on the broad dichotomous injury groupings denoting the presence or absence of one or more injuries in this cohort. The LCA technique used is an exploratory technique, assuming that latent classes do exist, with the number of classes defined prior to running the analysis. However, this study performed a number of LCA models to test for different number of classes, with and without covariates, using well-established fit statistics and theoretical understanding to decide the final number of classes. Since this is a retrospective study, future research involving a validation cohort from a developed country would be a worthwhile exercise for the evaluation of the performance of the models.
# Conclusions
The key latent injury classes of isolated head, isolated abdominal organ and multi-trauma injuries revealed by this research are important for understanding the patterns of injuries sustained in paediatric major trauma, and will inform injury prevention and treatment strategies. It is recommended that researchers consider the inclusion of these injury classes with the NISS to refine future risk-adjusted paediatric injury models when evaluating mortality and functional outcomes. Appendix B. Mortality and 6-month functional outcome logistic regression model confounders.
## Mortality
(1) |
Food Insecurity and Its Sociodemographic Correlates among Afghan Immigrants in Iran
# Introduction
In the history of the modern world, the situation in Afghanistan has resulted in one of the largest displacements of mankind in search of safety and a better life [bib_ref] My body is broken like my country: identity, nation, and repatriation among..., Tober [/bib_ref]. Migratory movements of the Afghans to Iran have a long history. Transitory migration of the Afghans to Iran motivated by economic differences has occurred since the nineteenth century. Also, Shiite Afghans have been making pilgrimages to Iran for several hundred years. However, the modern history of Afghan immigration to Iran started in 1979. Since then, Afghan immigration to Iran has been primarily motivated by the direct and indirect effects of war, insecurity, threat to female honour (namoos), unemployment, and poor dietary intake (e.g. low intake of fruits and vegetables), limited social capital, and depressive disorders [bib_ref] Acculturation, economics and food insecurity among refugees resettled in the USA: a..., Hadley [/bib_ref]. The dynamics of food insecurity reflect the composition and financial circumstances of families [bib_ref] Persistence and change in the food security of families with children, Hofferth [/bib_ref]. In case of immigrants, different factors are identified as being responsible for food insecurity, including economic constraints posed by poverty, low-wage employment, job insecurity, education, and marginal social position as well as the obligation to send money to family remaining in their country of origin in some cases. Also, many immigrants face additional challenges due to lack of valid immigration documents and fear of being deported. Therefore, immigrants are distinguished from many non-immigrants because they face additional pressure that may strain already-limited household food resources. Based on results of previous studies, immigrant families are more likely than native families to face food insecurity. Research also suggests that food insecurity is higher among lessacculturated immigrants [bib_ref] How are immigrants faring after welfare reform?, Capps [/bib_ref].
Despite the large volume and long trend of Afghan migratory movements to Iran, little information is available on nutritional status of Afghan immigrants in Iran [bib_ref] Food security assessment of urban Afghan refugee populations in Pakdasht, Sheykholeslam [/bib_ref]. Thus, the present study was undertaken to examine the extent of food insecurity and its correlates among Afghan immigrants in two metropolitan cities Tehran and Mashhad in 2010.
# Materials and methods
## Study design
This cross-sectional study was conducted in the framework of a larger study on the adaptation of Afghan immigrants in Iran. The study was performed in Tehran and Mashhad�two metropolitan cities of Iran�during February and March 2010. Based on 2006 Census, the majority of Afghan immigrants (72%) resided in urban areas of the country while only less than 3% were in refugee camps. The major provinces that host these immigrants include Tehran (32.7%), Khorasan Razavi (Mashhad) (13.3%), Isfahan (11.7%), and Sistan-Baluchistan (9.3%) (3). Selection of Tehran and Mashhad as the study sites was due to the sizeable population of Afghans in these two cities.
## Subjects
A sample of 310 adult females (aged 20 years and above) from Afghan households residing in Tehran (n=155) and Mashhad (n=155) was recruited. Multistage sampling was done through a two-stage procedure. In the first stage, stratified clustersampling procedure was used for selecting the lo-cation and blocks with high concentration of the Afghans in each city. In the second stage, chainreferral (snowball) sampling technique was used. Drawing a random sample of immigrants is not possible; thus, snowball sampling technique is the most widely-applied method of survey among immigrants.
The respondents were chosen from married women (housewives) who were directly involved in food preparation in the households.
## Data collection
Data were collected through face-to-face interviews by six trained Afghan female interviewers who were college students or graduates. The interviewers were trained prior to data collection in a oneday workshop conducted by the research team at the Faculty of Social Science in the University of Tehran. Through this workshop, the interviewers were trained on the purpose of the study, sampling procedures, considerations in administration of the questionnaire, and other field-related factors.
Data on sociodemographic characteristics, including age, sex, level of education, and occupational status of household head and spouse as well as family-size, expenditure, residency, and living conditions, were gathered through a structured questionnaire.
Household socioeconomic status (SES) was defined by composite indicators, including household assets (housing material, car, motorcycle, computer, colour television, freezer, vacuum cleaner, washing machine, and furniture) and average monthly expenditure per capita. We used principal component analysis (PCA) to construct the SES score and, based on the range of the score, the households were categorized into three SES categories: high, middle, and low.
In addition, socioeconomic position of the neighbourhood was also categorized into three categories: high, middle, and low, based on socioeconomic level of the area where the subjects resided. Neighbourhood index of socioeconomic development was determined using information from the 2006 Census in Iran on average education and occupational attainment by residents of that neighbourhood and their access to facilities.
## Measurement of food insecurity
Food security was measured at the household level. A locally-adapted HFIAS (Household Food Insecurity Access Scale) developed by USAID's Food and Nutrition Technical Assistance (FANTA) project was used in measuring food security in each household. The scale consisted of 9 items with 4 frequency options that classified households into secure, mild, moderate, and severe food-insecure. Steps taken in adaptation and validation of the scale is presented elsewhere [bib_ref] Validity of an adapted household food insecurity access scale in urban households..., Mohammadi [/bib_ref].
# Statistical analysis
Data were analyzed by SPSS software (version 16.0). Descriptive statistics were used in examining the association between food security status and each of the independent variables, using analysis of variance and χ 2 -test.
The likelihood of being food-insecure was determined by regression analyses, using binary logistic regression. Also, multinomial logistic regression was used in estimating the association between selected household characteristics and food security status. In this case, households were classified as 'food-secure', 'mild-to-moderately food-insecure', and 'severely food-insecure'.
# Results
## Characteristics of the sample
The respondents included 310 Afghan women with mean age of 35.3±11 years, who were either head of the household (n=25), or spouse (n=278) and, in a few cases (n=7), older unmarried girls in the household. The characteristics of respondents and their households are displayed in [fig_ref] Table 1: Sociodemographic [/fig_ref].
## Household food insecurity status
Distribution of responses to the nine questions in HFIAS is presented in [fig_ref] Table 2: Responses to nine questions in HFIAS applied among Afghan households in Iran,... [/fig_ref]. More than 40% of women reported worrying that food would run out while 16% had experienced actual lack of any food within the household, and less than 10% had gone to sleep hungry more than once. As shown in the figure, more than 60% Afghan households included in the study suffered from moderate-tosevere food insecurity while only 23% were foodsecure.
## Food insecurity and sociodemographic correlates
Bivariate analysis showed no significant association between the age of respondent as well as age of the household head and food security status while food security status was significantly correlated with the duration of residency in Iran, average household monthly expenditure per capita, and SES .
There was no relationship between household food insecurity and marital status, birth place, and city of residency . However, prevalence of food insecurity significantly increased with increasing household-size, in female-headed households, those households whose head and spouse had lower educational level, those belonging to the Sunni sect, those with illegal residential status, unemployed/low job status, those who did not own Binary and multinomial logistic regression analyses were used in investigating the association of demographic and socioeconomic factors with food insecurity. [fig_ref] Table 5: Sociodemographic factors relating to the likelihood of being food-insecure among Afghan households... [/fig_ref] presents the estimated odds ratios (OR) and 95% CI of binary logistic regression analysis. The dependent variable is a dichotomous measure of food security versus food insecurity. The model was initially fitted with fifteen factors, of which nine were found to be significantly associated with food insecurity. These included educational level of respondent, age and gender of household head, type of job, household monthly expenditure, housing tenure, SES, economic situation of the neighbourhood, and city context. No significant differences were observed in the prevalence of food insecurity by occupational status, religion, birthplace, legal status, household-size, and the duration of residency in Iran.
As shown in [fig_ref] Table 5: Sociodemographic factors relating to the likelihood of being food-insecure among Afghan households... [/fig_ref] , educational level was a protective factor against food insecurity. The probability of household food security was higher when household heads had a high school diploma and higher level of education compared to those with lower educational level. Also, labourers and farmers had more prevalence of food insecurity compared to persons with salaried occupations. With increase in age of the household head, the likelihood of being food-insecure increased. Female-headed households were more likely to be food-insecure than households headed by males.
The probability of food insecurity in the households with rented house was higher than those who owned their house. The prevalence of food insecurity was significantly higher in households in poor neighbourhoods than in those with high SES. In addition, the result showed that food insecurity among Afghan households in Mashhad was high compared to those in Tehran.
Multinomial logistic regression was used in estimating the association between selected individual/ household characteristics and household food security status. In this analysis, we classified household food security level as 'food-secure', 'mild-to-moderately food-insecure', and 'severely food-insecure'. Results of multinomial logistic regression analyses are displayed in [fig_ref] Table 6: Odds ratios [/fig_ref]. The first set of coefficients predicts severe food insecurity versus food security, the second set of coefficients compares mild-tomoderate food insecurity versus food security, and the third set of coefficients compares severe versus mild-to-moderate food insecurity.
Compared to food-secure households, low level of education, being Sunni, labourer, and farmer, having older age, and female gender of the household head, having less than US$ 50 monthly household expenditure per capita, having renting house, belonging to low social class, living in poor neighbourhood, and living in Mashhad increased the risk of severe food insecurity. In turn, living in Iran for 20-30 years decreased this risk.
When comparing mild-to-moderately food-insecure households with food-secure ones, the major predictors of food insecurity included low educational level, older age, and female gender of the household head as well as less than US$ 50 monthly expenditure per capita, having renting house, low-to-middle SES, living in neighbourhood with poor-to-middle SES, and living in Mashhad.
Comparison of severely food-insecure households with mild or moderately food-insecure group showed that being Sunni, unemployed, labourer and farmer, and having less than US$ 50 monthly expenditure per capita increased the risk of severe food insecurity while living in Iran for 20-30 years and living in neighbourhood with middle SES decreased the risk.
# Discussion
To the best of our knowledge, this is one of the first studies on food security status of Afghan refugees in Iran. The result indicated high prevalence of food insecurity among the studied households. The extent of food insecurity observed in this study is higher than those reported on immigrant Afghans in Pakdasht [bib_ref] Food security assessment of urban Afghan refugee populations in Pakdasht, Sheykholeslam [/bib_ref] , which is a low-income city, located south of Tehran and considerably higher than estimates on Iranian households (12) as well as Latino immigrant families in the USA [bib_ref] Experiences of Latino immigrant families in North Carolina help explain elevated levels..., Quandt [/bib_ref]. However, food insecurity levels observed among Afghan immigrants in Iran are comparable with those of migrant and seasonal farm worker (MSFW) households on the USA-Mexico border [bib_ref] The household food insecurity and health outcomes of U.S.-Mexico border migrant and..., Weigel [/bib_ref]. The Afghan immigrants face a high risk of food insecurity due to poverty, low human capital, limited access to resources and good job opportunities, and structural barriers to integration into Iranian society.
Analysis of influencing factors showed that immigrant households can be affected by food insecurity in different ways depending on their sociodemographic characteristics and economic situations. The major predictors identified for food insecurity in Afghan households in the present study included older age and female gender of household head, low educational level, being Sunni, low job status, low SES, having less than US$ 50 monthly expendi- Iranian households in the city of Tehran identified residency in medium-and low-SES districts, low education and job level of household head, lower income, expenditure, and facilities in the house as the major determinants of food insecurity among the studied population [bib_ref] Validity of an adapted household food insecurity access scale in urban households..., Mohammadi [/bib_ref]. In Pakdasht study, the low level of education of the household head and spouse (mother), sizeable number of young children in the family, and female gender of the head were also shown to be major predictors of household food insecurity [bib_ref] Food security assessment of urban Afghan refugee populations in Pakdasht, Sheykholeslam [/bib_ref]. We have identified old age, belonging to the Sunni sect, living in a rented house and in Mashhad to be additional risk factors for food insecurity among Afghan refugees.
One of the important findings of the present study is that food insecurity was inversely associated with the length of stay in Iran. Previous studies had shown higher prevalence of household food insecurity among the recent immigrant households in Canada [bib_ref] Food security and child hunger among recently resettled Liberian refugees and asylum..., Hadley [/bib_ref] and the USA (8-9). Longer length of residency has been shown to be related to increased acculturation, adaptation, and livelihood strategies.
Current evidence suggests the association between chronic household food insecurity and adverse health [bib_ref] food insecurity status: toward a refined definition, Coleman-Jensen [/bib_ref] [bib_ref] Selecting instruments for assessing psychological wellbeing in Afghan and Kurdish refugee groups, Sulaiman-Hill [/bib_ref] [bib_ref] Integrating ethnicity and migration as determinants of Canadian women's health, Vissandjee [/bib_ref] , including increased morbidity and mortality [bib_ref] Household food insecurity is associated with adult health status, Stuff [/bib_ref] [bib_ref] Food insufficiency and physical and mental health in a longitudinal survey of..., Siefert [/bib_ref] [bib_ref] Household food insufficiency is associated with poorer health, Vozoris [/bib_ref] and obesity [bib_ref] Food insecurity is associated with increased risk of obesity in California women, Adams [/bib_ref] [bib_ref] food insecurity, and nutritional outcomes in children and adults, Bhattacharya [/bib_ref] [bib_ref] Food insecurity is positively related to overweight in women, Townsend [/bib_ref] or underweight both in adults [bib_ref] Food insecurity is highly prevalent and predicts underweight but not overweight in..., Isanaka [/bib_ref] and children [bib_ref] The association of child and household food insecurity with childhood overweight status, Casey [/bib_ref]. Although we could not measure health or nutritional status of the subjects, such findings re-emphasize the importance of social policy for immigrants in the country.
# Limitations
This study had several limitations; the small sample-size and lack of random sample selection could prevent generalization to the total population of Afghan immigrants in Iran. Though not ideal, convenience sampling was necessary because no sampling frame is available for this sensitive and hard-to-reach population. Also, the high level of food insecurity observed among the Afghans in this study could be partly due to the selectivity of the Afghans in Iran; all are labourers, with low level of SES.
Income data were not collected in the study because of the difficulty for immigrants to estimate incomes that vary monthly. Assessment of dietary intake or other indicators of nutritional status were not possible in the frame of the present study.
# Conclusions
This research provides new insights into the prevalence and factors associated with food insecurity among immigrant and refugee households in Iran.
Despite their longstanding presence of the Afghans in Iran, they remain excluded from key aspects of social, political and economic life in the country. The Afghans in Iran are employed mostly in jobs requiring little or no skills because of structural barriers and low human capital, and they are mostly concentrated in low socioeconomic settings. The prevalence of food insecurity among Afghan immigrants is unacceptably high. Afghan refugees and immigrants are a potentially vulnerable group, and our results suggest a need for more thorough monitoring of their health and well-being. Policy-makers need to reconsider access to food programmes in light of the level of food insecurity and its short-and long-term consequences. Future studies are needed to evaluate and compare the results with those of the Afghan and Iranian counterparts. Also, further studies are needed to monitor levels of food insecurity in immigrant households and the strategies that they adopt for coping.
[fig] Figure: Food insecurity among Afghan households in Tehran and Mashhad [/fig]
[table] Table 1: Sociodemographic [/table]
[table] Table 2: Responses to nine questions in HFIAS applied among Afghan households in Iran, 2010 [/table]
[table] Table 3 Table 4: Association between some sociodemographic variables and food security status among Afghans in Iran, 2010 Frequency of different levels of food insecurity based on socioeconomic variables in Afghan households in Tehran and Mashhad, 2010 [/table]
[table] Table 5: Sociodemographic factors relating to the likelihood of being food-insecure among Afghan households in Tehran and Mashhad, 2010 (The results of binary logistic regression) [/table]
[table] Table 6: Odds ratios (from multivariate analysis) of independent predictors of food insecurity in Afghan households in Tehran and Mashhad, 2010 ture per capita, having renting house, living in poor neighbourhood, and living in Mashhad. A recent study on determinants of food insecurity among [/table]
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Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies*
Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.A cursory scan of scientific literature shows the increasing interest in the study of intrinsically disordered proteins, perhaps reflecting the discovery of the key role that disordered regions of proteins play in the central processes of recognition, cell signaling, and regulation. A more detailed analysis of the literature, however, reveals that the vast majority of this work is computational, theoretical, or structural, i.e. analysis and prediction of IDP 3 abundance (1, 2) and of the structural properties of disordered ensembles and assemblies (3-5). Biophysical studies have largely been carried out at equilibrium, investigating the dynamics of these disordered states (6, 7), their binding affinities, and how modulation in structure or binding affinities translates into function (8). Here we discuss just how powerful kinetic studies of the coupled folding and binding of IDPs have proved to be. They are essential for determining the mechanisms of binding (9), and also allow us to address some of the outstanding questions in the IDP field.
Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.
A cursory scan of scientific literature shows the increasing interest in the study of intrinsically disordered proteins, perhaps reflecting the discovery of the key role that disordered regions of proteins play in the central processes of recognition, cell signaling, and regulation. A more detailed analysis of the literature, however, reveals that the vast majority of this work is computational, theoretical, or structural, i.e. analysis and prediction of IDP 3 abundance (1, 2) and of the structural properties of disordered ensembles and assemblies [bib_ref] Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain, Wells [/bib_ref] [bib_ref] Constructing ensembles for intrinsically disordered proteins, Fisher [/bib_ref]. Biophysical studies have largely been carried out at equilibrium, investigating the dynamics of these disordered states [bib_ref] Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy, Soranno [/bib_ref] , their binding affinities, and how modulation in structure or binding affinities translates into function [bib_ref] Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells, Borcherds [/bib_ref]. Here we discuss just how powerful kinetic studies of the coupled folding and binding of IDPs have proved to be. They are essential for determining the mechanisms of binding [bib_ref] Only kinetics can prove conformational selection, Dogan [/bib_ref] , and also allow us to address some of the outstanding questions in the IDP field.
## How different are idps anyway? the importance of experimental conditions
A significant proportion of proteins lack a stable, well defined, three-dimensional structure [bib_ref] Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm, Wright [/bib_ref]. These proteins, termed IDPs, can display varying amounts of residual secondary structure. Their structural heterogeneity arises from their sequence composition, which differs markedly from that of folded proteins; Gly, Pro, and charged residues are over-represented, whereas hydrophobic amino acids, which typically form the core of folded proteins, are under-represented [bib_ref] Sequence complexity of disordered protein, Romero [/bib_ref] [bib_ref] Intrinsically disordered protein, Dunker [/bib_ref] [bib_ref] Why are "natively unfolded" proteins unstructured under physiologic conditions?, Uversky [/bib_ref]. These compositional differences form the basis for the identification of disordered regions using bioinformatics algorithms [bib_ref] PrDOS: prediction of disordered protein regions from amino acid sequence, Ishida [/bib_ref]. Contribution of charged residues to disorder profiles can be complex, as reflected by the importance of charge patterning in defining the extent of chain collapse [bib_ref] Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of..., Das [/bib_ref] [bib_ref] Net charge per residue modulates conformational ensembles of intrinsically disordered proteins, Mao [/bib_ref] [bib_ref] Charge interactions can dominate the dimensions of intrinsically disordered proteins, Müller-Späth [/bib_ref]. The increased conformational plasticity and altered physicochemical properties imparted by their sequence composition also change their responses to external factors such as ionic strength, temperature, and molecular crowders . Internal friction (roughness of the energy landscape) has been shown to be related to sequence composition, and may therefore be different for IDPs than denatured folded proteins [bib_ref] Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy, Soranno [/bib_ref]. However, what effects do these features have when it comes to IDP-ligand interactions? Do IDPs react similarly to changes in environment as their folded counterparts? These questions can be investigated mechanistically through kinetic studies. One paradigm of early IDP studies was that disorder facilitates high specificity, low affinity binding. Although it is true that on average IDPs form looser complexes with faster dissociation rate constants (k off ) and statistically similar association rate constants (k on ) compared with folded proteins, the available range of values for both is very wide [bib_ref] Slow, reversible, coupled folding and binding of the spectrin tetramerization domain, Shammas [/bib_ref] [bib_ref] Globular and disordered: the non-identical twins in protein-protein interactions. Front, Teilum [/bib_ref]. Thus, similar binding kinetics can be obtained for both folded and disordered proteins. It is likely that biophysical properties reflect the function of the folding and binding reaction [fig_ref] FIGURE 1: The thermodynamic and kinetic properties of IDPs vary over orders of magnitude,... [/fig_ref].
Electrostatic interactions can accelerate association for folded proteins by orders of magnitude [bib_ref] Rapid, electrostatically assisted association of proteins, Schreiber [/bib_ref] and cause dramatic ionic strength dependence of association rates (k on ), whereas dissociation is generally affected only marginally. Electrostatic steering has also been identified for coupled folding and binding of IDPs, where k on has been found to be beyond the expected "diffusion limit" but reduced at infinite ionic strength [bib_ref] Folding and binding of an intrinsically disordered protein: fast, but not "diffusion-limited, Rogers [/bib_ref] [bib_ref] Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain..., Shammas [/bib_ref]. Interestingly, the electrostatic rate enhancement for c-Myb binding to KIX (CREB binding domain of CBP), and PUMA binding to Mcl-1 are under 20-fold, much less than, for example, barnase binding to barstar (about 4 -5 orders of magnitude) [bib_ref] Rapid, electrostatically assisted association of proteins, Schreiber [/bib_ref]. Recent binding studies utilizing NCBD demonstrated larger rate enhancements from electrostatic steering for its IDP partners when compared with its folded partners [bib_ref] Binding rate constants reveal distinct features of disordered protein domains, Dogan [/bib_ref]. It is clear that, with many IDPs having an excess of charged residues, electrostatics is of crucial relevance. Another potential difference in the role of electrostatics for disordered protein interactions is their increased propensity to undergo post-translational modifications that can alter protein charge, e.g. phosphorylation [bib_ref] From sequence and forces to structure, function, and evolution of intrinsically disordered..., Forman-Kay [/bib_ref] [bib_ref] The importance of intrinsic disorder for protein phosphorylation, Iakoucheva [/bib_ref]. Such changes affect binding affin-ity [bib_ref] Cooperativity in transcription factor binding to the coactivator CREB-binding protein (CBP): the..., Goto [/bib_ref] [bib_ref] Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch, Bah [/bib_ref] and can be mediated both through altered longrange and local electrostatic forces and through more specific transition state effects.
Obtaining basal rate constants (k on in the absence of longrange electrostatics) is crucial for making mechanistic conclusions on the basis of k on , as exemplified for the case of c-Myb binding to KIX [bib_ref] Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants, Shammas [/bib_ref]. Here a longer version of c-Myb with increased residual structure associates faster under physiological ionic strength, suggesting that residual structure may be important in determining k on. However, the basal rate constants are identical within error, indicating that the change of charge and not the increase in residual helicity is responsible for the faster association at physiological ionic strength.
In addition to understanding the contribution from electrostatic interactions, kinetic studies of IDPs allow activation energies for coupled folding and binding reactions to be determined [bib_ref] Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain..., Shammas [/bib_ref] , giving further insight into the mechanisms by which this class of proteins achieves their functional roles. More fundamental studies of this kind are needed to determine whether IDPs really behave differently from their folded counterparts.
## Which comes first: binding or folding?
Kinetics are essential to answer this question, but even when kinetics have been determined, the answer can be difficult to obtain [bib_ref] Dynamics and mechanisms of coupled protein folding and binding reactions, Kiefhaber [/bib_ref] [bib_ref] Distinguishing induced fit from conformational selection, Gianni [/bib_ref] [bib_ref] Helical propensity in an intrinsically disordered protein accelerates ligand binding, Iešmantavičius [/bib_ref]. Practically speaking, kinetic studies involve monitoring changes in response of a probe, such as an intrinsic or extrinsic fluorophore, upon either (i) the rapid mixing of the IDP and its partner in a stopped-flow or continuous-flow format to observe complex formation [fig_ref] FIGURE 2: Kinetic experiments of coupled folding and binding reactions under pseudo-first order conditions [/fig_ref] , or (ii) sudden alteration of experimental conditions, e.g. temperature, leading to system relaxation back to equilibrium [bib_ref] T-jump infrared study of the folding mechanism of coiled-coil GCN4-p1, Wang [/bib_ref]. When the reaction timescales are appropriate, it is also possible to obtain kinetics from NMR experiments [bib_ref] Mechanism of coupled folding and binding of an intrinsically disordered protein, Sugase [/bib_ref] [bib_ref] Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and..., Arai [/bib_ref]. In mixing studies, it is common to arrange the conditions such that one protein is in excess over the other (typically described as Ͼ10-fold) (32) and its concentration remains relatively constant throughout the reaction. The rate equations then become pseudo-first order and can be readily solved to obtain a description of the reaction progress with time [bib_ref] Matrix and convolution methods in chemical kinetics, Pogliani [/bib_ref]. In the case of a simple two-state system, the kinetics are described by a single exponential decay function (or phase), with the associated observed rate constant being linearly dependent on the protein concentration [fig_ref] FIGURE 2: Kinetic experiments of coupled folding and binding reactions under pseudo-first order conditions [/fig_ref]. Experiments are performed at multiple concentrations, and the concentration dependence of the observed rates can then be used to extract fundamental rate constants for the system.
In coupled folding and binding studies, there is necessarily a conformational change as well as a binding step. A frequent question asked regards the order of these events. There are two extreme mechanisms that can be imagined [fig_ref] FIGURE 2: Kinetic experiments of coupled folding and binding reactions under pseudo-first order conditions [/fig_ref]. In the induced fit (IF) scheme, the IDP binds to its partner and subsequently folds. In the conformational selection (CS) scheme, the partner binds (selects) only the proteins in the IDP ensemble with the correct conformation. It can be possible to discriminate between these two situations through kinetic studies.
Unfortunately, an exact generalized description of reaction progress with time is impossible for both IF and CS schemes as there is no analytical solution to either set of rate equations. However, it is again possible to overcome this obstacle by arranging pseudo-first order conditions with the folded protein in excess, so the rate equations are simplified to linear equations that are readily solved. Kinetic traces then consist of up to two exponential decay phases [bib_ref] Matrix and convolution methods in chemical kinetics, Pogliani [/bib_ref] [bib_ref] Affinity, avidity, and kinetics of target sequence binding to LC8 dynein light..., Radnai [/bib_ref] [bib_ref] Fast Association and slow transitions in the interaction between two intrinsically disordered..., Dogan [/bib_ref] [fig_ref] FIGURE 2: Kinetic experiments of coupled folding and binding reactions under pseudo-first order conditions [/fig_ref] , which is a relatively simple functional form to fit. In both schemes, the observed rate constants and amplitudes of the two phases actually involve a defined mixture of the four fundamental rate constants, and their concentration dependences can appear very similar [bib_ref] Conformational selection or induced fit? A critical appraisal of the kinetic mechanism, Vogt [/bib_ref]. For example, at high protein concentrations, two rate constants may be observed: one that is apparently independent of protein concentration and corresponds to the unimolecular process, and another that is linearly dependent and corresponds to the binding process. Although this can make it difficult to discriminate between the two mechanisms, the ambiguity can be cleared up by performing similar experiments under reversed pseudo-first order conditions, i.e. by putting the IDP in excess. If the process is IF, then the observed rate constants will remain the same, but if the process is CS, then the observed rate constants will be different [bib_ref] Distinguishing induced fit from conformational selection, Gianni [/bib_ref]. Indeed because it can be difficult to practically obtain an excess of A* over B, 4 the kinetic trace may deviate from the exponential decay form in this case.
The discussion so far has described the situation when two phases are observed in kinetic traces. However, in practice, it is common to observe only one phase in these types of experi-ments, either because one of the rate constants is too fast, or because its amplitude is too low, to be reliably observed [bib_ref] Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain..., Shammas [/bib_ref] [bib_ref] Distinguishing induced fit from conformational selection, Gianni [/bib_ref] [bib_ref] Demonstration of a folding after binding mechanism in the recognition between the..., Dosnon [/bib_ref] [bib_ref] Interplay between partner and ligand facilitates the folding and binding of an..., Rogers [/bib_ref] [bib_ref] Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered..., Hill [/bib_ref]. The former may be likely in the case of IDP-partner interactions because IDPs often fold into relatively simple structures such as short ␣-helices upon binding, and helix (un)folding rate constants are known to be much higher than observed binding rate constants [bib_ref] Early events in protein folding, Ferguson [/bib_ref]. Thus, we dedicate the rest of this paragraph to a discussion of the kinetic features when folding and unfolding are very fast when compared with binding and unbinding. As in these cases there is always a fixed ratio of the folded and unfolded species, i.e. A*B and AB, or A and A*, the folded and unfolded forms are observed as only a single species by ensemble measurements, and association kinetics display a single observed rate constant that is linearly dependent upon the protein concentration. The kinetics then reduce to the simple two-state case described previously, with the observed rate constants being related to the fundamental rate constants. For CS processes, the observed association rate constant, which is given by the gradient of the straight line, is actually significantly lower than the microscopic association rate constant. This is essentially because few of the collisions will be with "reactive" IDP protein. It has been pointed out that very fast interactions, with observed k on Ͼ 10 7 M Ϫ1 s Ϫ1 in the absence of electrostatic enhancement, are therefore inconsistent with CS schemes [bib_ref] Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain..., Shammas [/bib_ref] [bib_ref] Dynamics and mechanisms of coupled protein folding and binding reactions, Kiefhaber [/bib_ref]. In contrast, the observed gradient for IF processes represents the binding rate constant and can be similar to those observed between pairs of folded proteins, whereas dissociation is slowed because it can only occur from the intermediate state. Observations so far have shown no significant differences in reported association rate constants for IDPs when compared with folded proteins [bib_ref] Slow, reversible, coupled folding and binding of the spectrin tetramerization domain, Shammas [/bib_ref] , which might suggest that IF is the preferred mechanism; however, both cover a large range of values, and it is possible that differences in electrostatic rate enhancements are masking an effect. It is often claimed that disorder might enhance association rates of IF mechanisms, through increasing capture radius ("fly-casting"); however, this is likely to make only a small contribution (Ͻ2-fold) and has yet to be experimentally demonstrated [bib_ref] Speeding molecular recognition by using the folding funnel: the fly-casting mechanism, Shoemaker [/bib_ref] [bib_ref] Kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical..., Huang [/bib_ref].
It is important to note that whereas reversing pseudo-first order conditions where two phases are reliably observed can discriminate between IF and CS, this is not always possible when (un)folding rate constants are high and the process is apparently two-state. In this case, reversing pseudo-first order conditions does not change the observed rate constant for either scheme. However, there are two circumstances where a CS mechanism is indicated. The first occurs if an observed rate constant decreases with protein concentration, which happens when conformational changes are slow when compared with unbinding [bib_ref] Conformational selection or induced fit? A critical appraisal of the kinetic mechanism, Vogt [/bib_ref]. The second occurs if observed kinetics deviate from single exponential behavior and/or the observed rate constant obtained with the folded partner in excess. This can occur if pseudo-first order conditions have not been achieved. Because pseudo-first order conditions with A over B are easily achieved for IF, but difficult to achieve with A* over B in CS (only a small proportion of unbound IDP is folded), this behavior suggests CS.
Although the majority of kinetic studies arrange pseudo-first order mixing conditions to achieve exponential decay kinetics, in the case of a two-state system (single phase observed with no populated intermediate), it is actually possible to solve the rate equations analytically [bib_ref] Slow, reversible, coupled folding and binding of the spectrin tetramerization domain, Shammas [/bib_ref] [bib_ref] Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain..., Shammas [/bib_ref]. If it is possible to perform experiments at concentrations such that k off makes a significant contribution to the observed kinetics, then both k on and k off can be estimated from a single mixing experiment [bib_ref] Slow, reversible, coupled folding and binding of the spectrin tetramerization domain, Shammas [/bib_ref] [bib_ref] Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain..., Shammas [/bib_ref].
Dissociation kinetic experiments can also be very informative. They typically involve dilution of a labeled preformed complex into a large excess of unlabeled partner protein, which ensures virtually irreversible dissociation of the labeled version [bib_ref] Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants, Shammas [/bib_ref] [bib_ref] Demonstration of a folding after binding mechanism in the recognition between the..., Dosnon [/bib_ref] [bib_ref] Interplay between partner and ligand facilitates the folding and binding of an..., Rogers [/bib_ref]. Care must be taken in these experiments because if the concentration of unlabeled competitor is not high enough, the observed dissociation rate constant will depend upon competitor concentration and will not be accurate. For two-state reactions, where A* or A*B are not significantly populated, the ratio k off /k on will equal the observed K d and k off matches the y axis intercept in the association kinetic graph.
Finally, it is worth noting that the viewpoint of pure IF or CS mechanisms is a likely oversimplification. Processes might contain elements of both mechanisms, e.g. selection of partially folded IDPs in the ensemble. It is also possible that both mechanisms exist in parallel, with flux through each depending upon experimental conditions including protein concentration [bib_ref] Conformational selection or induced fit: a flux description of reaction mechanism, Hammes [/bib_ref].
## What is the role of order within disorder?
Although largely unstructured, IDPs can contain regions of transient secondary structure. In the case of IDPs that undergo coupled folding and binding, the presence and abundance of the bound, folded conformation within the IDP ensemble are potentially important. For example, combining structural data from NMR with equilibrium measurements has indicated that increasing the proportion of unbound IDP with a structure that resembles the bound state enhances the binding affinity for the partner protein [bib_ref] Helical propensity in an intrinsically disordered protein accelerates ligand binding, Iešmantavičius [/bib_ref] [bib_ref] Conformational recognition of an intrinsically disordered protein, Krieger [/bib_ref]. Of course, increased unbound order and enhanced complex stability are not necessarily advantageous for the function of the IDP [bib_ref] Disorder and residual helicity alter p53-Mdm2 binding affinity and signaling in cells, Borcherds [/bib_ref].
Kinetic analysis is required to answer the key question from these studies. Is the increased complex affinity due to an enhanced k on or reduced k off ? Mechanistically, an increased k on upon increasing the order within the unbound ensemble might indicate that the reaction is proceeding via a CS mechanism. However, care must be taken in this analysis, as an increased k on would also be observed for IDPs where the rate-limiting folding step occurs after binding, i.e. the IF mechanism. Here, it is not the abundance of free structured IDP that is influencing the k on ; instead, increased structure may increase the k on by lowering the energy of the transition state for folding once bound.
A few studies have investigated the influence of residual structure on the kinetics of coupled folding and binding of IDPs. For association of c-Myb with KIX, increasing the residual structure of c-Myb, through the use of the helix stabilizer trifluoroethanol [bib_ref] A folding-afterbinding mechanism describes the recognition between the transactivation domain of c-Myb..., Gianni [/bib_ref] or modulation of peptide length (31), decreases k off without significantly altering k on (suggesting an IF mechanism). It has been suggested that positive correlation of the k on from the -value analysis for this system (51) with the predicted helicity indicates that the process may involve some form of CS (37) but, as described above, this apparent correlation could also be due to a lowering of the transition state barrier for folding. Mutation of surface residues in PUMA to pro-line, which destabilizes helices, was found to reduce its affinity for Mcl-1. Through kinetic analysis, it was shown that this reduction is due to an increase in k off , with no significant changes in k on [bib_ref] Coupled folding and binding of the disordered protein PUMA does not require..., Rogers [/bib_ref]. In contrast, the enhanced affinity for the CID domain of ACTR with NCBD upon increasing residual helical propensity was due to both due to an increase in k on and a decrease in k off [bib_ref] Helical propensity in an intrinsically disordered protein accelerates ligand binding, Iešmantavičius [/bib_ref].
So far, most studies show an increase in affinity upon increasing residual structure. However, the differing kinetic explanations behind the increases in affinity emphasize the importance of thorough kinetic analysis in describing mechanisms.
## Probing transition states
Analyzing an experimental system at residue level allows probing transition states or short-lived intermediates on a reaction pathway. Several studies in protein folding have applied site-directed mutagenesis along with biophysical measurements to understand folding mechanisms [bib_ref] ␣-Helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at..., Serrano [/bib_ref]. Such mutational analysis along with kinetics can also be applied to IDPs to study interactions with their partners in more detail.
Why is it important to look at transition states? Interactions between IDPs and their partners are complex reactions. The NMR techniques used to study these interactions can identify the unbound disordered IDP, the fully bound complex structure, and in some cases, stable intermediates [bib_ref] Mechanism of coupled folding and binding of an intrinsically disordered protein, Sugase [/bib_ref] [bib_ref] Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and..., Arai [/bib_ref]. However, it is particularly important to visualize the unpopulated transition states to understand the critical molecular contacts formed during these coupled folding and binding reactions. This can only be achieved through -value analysis, which maps the structure formation in the transition state by comparing rate constants for wild-type and mutant proteins [bib_ref] Mapping the transition state and pathway of protein folding by protein engineering, Matouschek [/bib_ref].
In protein folding studies, folding and unfolding rate constants are used to calculate -values. Analogously, for an IDP system, kinetic rate constants (k on and k off ) and K d are used to calculate the -values using Equation Point mutations in a protein may change k on and k off as shown in . The -value for each residue reports on the proportion of intermolecular or intramolecular native contacts it makes at the transition state. Where ϭ 1, these contacts are fully formed . Where ϭ 0, these contacts are as unformed in the transition state as they are in the unbound state . Intermediate -values reflect intermediate structure formation . Particular care has to be taken in interpreting values of association reactions because early contacts may be non-native [bib_ref] Interplay between partner and ligand facilitates the folding and binding of an..., Rogers [/bib_ref]. Conventionally, interfacial residues are mutated to Ala to probe for hydrophobic interactions (tertiary structure), and surface-exposed residues are mutated to Ala and Gly to probe for helix formation (secondary structure). Care must be taken if charged residues are mutated because, as we have seen, k on values are particularly sensitive to changes in electrostatics.
The few examples in the literature where -value analysis has been applied to IDPs are shown in [fig_ref] FIGURE 4: Illustrations of coupled folding and binding reaction transition states [/fig_ref]. In some cases, the IDP appears to be largely or partly unstructured at the transition state. For PUMA⅐Mcl-1, mutations to probe helix formation and hydrophobic interactions resulted in generally low -values, with values increasing slowly toward the N terminus, suggesting that the IDP has only embryonic structure at the N terminus [bib_ref] Interplay between partner and ligand facilitates the folding and binding of an..., Rogers [/bib_ref]. Low -values were also observed for helix formation and hydrophobic interactions in the S-protein⅐Speptide system, although we note that this is not an evolved folding upon binding system, so that the general principles of association may not be the same [bib_ref] Mapping backbone and side-chain interactions in the transition state of a coupled..., Bachmann [/bib_ref]. For NCBD⅐CID-ACTR, low -values were observed for intermolecular interactions, whereas higher -values were found for the N-terminal helix of both NCBD and CID-ACTR. Thus, although some structure is present at the N-terminal helices, the native hydrophobic interactions form after the rate-limiting transition state [bib_ref] The transition state structure for coupled binding and folding of disordered protein..., Dogan [/bib_ref]. In contrast, high -values were calculated for both the C terminus and the N terminus of c-Myb, implying that considerable native interactions are present in the transition state [bib_ref] Structure of the transition state for the binding of c-Myb and KIX..., Giri [/bib_ref] , perhaps surprising given that no change in k on was seen upon increasing residual structure [bib_ref] Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants, Shammas [/bib_ref] [bib_ref] A folding-afterbinding mechanism describes the recognition between the transactivation domain of c-Myb..., Gianni [/bib_ref]. Finally, analysis of the formation of the spectrin tetramerization domain from two disordered peptides revealed high -values in the C terminus of helix A and the N terminus of helix B. For helix C, tertiary -values were higher than for helix A and B. A mechanism was proposed whereby preformed helix C provides a template onto which helix A and B dock, thus allowing core contacts to form and further folding to proceed after binding [bib_ref] Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered..., Hill [/bib_ref].
In all of these studies, a general trend of binding before folding is inferred for the coupled folding and binding reactions.
Because there are few studies so far, it is not possible to come to a general conclusion about the mechanism of coupled folding and binding for IDPs. It is likely, as in protein folding [bib_ref] The present view of the mechanism of protein folding, Daggett [/bib_ref] , that there will be a spectrum of folding upon binding mechanisms, but where the interaction is very rapid, binding before folding seems, at present, to be most likely.
## Do folded partner proteins play a role?
Coupled folding and binding studies have tended to focus on IDPs, with less attention paid to folded partner proteins. Nevertheless, as we now describe, the studies that have been performed have indicated that they may have an important role to play. Truncations in the binding interface of the folded partner protein Mcl-1 reduce the affinity for the IDP PUMA, due to an increase in k off ; however, an unexpected increase in k on occurs for some residues. Although beneficial for affinity of the com- . Relationship between association and dissociation rate constants and -values for apparent two-state systems. Shown are energy diagrams (first column), observed association rate constants under pseudofirst order conditions (middle column), and observed dissociation rate constants (third column) for wild-type IDP (blue) and mutant IDP (red). A, ϭ 1, i.e. native interactions are formed in the transition state. k on is lower, and k off is unchanged. B, 0 Ͻ Ͻ1 structure is partially formed, resulting in changes in both k on and k off . C, ϭ 0, residue is as unstructured at the transition state as in the unbound state. k on is unchanged, and k off is increased. The rate constants k on and k off are controlled by energy barrier sizes (first column), and are determined from straight-line gradients in association mixing experiments (second column) and from high concentration asymptotes in out-competition dissociation mixing experiments (third column), respectively. [bib_ref] Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered..., Hill [/bib_ref]. In A, B, and C, the folded partners are shown in gray. In D and E, both partners are disordered; one is shown in gray, and one is shown in bronze. The residues in blue, magenta, and red represent high ( Ͼ 0.6), medium (0.25 Ն Յ0.6), and low ( Ͻ 0.25) -values, respectively. N and C denote the N and C termini of the IDP (note that in E the disordered regions are capped by folded domains).
plex, these residues are effectively inhibiting association. Spatial patterning of the association-inhibiting residues, together with analysis of the NMR ensemble of free Mcl-1, suggests that the hydrophobic binding grove of Mcl-1 undergoes a conformational rearrangement while binding PUMA [bib_ref] Interplay between partner and ligand facilitates the folding and binding of an..., Rogers [/bib_ref]. Closing of the grove around PUMA helps to maintain the complex.
The folded KIX domain of CBP is able to bind multiple transcription factors in vivo [bib_ref] CBP/p300 in cell growth, transformation, and development, Goodman [/bib_ref] , most of which are intrinsically disordered. Several studies noted positive cooperativity between ligands binding to its two binding sites [bib_ref] Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants, Shammas [/bib_ref] [bib_ref] Molecular recognition by the KIX domain and its role in gene regulation, Thakur [/bib_ref] , although the mechanism behind the cooperativity was not initially clear. Kinetic analysis revealed that both k on and k off were reduced when a ligand was already bound to the alternate site, and that the stabilization of the ternary complex was because the reduction in k off exceeded that for k on [bib_ref] Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants, Shammas [/bib_ref] [bib_ref] The mechanism of binding of the KIX domain to the mixed lineage..., Toto [/bib_ref]. A similar finding was reached independently using Gō-like molecular dynamics simulations [bib_ref] Prepaying the entropic cost for allosteric regulation in KIX, Law [/bib_ref]. Combined with NMR data showing a stiffening of the CBP KIX backbone upon ligand binding [bib_ref] Allosteric communication in the KIX domain proceeds through dynamic repacking of the..., Brüschweiler [/bib_ref] , this leads to the suggestion that binding of one ligand to CBP KIX changes the flexibility of the folded domain, reducing the entropic cost for ligand binding to the alternate site [bib_ref] Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants, Shammas [/bib_ref] [bib_ref] Prepaying the entropic cost for allosteric regulation in KIX, Law [/bib_ref]. Dynamics in the folded CBP KIX domain are therefore an important factor that is able to influence the binding of its IDP partners.
These two examples demonstrate the importance of structural and dynamical changes in the folded partner protein upon ligand (IDP) binding. Further kinetic studies will help to uncover whether these findings are system-dependent or widespread.
# Conclusions and outlook
IDPs have emerged as an important class of proteins. Their predicted abundance within the eukaryotic proteome has raised several questions. What are the advantages and disadvantages of being disordered? Why are IDPs more prominent in some processes than others? What is the functional relevance of disorder? Answering these questions is important in understanding IDPs at a fundamental and applied level, e.g. protein or drug design. Studies of IDPs have revealed that the conformational ensemble can be altered by external factors (e.g. salts, crowders), which must be taken into account when investigating coupled folding and binding reactions [bib_ref] Interplay between partner and ligand facilitates the folding and binding of an..., Rogers [/bib_ref] [bib_ref] Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered..., Hill [/bib_ref] [bib_ref] Structure of the transition state for the binding of c-Myb and KIX..., Giri [/bib_ref] [bib_ref] Mapping backbone and side-chain interactions in the transition state of a coupled..., Bachmann [/bib_ref] [bib_ref] The transition state structure for coupled binding and folding of disordered protein..., Dogan [/bib_ref]. Although more studies are required, the few that have been published indicate that the transition state of coupled folding and binding reactions is relatively unstructured. Nevertheless, residual structure appears to be an important factor that is able to influence complex affinity by modulating association and dissociation rate constants. Due to their prominence in cell signaling, IDPs have arisen as important biomedical targets. When compared with a folded protein, IDPs typically lack accessible binding pockets, making them more difficult to target with traditional small molecules. Development of new therapeutic strategies requires a thorough mechanistic understanding of coupled folding and binding reactions. Should the target be the unbound IDP, the partner protein, or the complex? Which rate constants should be altered to modulate binding affinities during therapeutic development? Through understanding the importance of electrostatics, residual structure, transition state interactions, and partner proteins, kinetic analysis can describe fundamental properties of IDPs, as well as their coupled folding and binding interactions.
[fig] FIGURE 1: The thermodynamic and kinetic properties of IDPs vary over orders of magnitude, and may be related to their function. Examples are given from studies in our laboratory(21,24,31). [/fig]
[fig] FIGURE 2: Kinetic experiments of coupled folding and binding reactions under pseudo-first order conditions. A, example kinetic traces for two-state and three-state processes, fit to single exponential and double exponential decay functions, respectively. a. u., arbitrary units. B, dependence of observed rate constants upon protein concentrations. Analytical solutions are presented for a two-state reaction (k on ϭ 40 M Ϫ1 s Ϫ1 , k off ϭ 10 s Ϫ1 ) and a three-state IF reaction (k ϩ ϭ 40 M Ϫ1 s Ϫ1 , k Ϫ ϭ 10 s Ϫ1 , k f ϭ 10 s Ϫ1 , k u ϭ 20 s Ϫ1 ). C, reaction schemes for two-state and three-state (IF and CS) processes. [/fig]
[fig] FIGURE 4: Illustrations of coupled folding and binding reaction transition states. -Values are mapped onto structures of the following complexes: A, PUMA⅐Mcl-1 (PDB: 2ROC) (43); B, c-Myb⅐CBP KIX (PDB: 1SBO) (51); C, S-peptide⅐S-protein (PDB: 1FEV) (55); D, ACTR⅐NCBD (PDB: 1KBH) (56; E, ␣⅐ spectrin tetramerization domain (PDB: 3LBX) [/fig]
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A Study of the Immunoloregulation of Double Filtration Plasmapheresis in Maintenance Hemodialysis Patients with Chronic Hepatitis C
Although a large number of drugs have been used to treat chronic hepatitis C (CHC), there still remains a great challenge to treat maintenance hemodialysis (MHD) patients with chronic hepatitis C. To clarify the immunnoloregulation of double filtration plasmapheresis (DFPP) in MHD patients with CHC, DFPP was performed in 20 MHD patients with CHC (HCVantibody positive, serum HCV RNA .500 IU/ml more than 6 months and HCV genotype 1b). The clinical data was collected and peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry at the time of hour 0, hour 3, day 3, day 7 and day 28 after the DFPP, respectively. Serum HCV particles could be removed partially by the DFPP. The titer of serum HCV RNA could remain in a lower level even 28 days after the treatment. Compared to MHD patients without HCV infection, the frequencies of innate immune cells were similar in MHD patients with CHC, while Th1/Th2 was elevated and the frequencies of regulatory T (Treg) cells were higher in those MHD patients with CHC. The frequencies of monocytes and natural killer (NK) cells remained after the DFPP in MHD patients with CHC. There were no significant changes of Th1, Th2 and Th1/Th2 in PBMC after DFPP. DFPP could reduce the frequencies of Th17 cells and Treg cells in PBMC from 7 days after DFPP in MHD patients with CHC. DFPP could partially remove the serum HCV particles mechanically. The titer of HCV RNA could remain in a lower level at least for 28 days probably due to the redistribution of the immunocytes in circulation.
# Introduction
Chronic hepatitis C (CHC) is the main cause of chronic liver disease in maintenance hemodialysis (MHD) patients who are at particular high risk for hepatitis C virus (HCV) infection. Among MHD patients, the prevalence of CHC varies greatly, from less than 5% to nearly 60% according to different areas of the world [bib_ref] Chronic viral hepatitis in hemodialysis patients, Tang [/bib_ref] [bib_ref] Liver damage in hemodialysis patients with hepatitis C virus viremia: a prospective..., Furusyo [/bib_ref] [bib_ref] Meta-analysis: Effect of hepatitis C virus infection on mortality in dialysis, Fabrizi [/bib_ref] [bib_ref] HCV infection and hemodialysis, Pol [/bib_ref] [bib_ref] The impact of renal transplantation on survival in hepatitis C-positive end-stage renal..., Knoll [/bib_ref]. The prevalence of HCV infection has declined in many dialysis centers, and yet it remains unacceptably high, ranging from 8% to 10% even in the industrialized countries [bib_ref] Hepatitis C and renal disease: an update, Meyers [/bib_ref]. It was recommended to monitor the markers of HCV routinely in MHD patients [bib_ref] KDOQI US commentary on the KDIGO clinical practice guideline for the prevention,..., Gordon [/bib_ref]. What's more, it has also been reported that HCV was associated with higher all-cause and cardiovascular mortality in MHD patients [bib_ref] Hepatitis C virus and death risk in hemodialysis patients, Kalantar-Zadeh [/bib_ref]. Over the past decades, several studies have pointed that the effective strategies of preventing and treating HCV infection in MHD patients could improve the prognosis of this population [bib_ref] Hepatitis C virus and death risk in hemodialysis patients, Kalantar-Zadeh [/bib_ref].
Combination of ribavirin (RBV) with peginterferon (PEG-IFN) is considered the gold standard of therapy in HCV-positive patients with normal renal function based on sustained virus response (SVR) up to 50% to 60% [bib_ref] Interferon treatment in hemodialysis patients with chronic hepatitis C virus infection: a..., Gordon [/bib_ref].The distribution of HCV genotypes were geographical different, and the predominant HCV genotype in China was genotype 1, with type 1b in particular [bib_ref] Update on epidemiology of hepatitis B and C in China, Cui [/bib_ref] , which was similar in MHD patients [bib_ref] Prevalence and risk factors of hepatitis C and B virus infections in..., Su [/bib_ref]. Unfortunately, SVR to standard therapy was much lower in patients with HCV genotype 1. Physicians are reluctant to use RBV in MHD patients given the fear of the drug-related side effects, particularly hemolytic anemia, which can be exacerbated in MHD patients [bib_ref] Hepatitis C virus infection and the dialysis patient, Fabrizi [/bib_ref]. The risk of severe side effects and the SVR limited the application of RBV and PEG-IFN in MHD patients. To date, it has been still difficult to treat CHC in MHD patients.
HCV clearance is mediated by T cells and the innate immune response. However, due to the progressive loss of kidney function, the function and interactions of the innate and adaptive immune systems in MHD patients are impaired and become much more complex [bib_ref] Immune cell dysfunction and inflammation in end-stage renal disease, Betjes [/bib_ref] [bib_ref] Immune dysfunction in uremia—an update, Cohen [/bib_ref]. Thus, it seems that improving the impairment of the innate and adaptive immune systems might provide novel treatment strategy for MHD patients with CHC. DFPP, a newly developed apheretic technique, selectively remove high molecular weight substances, has been proven to have several beneficial effects in immune systems. Recently, it has been reported that double-filtration plasmapheresis (DFPP) was effective for CHC. For CHC patients with high viral load, DFPP and IFN combination therapy produced a great reduction of viral load during the early stage of treatment and achieved a high SVR [bib_ref] Double filtration plasmapheresis and interferon combination therapy for chronic hepatitis C patients..., Fujiwara [/bib_ref]. However, as it stands, DFPP has also not been used in MHD patients with CHC and the underlying mechanisms of DFPP remain largely unknown.
In this study, single DFPP without IFN or RBV was given to MHD patients with CHC and the immune regulation of DFPP was focused. To clarify the immune regulation of DFPP in MHD patients with CHC, innate and adaptive immune cells in peripheral blood mononuclear cells (PBMCs) were monitored during the DFPP. It might provide the immunological mechanisms of a useful adjuvant therapy in MHD patients with CHC.
# Materials and methods
# Ethics statement
All of the following details of the study were approval by the responsible ethics committee of Nanjing Medical University (Permit Number: KY027). The written informed consent was supplied by the patients before the study.
## Study population
From October 2011 to April 2012, twenty MHD patients with CHC and 8 MHD patients without CHC from the Center for Kidney Disease of 2 nd Affiliated Hospital of Nanjing Medical University were recruited. MHD patients with CHC were defined as MHD patients with HCV-antibody positive and the titer of HCV RNA more than 500 IU/ml for 6 months or longer. The HCV genotype was genotype 1b in these 20 MHD patients with CHC. MHD patients without CHC were defined as MHD patients with HCV-antibody negative and the titer of HCV RNA less than 500 IU/ml. All participants were negative for hepatitis B surface antigen. Patients with platelet counts less than 100*10 9 /L and leukocyte counts less than 3*10 9 /L were excluded from the study.
## Dfpp
DFPP was performed once in MHD patients with CHC. For the DFPP, a Plasmaflo TM OP-08W (Asahi Kasei Medical, Tokyo, Japan) was used to separate the blood into plasma and cell components. The virus was then removed from the separated plasma by a second filter, Cascadeflo TM EC-50W (Asahi Kasei Medical, Tokyo, Japan) with an average pore size of 30 nm. The final volume of treated plasma was 50 ml/kg, and the treatment time was about 3 hours for the DFPP. All the patients were given the single DFPP. Blood samples were obtained at the time of hour 0, hour 3, day 3, day 7 and day 28 of the DFPP.
## Hcv rna measurement
The titer of serum HCV RNA was measured by real-time PCR with diagnostic kit for quantification of HCV RNA (PCR-Fluorescence Probing, Shanghai, China), which has a lower limit of detection of 500 IU/ml. Serum HCV RNA levels were measured by real-time PCR before the DFPP (hour 0), after the DFPP(hour 3) and 28 days after the therapy. The quantity of HCV RNA was converted to a log value at each virus measurement point.
## Isolation of pbmcs and flow cytometry
PBMCs were isolated from fresh heparin anti-coagulated blood by density gradient centrifugation using Ficoll-Hypaque (Sigma Chemical Co., St Louis, MO, USA). Multi-parameter flow cytometry was performed using a BD FACSAira instrument for detection of single fluorochromes and analyzed using FACSDiva software (BD Biosciences, San Jose, CA, USA).
Fluorochrome-labeled monoclonal antibodies (MAbs) specific for CD14-FITC, CD3-FITC CD16CD56-PE, CD3-PerCP-Cy5-5, CD8-APC, IFNc-FITC, IL-4-PE, CD4-PerCP-Cy5-5, CD25-FITC, CD127-PE were supplied by Beckman Coulter (San Diego, CA, USA). IL-17-PE MAb was purchased from eBiosciences (San Diego, CA, USA). For the detection of IL-17, IFNc and IL-4, PBMC (100 ml) was firstly activated with 50 ng/ml phorbol-12-myristate 13-acetate (PMA),1 mg/ml ionomycin and 1.7 mg/ml monensin ( all from Sigma Chemical Co., St Louis, MO, USA) for 4 hours. After staining for surface antigens, the remaining cells were permeabilized and stained with IFNc-FITC, IL-4-PE or IL-17-PE, respectively. The cells were fixed in 1% of paraformaldehyde if they could not be detected immediately.
The frequency of monocytes was expressed as a ratio of CD14+ cells to PBMCs, while natural killer (NK) cells was expresses as a ratio of CD3-(CD16CD56)+ cells to PBMCs. For subsets of T helper (Th) lymphocytes, the frequencies of Th1 and Th2 cells were expressed as a ratio of IFN+CD4+ cells to CD4+ lymphocytes and IL-4+CD4+ cells to CD4+ lymphocytes, respectively. The frequency of Th17 cells was expressed as a ratio of CD4+IL-17A+ cells to CD4+ lymphocytes. The frequency of regulatory T (Treg) lymphocytes was expressed as a ratio of CD4+CD25+CD127 2/low cells to CD4+ lymphocytes.
# Statistic analysis
The data was analyzed using SPSS for windows version 18.0. Clinical data are expressed as the mean 6 SD or frequency, as appropriate. Comparisons of continuous data of patients used the independent t test. Compared t test was used in the data at different time point after the DFPP to the data before the DFPP. A P value less than 0.05 was considered to represent a statistically significant difference.
# Results
## The clinical characteristics of mhd patients with chc and without chc
This study included 20 MHD patients with CHC and 8 MHD patients without CHC. Baseline characteristics of participants were summarized in [fig_ref] Table 1: The characteristics of MHD patients without and with CHC [/fig_ref]. The age of MHD patients with CHC was 50.762.3 years old and male-to-female ratio was 12:8; while the age of MHD patients without CHC was 41.7610.9 years old and male-to-female ratio was 5:3. The duration of hemodialysis in MHD patients with CHC was longer than those without CHC, with 11.066.5 years compared to 6.465.4 years (P,0.05). Hemoglobin (HGB) was similar in these two groups, with 110.567.9 g/L in MHD patients without CHC and 108.2615.2 g/L in patients with CHC. Liver function in MHD patients without CHC was all within the normal range, while 4 patients with CHC had mild abnormal liver function. Serum alanine transaminase (ALT) was 37.3628.0 U/L in patients with CHC compared to 20.367.8 U/L in patients without CHC. There was no difference in serum albumin (ALB) between MHD patients with and without CHC. The titer of serum HCV RNA in MHD patients with CHC before DFPP was 0.53*10 6 IU/ml -50.4*10 6 IU/ml.
## The safety of dfpp
When DFPP was performed in those 20 MHD patients with CHC, 4 of 20 (20.0%) cases experienced some adverse events. A drop in blood pressure was observed in 2 patients, but recovered after giving intravenous 100 ml-200 ml saline solution. Minor disorder was observed in 2 patients, which was temporary and recovered without any treatment. All the 20 patients completed the treatment smoothly. Clinical markers were monitored during the DFPP. There was no change in HGB and the platelet count. The fibrinogen levels fell significantly from 2.6860.76 g/L to 1.3460.7 g/L after DFPP. Serum ALB was reduced from 42.962.1 g/L to 34.563.1 g/L after DFPP. Serum immunoglobulin could also be removed by DFPP.The reduction of fibrinogen, ALB and immunoglobulin could recover to the initial levels within one week after the completion of DFPP. There was no bleeding and infection or other adverse events within one month after the DFPP.
Single DFPP could maintain the titer of serum HCV RNA in a lower level for at least 28 days Serum HCV RNA was used to evaluate the efficiency of DFPP. The titer of serum HCV RNA was monitored before and after the DFPP. The titer of serum HCV RNA could be reduced from 0.53*10 6 IU/ml -50.4*10 6 IU/ml to 0.20 *10 6 IU/ml -21*10 6 IU/ml in MHD patients with CHC by single DFPP. The titer of serum HCV RNA reduced rapidly after the therapy, which was accordant to the reported. [bib_ref] Double filtration plasmapheresis and interferon combination therapy for chronic hepatitis C patients..., Fujiwara [/bib_ref] The log HCV RNA before the DFPP was 6.8860.51 IU/ml, while which was lower after the DFPP with log HCV RNA 6.4360.67 IU/ml (P,0.001) [fig_ref] Figure 1: Viral dynamics of HCV RNA during the DFPP in MHD patients with... [/fig_ref]. There was another important phenomenon we were interested. The level of serum HCV RNA was monitored 28 days after the DFPP. The log HCV RNA on day 28 after DFPP was 5.9261.07 IU/ml, which was still lower than the titer before DFPP (P = 0.003) [fig_ref] Figure 1: Viral dynamics of HCV RNA during the DFPP in MHD patients with... [/fig_ref]. It was demonstrated that the titer of serum HCV RNA was maintained in a lower level even 28 days after the therapy.
The frequencies of monocyte in PBMC in MHD patients with CHC maintained during one single DFPP Monocyte is one of the important parts of the innate immune system in human, which play multiple roles in immune function. The classical monocyte is characterized by high level expression of the CD14 cell surface receptor. The frequency of monocyte was expressed as a ratio of CD14+ cells to PBMC [fig_ref] Figure 2: The frequencies of monocytes in PBMC after a single DFPP [/fig_ref]. The frequency of monocyte in PBMC was evaluated in MHD patients with or without CHC. It was found that the frequency of monocytes was 3.9961.52% in patients without CHC and 3.9661.81% in patients with CHC (P = 0.983) [fig_ref] Figure 2: The frequencies of monocytes in PBMC after a single DFPP [/fig_ref]. The tendency of monocyte in PBMC was monitored in MHD patients with CHC during the DFPP. It was shown that the frequencies of monocytes in PBMC maintained during the DFPP, with 3.9761.88% at hour 3, 3.9261.80% on day 3, 3.9061.20% on day 7 and 3.5161.52% on day 28, respectively (P.0.05) [fig_ref] Figure 2: The frequencies of monocytes in PBMC after a single DFPP [/fig_ref].
## The frequencies of nk cells remained unchanged during single dfpp
NK cells are a type of cytotoxic lymphocytes critical to the innate immune system. The frequency of NK cells was expressed as a ratio of CD3-CD16+CD56+ cells to PBMC [fig_ref] Figure 3: The percentages of NK cells in PBMC after the DFPP [/fig_ref]. The frequency of Th17 cells decreased from day 7 after the DFPP Th17 cells have been identified as a unique subset of T helper cells, which are CD4+ T cells that are defined by the production of IL17. The frequency of Th17 cells in PBMC was evaluated in patients during the DFPP, which was expressed as a ratio of IL-17+CD4+ cells to CD4+ lymphocytes [fig_ref] Figure 5: The changes of Th17 cells in PBMC after the DFPP [/fig_ref]. The frequency of Th17 cells was 2.0061.19% in MHD patients with CHC and 1.5060.91% in those patients without CHC, although there was no significant difference (P = 0.428) [fig_ref] Figure 5: The changes of Th17 cells in PBMC after the DFPP [/fig_ref]. The tendency of Th17 cells in PBMC was monitored during the DFPP. It was found that accompanied with the reduction of the titer of serum HCV RNA, the frequency of Th17 cells decreased from day 7 (1.6760.73%) to day 28 (1.4760.59%) after the therapy (P = 0.032) [fig_ref] Figure 5: The changes of Th17 cells in PBMC after the DFPP [/fig_ref].
The frequency of Treg cells was higher in MHD patients with CHC, which was reduced from 7 days after the DFPP Treg cells are a component of the immune system that suppress immune responses of other cells, which come in many forms with the most well-understood being those that express CD4, CD25, and Foxp3 (CD4+CD25+Foxp3+ regulatory T cells). It has recently been suggested that a lack of CD127 expression can be used to identify human CD4+ regulatory T cells, especially when combined with CD25. It has been reported that CD4+CD25+CD127 2/low phenotype is a good and easy-toperform surrogate identification strategy for FOXP3+ regulatory T cells in HIV-1 infected subjects [bib_ref] CD4+CD25+CD127-assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV-1..., Saison [/bib_ref]. The frequency of Treg cells in this study was expressed as a ratio of CD4+CD25+CD127 2/low cells to CD4+ lymphocytes. The ratio of CD4+CD25+ cells to CD4+ lymphocytes was also analyzed [fig_ref] Figure 6: The tendencies of Treg cells in PBMC after a single DFPP [/fig_ref].
The frequency of CD4+CD25+ cells in MHD patients with CHC was higher than that in MHD patients without CHC, 3.1761.68% vs 1.7860.86% (P = 0.026) [fig_ref] Figure 6: The tendencies of Treg cells in PBMC after a single DFPP [/fig_ref]. The frequency of CD4+CD25+CD127 2/low Treg cells was also higher in patients with CHC than in those without CHC, 2.2261.47% vs 1.3260.78% (P = 0.019) [fig_ref] Figure 6: The tendencies of Treg cells in PBMC after a single DFPP [/fig_ref]. The tendency of Treg cells was monitored during the DFPP. Accompanied with the reduction of serum HCV particles, the frequency of Treg cells decreased from day 7 to day 28 after the DFPP. The frequency of CD4+CD25+ Treg cells was 2.3261.07% on day 7 and 2.1560.78% on day 28 (P = 0.027) after the DFPP [fig_ref] Figure 6: The tendencies of Treg cells in PBMC after a single DFPP [/fig_ref] , while the frequency of CD4+CD25+CD127 2/low Treg cells was lower on day 7 with 1.7560.95% after the DFPP (P = 0.034) [fig_ref] Figure 6: The tendencies of Treg cells in PBMC after a single DFPP [/fig_ref].
# Discussion
More than 170 million people worldwide are chronically infected with HCV [bib_ref] Viral hepatitis C, Poynard [/bib_ref]. MHD patients are at particular high risk for bloodborne infections because of prolonged vascular access and potential for exposure to contaminated equipment. The prevalence of HCV in MHD patients exceeds 50% in some developing countries [bib_ref] Patterns of hepatitis C prevalence and seroconversion in hemodialysis units from three..., Fissell [/bib_ref] [bib_ref] High prevalence of and risk factors for hepatitis C in haemodialysis patients..., Huraib [/bib_ref]. Although it has been documented that CHC among MHD patients was mild in disease activity and was not so progressive compared to the controls with CHC [bib_ref] Natural history of chronic hepatitis C in patients on hemodialysis: case control..., Okuda [/bib_ref] , in MHD patients, HCV infection exhibited distinct clinical patterns, including associations with higher dialysis treatment vintage, and is associated with higher mortality [bib_ref] Hepatitis C virus and death risk in hemodialysis patients, Kalantar-Zadeh [/bib_ref].
Combination of RBV with PEG-IFN is considered the gold standard of therapy in HCV-positive patients with normal renal function [bib_ref] Interferon treatment in hemodialysis patients with chronic hepatitis C virus infection: a..., Gordon [/bib_ref]. Although this therapeutic schedule was also recommended in MHD patients with HCV, the risk of severe life-threatening side effects in this population could not be ignored [bib_ref] Hepatitis C virus infection and the dialysis patient, Fabrizi [/bib_ref] [bib_ref] Interferon monotherapy of chronic hepatitis C in dialysis patients: meta-analysis of clinical..., Fabrizi [/bib_ref]. The high cost of the antiviral treatment, the risk of severe side effects, and the lack of data on the impact of therapy and SVR response on mortality in MHD patients limited the application in this population [bib_ref] Hepatitis C infection and chronic renal diseases, Perico [/bib_ref]. There is still a relative lack of treatment of CHC in MHD patients which needs further research. Finding better therapeutic measures to MHD patients with CHC has been a great challenge in improving the long-term prognosis of dialysis patients.
Immunological cell dysfunction is an important clinical feature in MHD patients, which results in an increased susceptibility to bacterial and viral infection. It has been reported that the absolute number of NK cells in MHD patients is markedly decreased (by 20-32%) [bib_ref] Natural killer cell alterations correlate with loss of renal function and dialysis..., Vacher-Coponat [/bib_ref]. Monocytes in MHD patients have an activated profile with increased secretion of proinflammatory cytokines [bib_ref] Uremia impairs monocyte and monocyte-derived dendritic cell function in hemodialysis patients, Lim [/bib_ref]. The injury of T cell-mediated immune function is an important characteristic in MHD patients [bib_ref] Uremia causes premature ageing of the T cell compartment in end-stage renal..., Meijers [/bib_ref]. The effector T cells decreased severely and the inhibitory Treg cells reduced in MHD patients [bib_ref] Immune dysfunction in uremia—an update, Cohen [/bib_ref] [bib_ref] Dialysis treatment and regulatory T cells, Libetta [/bib_ref]. In patients with CHC, HCV has several means of inhibiting innate immune mechanisms, including inhibiting type I interferon responses, raising the activation threshold of NK cells activation [bib_ref] Evolutionary struggles between NK cells and viruses, Lanier [/bib_ref] [bib_ref] Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits..., Tseng [/bib_ref]. It has been reported that the pro-inflammatory cells Th17 cells were increased with severity of liver inflammation in patients with CHC [bib_ref] Th17 cells are increased with severity of liver inflammation in patients with..., Chang [/bib_ref]. Higher frequency of Tregs was identified in patients with CHC compared with controls, which might inhibit Th1 and Th2 cell responses either indirectly by modulating antigen-presenting cell function or directly by cell-cell contact [bib_ref] Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T..., Ebinuma [/bib_ref] [bib_ref] Regulatory T cells and infection: a dangerous necessity, Belkaid [/bib_ref]. The disturbance of immune function is more noticeable in MHD patients with CHC. Data in this study also demonstrated that, the frequency of Treg cells was higher and Th1/Th2 was increased in MHD patients with CHC compared to those MHD patients without CHC.
DFPP, a newly developed apheretic technique, selectively remove high molecular weight substances, including immunoglobulins and immune complexes, has been widely used to eliminate auto-antibodies from plasma. Recently, DFPP combined with IFN and RBV has been used in CHC patients with high viral load and living donor liver transplant recipients with hepatitis C. All these clinical studies demonstrated that DFPP appeared to be effective in reducing viremia and preventing HCV recurrence in patients. DFPP combined with IFN and RBV is indicated more for relapse patients than for NVR patients [bib_ref] Effects of doublefiltration plasmapheresis combined with interferon plus ribavirin for recurrent hepatitis..., Takada [/bib_ref] [bib_ref] Double filtration plasmapheresis in combination with interferon therapy for chronic hepatitis C, Levent [/bib_ref] [bib_ref] Efficacy and safety of double filtration plasmapheresis in combination with interferon therapy..., Kaneko [/bib_ref]. Kondo etc observed 12 CHC patients treated with DFPP/PEG-IFN/RBV therapy. The rapid reduction of HCV-Core antigens and changes in the distribution of lymphoid cells could contribute to the favorable immunological response during the DFPP/Peg-IFN/ RBV therapy [bib_ref] Double filtration plasmapheresis could induce a favorable immune response for chronic hepatitis..., Kondo [/bib_ref] [bib_ref] Rapid reduction of hepatitis C virus-Core protein in the peripheral blood improve..., Kondo [/bib_ref]. However, the effect of DFPP in MHD patients with CHC has not been reported. Thus far, the immunological mechanism underlying the efficacy of DFPP besides the reduction of HCV RNA remains poorly understood. The understanding of its underlying mechanisms may identify novel avenues for treatment MHD patients with CHC.
In order to exclude the interference of IFN and RBV, single DFPP without antivirus drugs was performed in MHD patients with CHC. It was found that besides the rapid reduction of HCV particles, the titer of serum HCV RNA maintained in a lower level even 28 days after the therapy. Main immunocytes in PBMCs in MHD patients with CHC were monitored during the DFPP. Single DFPP could not affect the frequencies of innate immune cells such as monocytes and NK cells. There were no significant changes in the frequencies of Th1 cells, Th2 cells and Th1/Th2 during the therapy. The frequency of Th17 cells decreased from day 7 to day 28 after the DFPP. It was indicated that the systemic inflammatory reaction was ameliorated after the partial removal of HCV particles. An increased frequency of Treg cells at the onset of HCV infection was suggested to predict a chronic outcome of the infection [bib_ref] Elevated CD4+/CD25+ T cell frequency and function during acute hepatitis C presage..., Perrella [/bib_ref]. In this study, we found that the frequencies of Treg cells decreased from day 7 after the DFPP. The decrease of Treg cells after the DFPP indicated that the immune tolerance was partially released accompanied with the reduction of HCV particles. To some extent, the decrease of Treg cells might contribute to the persistent lower titer of serum HCV RNA.
In conclusion, it was a preliminary study focusing on the improvement of immunological function in PBMCs after the DFPP in MHD patients with CHC. The application of DFPP with or without anti-virus drugs in MHD patients with CHC still needs further studied.
[fig] Figure 1: Viral dynamics of HCV RNA during the DFPP in MHD patients with CHC. A. The titers of HCV RNA before and after the DFPP. B. The titers of HCV RNA during 28 days after DFPP. The Y axis indicates the log HCV RNA. The X axis indicates the time course of the therapy. 0 h means before the DFPP, 3 h means after the DFPP, 28 d means 28 days after the DFPP. doi:10.1371/journal.pone.0082524.g001 [/fig]
[fig] Figure 2: The frequencies of monocytes in PBMC after a single DFPP. A, B. Monocyte was expressed as CD14+ cells in PBMCs. C. The frequencies of monocytes in MHD patients with CHC compared to those without CHC. D. The frequencies of monocytes in MHD patients with CHC during the DFPP. doi:10.1371/journal.pone.0082524.g002There were no significant changes of Th1, Th2 and Th1/ Th2 in PBMC during the DFPP The frequencies of Th1 and Th2 cells were expressed as a ratio of IFN-c+CD4+ cells to CD4+ lymphocytes and a ratio of IL-4+CD4+ cells to CD4+ lymphocytes, respectively(Figure 4 A-D). There was no significant difference of the frequencies of Th1 and Th2 cells in MHD patients with or without CHC(Figure 4 E, G). Th1/Th2 was enhanced in MHD patients with CHC compared to those MHD patients without CHC, 16.768.9 vs 6.262.6 (P = 0.048)(Figure 4 I). The frequencies of Th1 cells, the frequencies of Th2 cells and the ratio of Th1 to Th2 in MHD patients with CHC remained unchanged during the DFPP, respectively(Figure 4 F, H, J). It was demonstrated that there was no significant change of Th1 cells and Th2 cells during the DFPP, although Th1/Th2 was enhance in MHD patients with CHC. [/fig]
[fig] Figure 3: The percentages of NK cells in PBMC after the DFPP. A, B. NK cell was expressed as CD3-CD16+CD56+ cells in PBMCs. C. The frequencies of NK cells in MHD patients with CHC compared to those without CHC. D. The frequencies of NK cells in MHD patients with CHC during the DFPP. doi:10.1371/journal.pone.0082524.g003 [/fig]
[fig] Figure 4: The tendencies of Th1 and Th2 cells in PBMC after the DFPP. A-D. Th1 cells was expressed as IFN-c+CD4+ cells; Th2 cells was expressed as IL-4+CD4+ cells. E, F The frequencies of Th1 cells in MHD patients with CHC during the DFPP. G, H The frequencies of Th2 cells in MHD patients with CHC during the DFPP. I, J The ratio of Th1 to Th2 cells in MHD patients with CHC. doi:10.1371/journal.pone.0082524.g004 [/fig]
[fig] Figure 5: The changes of Th17 cells in PBMC after the DFPP. A-C. Th17 cell was expressed as CD17+CD4+ cells in PBMC. D. The frequency of Th17 cells in MHD patients with CHC compared to those without CHC. E. The frequencies of Th17 cells in MHD patients with CHC during the DFPP. doi:10.1371/journal.pone.0082524.g005 [/fig]
[fig] Figure 6: The tendencies of Treg cells in PBMC after a single DFPP.A-D. Treg cell was expressed as CD4+CD25+ CD127 low/2 cells in PBMC. E, F. The frequency of CD4+CD25+ Treg cells in MHD patients with CHC during the DFPP. G, H. The frequencies of CD4+CD25+ CD127 low/2 Treg cells in MHD patients with CHC during the DFPP doi:10.1371/journal.pone.0082524.g006 [/fig]
[table] Table 1: The characteristics of MHD patients without and with CHC. [/table]
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A sacrificial layer strategy for photolithography on highly hydrophobic surface and its application for electrowetting devices
Patterning micro-structures on highly hydrophobic surface by photolithography is usually inevitable for fabricating devices based on electrowetting effects. The key challenges for such photolithography processes are how to coat photoresist uniformly and maintain the hydrophobicity of the highly hydrophobic surface, which are usually two contradict aspects. Moreover, the patterned microstructure must adhere to the highly hydrophobic surface excellently, which is critical for device application. However, a simple and robust fabrication process that fulfills all the above requirements was seldom reported. In this paper, we developed a sacrificial layer photolithography strategy on highly hydrophobic surface. Photoresist is easily coated uniformly all over the substrate by introducing a sacrificial layer between the photoresist and the highly hydrophobic surface. The hydrophobicity of the exposed hydrophobic surface was maintained and the adhesion of the microstructures to the substrate is excellent. An electrowetting display sample was demonstrated by this fabrication strategy, which showed dynamic image displaying with response time less than 40 ms. The strategy is applicable to both rigid and flexible substrate and manufacturing compatible. We believe our developed photolithography process is important for research and development of devices based on electrowetting effect.Electrowetting (EW) is an electromechanical effect which generally refers to changing the contact angle of liquid to a solid surface by application of an electric field 1, 2 . It has become one of the most powerful tools for manipulating tiny amounts of liquids in microsystems. The applications of electrowetting range from 'lab-on-a-chip' devices 3-5 to adjustable liquid lenses 6-8 and new kinds of electronic displays 9-12 . In these applications, the microsystems are usually fabricated by photolithography on a solid dielectric layer with very low surface energy (highly hydrophobic) and the liquid was confined on this layer by relatively hydrophilic vertical grid or walls 13 . Bottom electrodes were patterned below the hydrophobic dielectric layer, and a top substrate with common or separated electrodes was used to seal the liquid in between. The liquid was manipulated by the electric field that generated between the top and bottom electrodes, as schematic diagramed inFig. 1d. Usually lower surface energy of the hydrophobic dielectric layer leads to better manipulation of the liquid 2 . However, this also causes more difficulties for fabricating the hydrophilic grid or walls by photolithography to confine the liquid 13-15 .There are many reports on fabrication of devices based on electrowetting effect. In these reports, highly hydrophobic dielectric materials (fluoropolymer such as Teflon TM AF 16 , CYTOP 13 , Fluoropel 13 , etc.) were usually used for the low surface energy layer. SU-8 negative-tone photoresist is a common choice for the hydrophilic grid. The SU-8 is wet with aqueous liquid and does not dissolve or degrade over time in the presence of water, oil or light 13 . More importantly, SU-8 resist is photo-patternable with easily controlled grid thickness. For the purpose of EW devices, patterning the SU-8 grid on the fluoropolymer must not foul the hydrophobicity of the Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 7: 3983 |
surface, and the SU-8 grid must adhere to the fluoropolymer with such strength that it survives in a tape test or a finger-nail scratch test [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref]. These requirements are very challenging for the EW devices fabrication. Patterning microstructures on fluoropolymer is not an easy task, because photoresists are very difficult to coat on the surface of fluoropolymer due to its high hydrophobicity [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref] [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref] [bib_ref] The application of fixed hydrophobic patterns for confinement of aqueous solutions in..., Li [/bib_ref]. High viscous photoresists [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref] [bib_ref] The application of fixed hydrophobic patterns for confinement of aqueous solutions in..., Li [/bib_ref] are possible to be coated on fluoropolymer directly. The hydrophobicity was not compromised in this way, however, coating results of the resist was not satisfying [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref] [bib_ref] A new fabrication process for uniform SU-8 thick photoresist structures by simultaneously..., Lee [/bib_ref]. Photoresist at the edge area of the wafer shrinks towards the center during soft bake of it, which results in serious edge bead and leaving a relatively large blank edge area without resist [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref]. Furthermore, the adhesion of the patterned resist at the end of processing was very poor. The fabricated grid may even be blown off the substrate by a nitrogen gun (Supporting information S1). To facilitate the resist coating, various leveling agents (e.g. FluoroN of Cytonix Corp [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref]. or Zonyl ® FSN [bib_ref] The application of fixed hydrophobic patterns for confinement of aqueous solutions in..., Li [/bib_ref] or oxygen plasma [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref] were employed to treat the fluoropolymer, which allows the SU-8 to be spin coated on the fluoropolymer surface with pretty good coverage. However, either the surface hydrophobicity was found to be degraded [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref] [bib_ref] The application of fixed hydrophobic patterns for confinement of aqueous solutions in..., Li [/bib_ref] or the adhesion was still not good enough [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref].
In spite of pretty good micro-fabrication results of SU-8 grid on fluoropolymer were presented in many reports [bib_ref] Agile wide-angle beam steering with electrowetting microprisms, Smith [/bib_ref] [bib_ref] A 3.5-inch Bendable Active Matrix Electrowetting Display, Chen [/bib_ref] [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref] [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref] , the complete description of the details of the fabrication process was seldom released, which made the results difficult to repeat by other groups [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref]. Therefore, a robust and general fabrication process for electrowetting devices is important for its development and application. In this work, we report a simple and robust fabrication process of SU-8 grid on fluoropolymer surface. SU-8 resist can be coated and patterned uniformly throughout the surface of the fluoropolymer. The patterned SU-8 grid adhered to the substrate every well and the hydrophobicity of the exposed fluoropolymer was almost not influenced. An electrowetting display (EWD) samples were fabricated following our proposed process flow, which demonstrates the potential application of the fabrication process.
# Results and discussion
There are several types of commercial available fluoropolymer usually used for electrowetting or micro-fluid devices as mentioned before [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref] [bib_ref] The application of fixed hydrophobic patterns for confinement of aqueous solutions in..., Li [/bib_ref] [bib_ref] Amorphous fluoropolymers as insulators for reversible low-voltage electrowetting, Seyrat [/bib_ref]. In this work, CYTOP (Asahi, CTL-809M) was used as the hydrophobic dielectrics, and the solution was diluted to a concentration about 3%. The CYTOP solution was spin coated onto the silicon wafer at a speed of 1000 rpm for 1 minute. Then it was hard baked on hotplate at 180 °C for 20 minutes to evaporate the solvent. The thickness of the obtained CYTOP film was about 115 nm, which is very uniform except for the very edge area of wafer due to the edge bead effect [fig_ref] Figure 2: Optical images of the photolithography process [/fig_ref]. The water contact angel (CA) of the obtained CYTOP film was measured to be ∼115° in average. Such surface is too hydrophobic for spin coating of photoresist (Supporting information S1).
As mentioned above, leveling agent or oxygen plasma treatment will facilitate coating of SU-8 on CYTOP, however, with the cost of hydrophobicity [bib_ref] The Structure and Manufacturing Process of Large Area Transparent Electrowetting Display, Ku [/bib_ref] [bib_ref] The application of fixed hydrophobic patterns for confinement of aqueous solutions in..., Li [/bib_ref] , which degrades the performance of the EW devices. To coating SU-8 on CYTOP, we introduced a sacrificial layer strategy as shown in [fig_ref] Figure 1: Process flow of the sacrificial layer photolithography strategy on highly hydrophobic substrate [/fig_ref]. A layer of SiO 2 (∼60 nm) was deposited onto hard baked CYTOP by e-beam evaporation. This layer was used as sacrificial layer in the following fabrication steps. The evaporated SiO 2 covered the CYTOP surface uniformly as shown in Figs 2b and 3b. Then SU-8 3010 (Micro Chem) was spin coated on the SiO 2 layer, and pre-baked at 95 °C on hot-plate for 3 min. Benefiting from the SiO 2 sacrificial layer, either high or low viscous photoresist was easily coated on the substrate with 100% coverage [fig_ref] Figure 2: Optical images of the photolithography process [/fig_ref] and Supporting information S1). Then the designed patterns were easily transferred to the substrate by conventional photolithography [fig_ref] Figure 2: Optical images of the photolithography process [/fig_ref]. After exposure, the SU-8 resist was post-baked at 95 °C for another 3 min., followed by developing and rinsing. The exposed SiO 2 was removed by HF etching (1:25 diluted, 30s) (Supporting information [fig_ref] Figure 1: Process flow of the sacrificial layer photolithography strategy on highly hydrophobic substrate [/fig_ref]. Then the sample was rinsed in deionized water and blown dry by nitrogen. Finally, the fabricated SU-8 grid was hard baked at 150 °C for 30 min. to fully crosslink the resist and evaporate the solvent. Following these procedures, SU-8 was easily coated and patterned on both rigid and flexible substrates uniformly [fig_ref] Figure 2: Optical images of the photolithography process [/fig_ref].
The thickness uniformity of the coated photoresist was measured at different locations around the substrate, and the results were shown in . The thickness fluctuation of both high (SU-8 3010) and low (S1813, Microposit) viscous resist are less than ± 3%, which is consistent with the uniformity specification of our spin coater.
As mentioned before, the adhesion of the fabricated SU-8 grid to the fluoropolymer and the hydrophobicity of the fluoropolymer are critical for the EW devices. To characterize the adhesion, standard tape test 21 was carried out [fig_ref] Figure 4: Tape test and bending test for adhesion [/fig_ref]. We firstly tested the adhesion of the evaporated SiO 2 film to the CYTOP (Supporting information S2). As shown in [fig_ref] Figure 4: Tape test and bending test for adhesion [/fig_ref] , the squares cut in the as-deposited SiO 2 layer was not peeled off the CYTOP by the adhesive tape. Since there is an annealing step in our process, so the adhesion of SiO 2 to CYTOP was also tested after 150 °C annealing treatment, and the result was shown in [fig_ref] Figure 4: Tape test and bending test for adhesion [/fig_ref]. These results demonstrate the SiO 2 sacrificial layer adheres to the CYTOP surface very well. The finally used actual grid structure for EWD devices is the SU-8 on SiO 2 sacrificial layer as shown in [fig_ref] Figure 1: Process flow of the sacrificial layer photolithography strategy on highly hydrophobic substrate [/fig_ref] ,d. So tape test was carried out on the fabricated SU-8/ SiO 2 /CYTOP stack. [fig_ref] Figure 4: Tape test and bending test for adhesion [/fig_ref] shows the optical images of the tested SU-8/SiO 2 /CYTOP stack on Si substrate. The structure was not found affected by peeling of the tape, which demonstrates the adhesion of the SU-8/SiO 2 / CYTOP stack is also very good. The adhesion of the grid structure on flexible substrate (PET) was characterized by a bending test as shown in [fig_ref] Figure 4: Tape test and bending test for adhesion [/fig_ref] (Supporting information S2 and Video-1). After 10,000 times of bending, the fabricated grid structure still kept its integrity and no peeling off the PET substrate was found even in the most strained area [fig_ref] Figure 4: Tape test and bending test for adhesion [/fig_ref]. These results suggest the adhesion of our fabricated structure can fulfill the requirement of EW devices on both rigid and flexible substrates.
The hydrophobicity change of CYTOP was monitored during the fabrication process, as shown in [fig_ref] Figure 5: Contact angle measurement results during the photolithography process [/fig_ref]. The CA of the as-coated CYTOP is ~115°. Since the CYTOP will exposed to HF during the SiO 2 etching, thus the as-coated CYTOP sample was put into HF solutions for 1 min. to test the influence of the HF etching on the CA of CYTOP. The measurement indicates HF has no effect on the hydrophobicity of the surface of CYTOP. After SiO 2 deposition, the measured CA of the SiO 2 surface is ~60°2 2 . It is very easy for photoresist, either high or low viscosity, coating on such surface, which gives more choice for the grid materials of the EW devices (Supporting information S1). After the SiO 2 layer was etched away by HF, the CA of the exposed CYTOP is ~103°. This indicates the hydrophobicity of the CYTOP was slightly affected by the e-beam deposition of SiO 2 . The evaporated SiO 2 bombarded the CYTOP surface, which changes the surface energy of CYTOP and results in excellent adhesion between SiO 2 and CYTOP. Though the SiO 2 evaporation affects the CYTOP slightly, we still want to minimize this effect. Therefore thermal anneal was carried out. As shown in [fig_ref] Figure 5: Contact angle measurement results during the photolithography process [/fig_ref] , 120 °C anneal will recover the CA to ~112°, and 150 °C anneal improve the CA to 113°, almost fully recovered. As we specified before, 150 °C hard bake, was used after patterning SU-8 grid. This commonly used hard bake procedure not only hardened the SU-8 grid, but also recovered the hydrophobicity of the exposed CYTOP surface in our process. Now we concluded that the SU-8 grid can be patterned on CYTOP with excellent adhesion and no compromise of its hydrophobicity by using our proposed process flow, which is very promising for EWD devices fabrication. To demonstrate the application of our proposed fabrication process, an electrowetting display sample with 10 × 10 pixel array was fabricated as shown in. Each pixel contains an array of 100 μm × 300 μm sub-pixels in 30 rows and 12 columnsand Supporting information S3). The lateral thickness of the SU-8 wall in one pixel is 30 μm. Since the purpose is to demonstrate the simple and robust fabrication process, these pixels were not fabricated on a thin film transistor (TFT) backplane. The 100 pixels were controlled individually by a homemade external circuit (Supporting information S3).The oil in the grid of one pixel contracted when a 13 V voltage was forced to the electrode of this pixel. Once the voltage was switched off, the oil fully recovered. The contraction and recovery of the oil was recorded by a CCD camera with video frequency of 25 frames/s (Supporting information S3 and Video-2). Both the contraction and recovery of the oil finished within one video frame which indicates the response time of the EWD is less than 40 ms. The EWD performance was mainly decided by the hydrophobicity of the surface, thus the measured EWD effect confirmed that the hydrophobicity of the CYTOP was well maintained in our fabrication process. By controlling the input signals of the pixels one by one, dynamic images of number 1 to 9 were displayed (Video-3). These results indicate the fabricated EWD is pretty good and possible for video frequency image display when combined with a TFT backplane. The fabrication of a EWD sample on a TFT backplane by our proposed simple and robust process is ongoing in our group.
In the above fabrication process, SiO 2 was used as the sacrificial layer. However, the sacrificial layer was not limited to SiO 2 . Other kinds of oxides or metals (e.g. Ti or Au) can also be deposited onto CYTOP for sacrifice. This gives us additional freedom for the device fabrication. Since the process is simple and the hydrophobicity of the exposed surface of the fluoropolymer was maintained with excellent adhesion of the stack, we believe this strategy is easily repeated in different labs and can be generalized to large area substrates. Furthermore, the sacrificial layer strategy is compatible with the existing manufacturing process of EWD, which is good for cost control.
In summary, a simple, robust and manufacturing compatible photolithography process on highly hydrophobic surface was developed in this paper. By introducing a sacrificial layer between the photoresist and the fluoropolymer layers, the fabricated patterns was found adhere to the highly hydrophobic surface excellently and the hydrophobicity of the exposed highly hydrophobic surface was maintained. EWD devices were fabricated following our developed fabrication process, which shows dynamic image displaying with response time less than 40 ms. These results demonstrate our developed fabrication process is very promising for EWD manufacturing.
# Methods
Spin coating of CYTOP and photoresist was using a CEE ® -200 (Brewer Science ® ) spin coater. SiO 2 was deposited by e-beam evaporator (K. J. Lesker AXXIS). Contact angle was measured by Dataphysics OCA20. Film thickness was measured by HORIBA UVISEL FUV ellipsometer and AMBiOS XP-1 profilometer. The adhesion of the fabricated microstructure to the substrate was characterized by using a standard tape test knit and a homemade bending equipment (Supporting information S2). EWD samples were fabricated on ITO coated glass substrate. The bottom ITO electrodes were first patterned by photolithography and wet etching. Then the SU-8 grid was fabricated following our sacrificial layer strategy. The colored oil (dodecane) was dosed by the reported self-dosing process [bib_ref] A full description of a simple and scalable fabrication process for electrowetting..., Zhou [/bib_ref] [bib_ref] Scalable fabrication of electrowetting displays with self-assembled oil dosing, Sun [/bib_ref] and then sealed under water using epoxy. The sample was tested for image display on a homemade test fixture (shown in [fig_ref] Figure 1: Process flow of the sacrificial layer photolithography strategy on highly hydrophobic substrate [/fig_ref] and Supporting information S3).
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).
[fig] Figure 1: Process flow of the sacrificial layer photolithography strategy on highly hydrophobic substrate (a-c), and schematic structure of the EWD (d). [/fig]
[fig] Figure 2: Optical images of the photolithography process. (a) CYTOP coated Si wafer, which was hard baked at 180 °C. (b) After SiO 2 deposition. (c) After spin coating of SU-8 3010 resist. (d) After photolithography and SiO 2 etching.(e) Typical zoom-in image of (d). The same process on PET (f) and corresponding zoom-in image (g). Also see Supporting information S1. [/fig]
[fig] Figure3.: Film thickness measurement around the 4-inch substrate of (a) CYTOP, (b) SiO 2 on CYTOP, and the coated high (c) and (d) low viscous photoresists. The thickness fluctuations of these films are less than ± 3% with 3 mm edge exclusion zone. [/fig]
[fig] Figure 4: Tape test and bending test for adhesion. (a) Schematic illustration of tape test. (b) Typical images of tape test of SiO 2 on CYTOP without and (c) with 30 min. annealing at 150 °C. (d) Typical tape test results of SU-8/SiO 2 /CYTOP stack on Si wafer. (e) Bending test for structures on flexible substrate. (f) Images of the SU-8/SiO 2 /CYTOP stack on PET which was taken at the most strained area (arrow in (e)) after bending test. Also see Supporting information S2. [/fig]
[fig] Figure 5: Contact angle measurement results during the photolithography process. [/fig]
[fig] Figure 6: (a) Photo of the fabricated EWD sample with 10 × 10 pixel array. (b) Zoom-in image of one pixel when switched off and (c) on. (d∼l) Photos of the displayed numbers 1 to 9 respectively. Also see Supporting information S3. [/fig]
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Designed for life: biocompatible de novo designed proteins and components
A principal goal of synthetic biology is the de novo design or redesign of biomolecular components. In addition to revealing fundamentally important information regarding natural biomolecular engineering and biochemistry, functional building blocks will ultimately be provided for applications including the manufacture of valuable products and therapeutics. To fully realize this ambitious goal, the designed components must be biocompatible, working in concert with natural biochemical processes and pathways, while not adversely affecting cellular function. For example, de novo protein design has provided us with a wide repertoire of structures and functions, including those that can be assembled and function in vivo. Here we discuss such biocompatible designs, as well as others that have the potential to become biocompatible, including non-protein molecules, and routes to achieving full biological integration.
# Introduction
Designed biomolecular and synthetic components that reproduce or even supersede the functions and activities of natural proteins and enzymes promise to revolutionize synthetic biology, industrial biotechnology and medicine [bib_ref] The coming of age of de novo protein design, Huang [/bib_ref] [bib_ref] Designing artificial enzymes by intuition and computation, Nanda [/bib_ref]. While Nature has undoubtedly provided us with a rich diversity of natural biomolecules, they might not be well adapted for a selected purpose or environment outside of their preferred cellular milieu [bib_ref] Biocatalysis in organic chemistry and biotechnology: past, present, and future, Reetz [/bib_ref] [bib_ref] Role of biocatalysis in sustainable chemistry, Sheldon [/bib_ref]. With artificial components such as de novo proteins, their sequence, structure and biophysical characteristics are selected solely by the designer, conferring, for instance, greater thermal and chemical stability than their natural equivalents and rendering them better suited to our requirements. Chemical activities unknown in Nature may be imposed upon them [bib_ref] Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction, Siegel [/bib_ref] , imparting unique reactivities that may be integrated into the biochemical processes of living organisms. Such endeavours not only result in useful biomolecular or artificial componentry, but also provide powerful, fundamental insights into the engineering of natural biomolecules.
This review focuses on artificial components that are, or have the potential to become, biocompatible. To be defined as biocompatible a component must efficiently perform an intended role in vivo and at least be able to interact in a complementary manner with cells or natural biomolecular components. To satisfy a more stringent definition of biocompatibility, the components-or the biosynthetic pathway that produces them-should be genetically encoded or imported directly into the cell, and they should be fully assembled and functional in vivo, without any significant deleterious effects. For cofactor-dependent proteins and enzymes, this inevitably requires post-translational insertion of small molecules such as hemes and flavins to impart the desired functionality.
With such biocompatible components, there is then an opportunity to design systems where natural and synthetic components work synergistically to expand the range of possibilities offered by entirely natural or entirely synthetic systems [bib_ref] Synthetic biology through biomolecular design and engineering, Channon [/bib_ref]. Synthetic molecules that can be produced by living organisms also present the possibility of 'eco-friendly' manufacturing, negating the need for expensive synthetic processes [bib_ref] Role of biocatalysis in sustainable chemistry, Sheldon [/bib_ref].
Translating a particular function from a natural protein to a synthetic element is a challenge, and achieving biocompatibility is a further hurdle due to the immense complexity, diversity and specificity of cellular processes [bib_ref] Constructing manmade enzymes for oxygen activation, Armstrong [/bib_ref]. Currently, the components that most fulfil these requirements are de novo designed proteins, although there are other chemical entities that, with further development, could become biocompatible. Here we will discuss recent developments in the design of de novo proteins and non-natural elements that reproduce natural biomolecular functions, with a particular focus on biocompatibility. This review is not intended to be exhaustive, but key examples have been selected to illustrate the topics covered. We will also look to the future and highlight research that lays the groundwork towards the use of synthetic elements in vivo.
## Protein scaffold design
Before function can be conferred onto an artificial protein, robust yet mutatable protein scaffolds must be designed. These proteins may directly mimic existing, natural structures, or adopt completely new folds. Simplicity and tolerance to mutations are key to designing a protein scaffold; a protein that is highly tolerant to mutation while largely retaining its tertiary structure allows the designer to alter or improve function in a tractable and facile manner. A well-defined structure and amenability to characterization techniques are obviously ideal in such a process, though the ability to design and predict de novo protein structures with atomic detail remains a significant challenge [bib_ref] The coming of age of de novo protein design, Huang [/bib_ref]. It is also not always easy to predict how changes to a protein's amino acid sequence might affect its stability, structure and function, therefore simplicity is key. For this reason, the redesign of natural proteins may not be the simplest approach to achieving new function, although this has been a fruitful area of research.
The complex network of interactions found in natural proteins has arisen through millennia of natural selection. These networks arise as amino acids within the protein become irreversibly co-dependent, resulting in a Muller's ratchet-like accumulation of fragility and resistance to modification and change [bib_ref] The relation of recombination to mutational advance, Muller [/bib_ref] [bib_ref] Engineering enzymes, Dutton [/bib_ref]. It is usually challenging to wholesale identify the functional roles of any one amino acid, or to discern which specific amino acids support a given function. Evolutionarily naive, de novo designed proteins can eliminate this problem and provide a simple framework on which to build function [bib_ref] Engineering oxidoreductases: maquette proteins designed from scratch, Lichtenstein [/bib_ref]. Ultimately, a protein is designed and constructed in which the roles of each individual amino acid are more easily determined and controlled, and a more tractable design process can thus be implemented.
Given the relative simplicity of designing helical peptides and small helical bundle proteins, there are now many examples of functional de novo designed proteins whose scaffolds are constructed from alpha helices [bib_ref] Engineering oxidoreductases: maquette proteins designed from scratch, Lichtenstein [/bib_ref] [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref] [bib_ref] De novo protein components for oxidoreductase assembly and biological integration, Watkins [/bib_ref]. The design principles for assembling helices are elementary: two turns of an alpha helix can be formed by a heptad of amino acids with helical-forming propensities (e.g. alanine, leucine, glutamic acid), and repeating heptads of such residues will extend the helix length as required [bib_ref] Characterization of a helical protein designed from 1st principles, Regan [/bib_ref]. To form larger oligomeric helical assemblies such as unlinked coiled coils, a defined hydrophobic/hydrophilic periodicity is imprinted on the heptads, dictating the size and orientation of the hydrophobic face, which in turn defines the oligomeric state of the assembled peptides [bib_ref] Alpha-helical coiled coils and bundles-how to design an alpha-helical protein, Cohen [/bib_ref]. These coiled coils are often used in de novo protein design and consist of bundles of two or more helices that form a rope-like superhelical structure with well-defined, interhelical knobs-into-holes packing [bib_ref] The packing of alpha-helicessimple coiled-coils, Crick [/bib_ref] [bib_ref] The design of coiled-coil structures and assemblies, Woolfson [/bib_ref] [bib_ref] New currency for old rope: from coiled-coil assemblies to alpha-helical barrels, Woolfson [/bib_ref]. The folding of coiled coils, and soluble proteins in general, is driven by the favourable entropy change when water is expelled from the interior of the folding protein [bib_ref] Characterization of a helical protein designed from 1st principles, Regan [/bib_ref] [bib_ref] Passing the 1st milestone in protein design, Kim [/bib_ref]. Simple design principles similar to those employed in coiledcoil heptad repeat patterns can also be used to construct elementary 4-helix bundles that form discrete and stable scaffolds that do not necessarily conform to coiled-coil structural parameters [bib_ref] Characterization of a helical protein designed from 1st principles, Regan [/bib_ref] [bib_ref] Protein design by binary patterning of polar and nonpolar amino-acids, Kamtekar [/bib_ref]. Most published de novo coiled coils are parallel with respect to each other, and because their N-termini are co-located, it is not possible to genetically loop them together with short peptide sequences for expression as a single-chain protein. However, in designs with helices that assemble in an antiparallel manner, the helices can be linked through simple loops containing residues with low helicalforming propensities, such as glycine and serine [bib_ref] Protein design by binary patterning of polar and nonpolar amino-acids, Kamtekar [/bib_ref] [bib_ref] Elementary tetrahelical protein design for diverse oxidoreductase functions, Farid [/bib_ref]. Therefore, in these cases, a single-chain helical bundle can be genetically encoded that not only facilitates in vivo protein expression, but also allows the cross-bundle sequence symmetry to be broken [bib_ref] Elementary tetrahelical protein design for diverse oxidoreductase functions, Farid [/bib_ref] [bib_ref] Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport..., Anderson [/bib_ref].
Even within a simple a-helix bundle, protein backbones can have highly variable geometry in which each amino acid can adopt many different side chain conformations. To remedy this, recent research by the Baker group focused on the design of protein interfaces with regular networks of hydrogen bonds that specifically interact in a modular way, similar to the base-pairing of DNA [bib_ref] De novo design of protein homo-oligomers with modular hydrogen bond network-mediated specificity, Boyken [/bib_ref]. The simplicity of a-helix bundle proteins is in many ways an advantage over more complex structures. However, the design of larger structures, including those that involve b-sheets, may allow us to access a wide range of functional capabilities. Existing de novo protein designs form a diverse range of structures, some of which are shown in figure 1.
The use of repeat sequences in protein design brings the advantage of modularity and allows the construction of larger, more complex scaffolds. Repeat proteins are prevalent in Nature, and present attractive targets for protein design. For example, repeat five-residue (RFR) beta-solenoids can tolerate substantial variations including mutations to the loop regions that link together the individual beta-helix subunits [bib_ref] Synthetic beta-solenoid proteins with the fragment-free computational design of a betahairpin extension, Macdonald [/bib_ref]. To capitalize on the potential of these versatile scaffolds, MacDonald et al. have developed computational methods which were used to calculate de novo backbones without using existing sequences of natural proteins [bib_ref] De novo backbone scaffolds for protein design, Macdonald [/bib_ref] [bib_ref] Validating a coarse-grained potential energy function through protein loop modelling, Macdonald [/bib_ref] [bib_ref] High-quality protein backbone reconstruction from alpha carbons using Gaussian mixture models, Moore [/bib_ref]. The authors then created a set of genetically encodable, de novo RFR-fold proteins with variable loops, and even whole protein insertions in the loop regions [bib_ref] Synthetic beta-solenoid proteins with the fragment-free computational design of a betahairpin extension, Macdonald [/bib_ref] (figure 1e).
The TIM-barrel fold is possibly the most prevalent protein topology found in natural enzymes, comprising eight a-helices surrounding eight b-strands in a closed toroid [bib_ref] The TIM-barrel fold: a versatile framework for efficient enzymes, Wierenga [/bib_ref]. Despite the structural similarity of TIM-barrel enzymes, there is very little sequence conservation across the many superfamilies that adopt this topology [bib_ref] De novo design of a fourfold symmetric TIM-barrel protein with atomic-level accuracy, Huang [/bib_ref] [bib_ref] One fold with many functions: the evolutionary relationships between TIM barrel families..., Nagano [/bib_ref]. While the TIM-barrel structure appears complex, there is much interest in the design of this topology de novo, owing to the functional diversity that might be tangibly available to the designer. With recent advances in computational protein design, the Baker group has created a series of genetically encodable TIM-barrel variants. One variant, sTim-11 (figure 1f ), was crystallized to reveal excellent structural fidelity to the original design. For simplicity, the group aimed to design a structure with fourfold symmetry, the maximum possible in this design. sTim-11 features four repeating motifs forming a structure which is thermostable and reversibly folds after denaturation by guanidinium chloride and temperature, providing a unique structure for the precise placement of catalytic amino acids [bib_ref] De novo design of a fourfold symmetric TIM-barrel protein with atomic-level accuracy, Huang [/bib_ref].
While the vast majority of designed proteins are soluble, natural membrane proteins have an array of functions that are worth replicating in de novo designed proteins, including receptors, transport in and out of the cell, and roles in photosynthesis. However, the design of de novo membrane protein scaffolds is hindered by the relatively small proportion of solved membrane protein structures compared with soluble proteins. In contrast to soluble proteins, designing a membrane protein that assembles, localizes and functions as intended is significantly more challenging. While the basic design principles for de novo designed membrane proteins are well established [bib_ref] De novo design of integral membrane proteins, Whitley [/bib_ref] , in practice it is often the case that the protein is incorporated into inclusion bodies [bib_ref] First principles design of a core bioenergetic transmembrane electron-transfer protein, Goparaju [/bib_ref] (which is not ideal for in vivo function), or that their low yields [bib_ref] De novo construction of redox active proteins, Moser [/bib_ref] and poor solubility can complicate downstream study.
Despite these difficulties, there have been significant advances in de novo membrane protein design in recent years, and achieving full, functional, biocompatibility is in sight. Many de novo membrane protein designs are made via peptide synthesis (see §4.5 De novo designed membrane pores), although amphiphilic maquettes can be expressed in Escherichia coli and human embryonic kidney cells (see §4.2 Light-responsive artificial proteins) [bib_ref] First principles design of a core bioenergetic transmembrane electron-transfer protein, Goparaju [/bib_ref]. Recent research by the Baker group has led to the design of de novo multipass membrane proteins that locate to the membrane of E. coli and human kidney cells, with crystal structures revealing fidelity to the intended design [bib_ref] Accurate computational design of multipass transmembrane proteins, Lu [/bib_ref].
For a review of de novo designed protein structures see Huang et al. [bib_ref] The coming of age of de novo protein design, Huang [/bib_ref]. Polymeric de novo peptides, such as the catalytic beta amyloids designed by the Korendovych group, are probably incompatible with the cell and therefore beyond the remit of this review; for a review on this topic and other catalytic peptide assemblies, see [bib_ref] Catalytic peptide assemblies, Zozulia [/bib_ref].
Function can be incorporated into a de novo protein design through the use of cofactors; however, designing a highly specific cofactor-binding site is not always straightforward. Amino acid side chains can directly coordinate metal ions [bib_ref] Computational approaches for de novo design and redesign of metal-binding sites on..., Akcapinar [/bib_ref] , but when the metal ion is part of a larger structure, such as heme, or in the case of other bulky molecules such as flavin, the situation becomes more complex. While basic design principles have been uncovered, progress in this area has been slow. Research by the Koder and Noy groups involved the scanning of databases of natural proteins to identify consensus sequences and geometric properties for heme and chlorophyllbinding sites using histidine residues [bib_ref] Geometric constraints for porphyrin binding in helical protein binding sites, Negron [/bib_ref] [bib_ref] Design principles for chlorophyll-binding sites in helical proteins, Braun [/bib_ref]. While there are computational methods in place for the design of cofactorbinding sites (for metal-binding sites, see [bib_ref] Computational approaches for de novo design and redesign of metal-binding sites on..., Akcapinar [/bib_ref] , further progress is required. Furthermore, when trying to replicate the function of, for example, light-harvesting proteins which bind multiple interacting cofactors, the situation becomes more complicated still. Not only must the cofactors be specifically bound, in the correct orientations with the correct properties, but also their interactions and properties must be tuned.
## Fully biocompatible de novo designed proteins
## Life-sustaining functions
The Hecht group has explored whether proteins with life-sustaining properties can spontaneously arise from combinatorial libraries of de novo designed proteins. These libraries [bib_ref] Synthetic beta-solenoid proteins with the fragment-free computational design of a betahairpin extension, Macdonald [/bib_ref]. (f ) sTIM-11 [bib_ref] De novo design of a fourfold symmetric TIM-barrel protein with atomic-level accuracy, Huang [/bib_ref]. PDB: 5BVL.
were created using a simple binary code strategy, where amino acids are considered as either polar or non-polar in a repeating pattern, ensuring the resulting proteins were folded into stable, discrete 3D structures [bib_ref] Protein design by binary patterning of polar and nonpolar amino-acids, Kamtekar [/bib_ref]. The libraries were screened for function through expression in various E. coli auxotroph strains. Despite the stochastic nature of their sequence selection, several de novo proteins were capable of rescuing specific knockout strains [bib_ref] De novo designed proteins from a library of artificial sequences function in..., Fisher [/bib_ref]. Some of these de novo sequences have been demonstrated to act on gene regulation [bib_ref] A protein constructed de novo enables cell growth by altering gene regulation, Digianantonio [/bib_ref] , in one case allowing the cells to grow on toxic levels of copper [bib_ref] A de novo protein confers copper resistance in Escherichia coli, Hoegler [/bib_ref]. One particular de novo protein, SynGltA, could rescue a citrate synthase gene deletion mutant unable to catalyse the first reaction of the tricarboxylic acid cycle. It was found that SynGltA does not reproduce the catalytic activity of citrate synthase, but instead upregulates a pathway which includes the promiscuous enzyme methylcitrate synthase, producing sufficient citrate to rescue growth [bib_ref] A non-natural protein rescues cells deleted for a key enzyme in central..., Digianantonio [/bib_ref]. This highlights the potential roles de novo proteins might have in 'rewiring' gene pathways and metabolism in auxotrophs [bib_ref] A non-natural protein rescues cells deleted for a key enzyme in central..., Digianantonio [/bib_ref]. In addition, a de novo catalytic protein, Syn-F4, has been developed from the library protein, Syn-IF. Syn-IF rescued two different E. coli auxotroph strains, but did not appear to have a catalytic role [bib_ref] Divergent evolution of a bifunctional de novo protein, Smith [/bib_ref]. Following random mutagenesis and selection of protein variants that could more rapidly rescue the auxotroph strain, the variant Syn-F4 was found to have catalytic activity in vivo, namely the enantioselective hydrolysis of ferric enterobactin (figure 2d) [bib_ref] A de novo enzyme catalyzes a lifesustaining reaction in Escherichia coli, Donnelly [/bib_ref].
## Therapeutic functions
Man-made biocompatible entities offer opportunities for designing therapeutic and diagnostic agents to combat disease, an avenue the Baker group has explored [bib_ref] Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site, Strauch [/bib_ref] [bib_ref] Massively parallel de novo protein design for targeted therapeutics, Chevalier [/bib_ref] [bib_ref] A Computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis..., Procko [/bib_ref]. Mimics of pro-apoptotic proteins have been used as treatments against diseases in which apoptosis is dysregulated, such as cancer. One such approach is to mimic proteins that can inhibit, through binding, BCL2 family pro-survival proteins that are expressed in many cancers. Members of this family have very high sequence homology and structures, so specific BCL2 binding is a challenge. The Baker group has created de novo proteins which bind to the BH3 binding groove of certain pro-survival proteins. Initially, a 3-helix bundle protein, BINDI, was designed as an inhibitor of BHRF1, an Epstein-Barr BCL2 homologue [bib_ref] A Computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis..., Procko [/bib_ref]. This scaffold was subsequently modified to produce variants that could each bind one of the six human pro-survival BCL2 family proteins (figure 3a), and can be expressed in human cancer cell lines. Rosetta Monte Carlo sequence design was used to design the proteins, which have three helices, one with a central BH3 motif. The two remaining helices were designed to aid specificity and stability. . A selection of catalytic de novo proteins. (a) Representation of the structure of a de novo protein which performs carbonic anhydrase activity. The solution nuclear magnetic resonance structure of the a3D scaffold (PDB: 2A3D [bib_ref] Solution structure and dynamics of a de novo designed three-helix bundle protein, Walsh [/bib_ref] , was modified to bind zinc (grey) and hydrate CO 2 [bib_ref] A de novo designed metalloenzyme for the hydration of CO 2, Cangelosi [/bib_ref]. (b) Molecular dynamics simulation model of C45, which can catalyse the oxidation of a range of small molecules, including 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref]. (c) The de novo protein Dft2 was modified to bind manganese and perform catalase activity [bib_ref] Design of dinuclear manganese cofactors for bacterial reaction centers, Olson [/bib_ref] [bib_ref] Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins, Olson [/bib_ref]. The crystal structure shown is that of the variant, P0 (PDB: 5C39); variants with two and three manganese-binding sites exhibit higher activity. (d) A 4-helix bundle library protein, Syn-F4, which performs enantioselective hydrolysis of ferric enterobactin (FeEnt) [bib_ref] A de novo enzyme catalyzes a lifesustaining reaction in Escherichia coli, Donnelly [/bib_ref]. As no structure is available of this protein, the structure shown is a representative 4-helix bindle from the Hecht lab (PDB: 2JUA). (e) Crystal structure of the heptameric coiled-coil CC-Hept-I18C-L22H-I25E with hydrolase activity towards p-nitrophenyl acetate ( pNPA). Catalytic triad residues are shown. (PDB: 5EZC) [bib_ref] Installing hydrolytic activity into a completely de novo protein framework, Burton [/bib_ref].
rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472
The de novo protein scaffold contacts regions of the BCL2 proteins that differ in sequence between family members. Following computational design, two of the designed proteins had high specificity and affinity for their targets, the remaining four provided good starting targets for in vitro optimization. Thus, these de novo proteins form a set of molecular probes which can be used for various purposes, including determining which of the BCL2 proteins are involved in individual cancers, and aiding understanding of the mechanisms of mitochondrial apoptotic pathways. The development of increasingly higher-throughput and computational methods has been a great aid to the field of de novo protein design, particularly when it comes to designing therapeutics [bib_ref] Accurate de novo design of hyperstable constrained peptides, Bhardwaj [/bib_ref]. In a recent study, the Baker group has developed a high-throughput computational method using Rosetta to design small protein binders of specific therapeutic targets, in this case influenza A H1 haemagglutinin (figure 3b) and botulinum neurotoxin B [bib_ref] Massively parallel de novo protein design for targeted therapeutics, Chevalier [/bib_ref]. This method allowed the simultaneous study of thousands of small protein designs, in which target binding and protein folding were characterized. Of the 22 600 starting proteins, more than 10% exhibited high-affinity binding to their targets; one protein was effective against influenza infection in mice, even when administered post-exposure. The proteins function in a similar way to neutralizing antibodies, which bind to and neutralize the activity of the target infectious agent or antigen, often preventing its entrance into the cell. The designs are highly specific, stable (including thermostable) and do not provoke an immune response. Although these approximately 40 residue proteins are genetically encodable, they can also be chemically synthesized, presenting the opportunity of chemical modification to expand their function. This high-throughput method is a promising approach to the production of new therapeutics and diagnostic tools for a wide range of targets [bib_ref] Massively parallel de novo protein design for targeted therapeutics, Chevalier [/bib_ref].
There is much interest in the development of protein cages, both natural and artificial, for the delivery of therapeutic molecules [bib_ref] Natural and artificial protein cages: design, structure and therapeutic applications, Heddle [/bib_ref] , for example by mimicking the function of a virus. Synthetic nucleocapsids designed by the Baker group can be fully assembled with their mRNA genome in E. coli cells and, following injection, persist in the circulation of mice for several hours [bib_ref] Evolution of a designed protein assembly encapsulating its own RNA genome, Butterfield [/bib_ref]. Computationally designed combinatorial libraries were produced and subjected to rounds of directed evolution to select for certain properties common to natural viruses: a well-packaged genome, resistance to nucleases and persistence in in vivo blood circulation. These nucleocapsids provide a tailorable platform for future applications such as therapeutic purposes, while minimizing the complexity found in naturally evolved viruses.
## Interaction of de novo proteins with cellular machineries in vivo
To be fully biocompatible, a de novo protein must not only be expressed in vivo, but also interact productively with natural cellular machineries, while undesirable interactions are minimized. The functionalities of natural systems may be harnessed, for example, by transporting man-designed elements to the desired location within the cell. In recent research the E. coli twin-arginine translocation (TAT) apparatus, whose quality control mechanism will only allow the export of fully folded proteins across the cytoplasmic membrane, could 'read' the folding state of a completely artificial heme-binding protein and translocate it to the periplasm [bib_ref] Probing the quality control mechanism of the Escherichia coli twinarginine translocase with..., Sutherland [/bib_ref]. While the bacterial Sec system has been proved capable of transporting de novo proteins in an unfolded state [bib_ref] Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport..., Anderson [/bib_ref] , the TAT system may be able to transport other de novo proteins that must fold in the cytoplasm prior to translocation.
Beyond transport, there are other cellular processes that may be exploited for the modification of de novo designed proteins, such as the rich diversity of natural post-translational modifications. Cells can selectively insert cofactors, for example, using accessory proteins and enzymes in what can be quite complex, multistep pathways. Amino acids from the polypeptide backbone may be modified to produce in situ cofactors, such as the covalently attached quinones (e.g. the topaquinone cofactor found in natural copper amine oxidases [bib_ref] Generation of the topa quinone cofactor in bacterial monoamine-oxidase by cupric iondependent..., Matsuzaki [/bib_ref] , which are often formed through the modification of tyrosine or tryptophan. Other modifications include catalytic activation or suppression through phosphorylation, and glycosylation, in which carbohydrate is covalently attached to the protein to aid stability and modulate activity.
In recent research, the Anderson group has designed and characterized an artificial heme C-containing oxidoreductase, C45, which makes use of the native E. coli cytochrome c maturation (Ccm) machinery to assemble the protein in its active form in vivo [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref]. The natural oxidoreductases are a functionally diverse superfamily of enzymes, performing a plethora of chemical transformations, and there has been much interest in replicating and exploiting their functions through de novo protein design [bib_ref] De novo protein components for oxidoreductase assembly and biological integration, Watkins [/bib_ref] [bib_ref] Design and synthesis of multi-heme proteins, Robertson [/bib_ref] [bib_ref] A suite of de novo c-type cytochromes for functional oxidoreductase engineering, Watkins [/bib_ref]. Owing to their catalytic power and potential utility in chemical synthesis and biotechnology, there has been particular interest in the oxygenases and peroxidases, catalysing the controlled insertion of oxygen into carbon-hydrogen bonds and the coupled oxidation of small molecules/reduction of hydrogen peroxide, respectively. Within these enzyme classes, many contain catalytically versatile heme cofactors that, despite the wide range of chemistries displayed by heme-containing enzymes, use essentially the same reactive intermediates to facilitate the diverse array of chemical transformations [bib_ref] Heme enzyme structure and function, Poulos [/bib_ref]. While artificial peroxidases have been developed [bib_ref] Peroxidase activity of de novo heme proteins immobilized on electrodes, Das [/bib_ref] [bib_ref] Peroxidase activity in heme proteins derived from a designed combinatorial library, Moffet [/bib_ref] , heme-containing oxygenases have proved more difficult. Heme can be spontaneously and non-covalently incorporated into proteins that are expressed in vivo [bib_ref] An Escherichia coli expression-based method for heme substitution, Woodward [/bib_ref] and it is possible to build a simple heme-binding site within a de novo protein scaffold using histidine residues to coordinate the heme iron [bib_ref] Design and synthesis of multi-heme proteins, Robertson [/bib_ref]. However, covalently appending heme to the protein backbone ensures secure and practically irreversible attachment of the cofactor, and can facilitate the design of more sophisticated de novo assemblies [bib_ref] Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport..., Anderson [/bib_ref]. C45 has arisen from the iterative improvement of oxygen-binding c-type cytochrome maquettes which contain a CXXCH motif on the protein backbone for heme C insertion by the cytochrome c maturation (Ccm) apparatus [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref] [bib_ref] Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport..., Anderson [/bib_ref] [bib_ref] A suite of de novo c-type cytochromes for functional oxidoreductase engineering, Watkins [/bib_ref]. Unlike these catalytically inactive maquettes with bis-histidine heme ligation, C45 contains a monohistidine ligated heme, allowing molecules such as hydrogen peroxide to bind in the vacant heme coordinate site and become activated towards simple substrate oxidation reactions. C45 is catalytically promiscuous and can oxidize a range of small molecules (figure 2b) [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref]. With regard to catalytic efficiency, C45 matches the activity of natural peroxidases against certain substrates. As C45 is produced in its functional form in E. coli, it can probably perform this type of peroxidase catalysis in vivo.
## De novo designed proteins: in vitro assembly and function
Many de novo proteins can be genetically encoded as singlechain proteins that can be expressed by living cells [bib_ref] Elementary tetrahelical protein design for diverse oxidoreductase functions, Farid [/bib_ref] [bib_ref] Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport..., Anderson [/bib_ref]. However, the designs may often incorporate functional elements that are not synthesized or constructed by the host cell and must currently be added in vitro [bib_ref] Elementary tetrahelical protein design for diverse oxidoreductase functions, Farid [/bib_ref]. Therefore, many genetically encodable artificial proteins are expressed in E. coli and subsequently purified for further assembly, study and analysis [bib_ref] De novo design of a nonnatural fold for an iron-sulfur protein: alpha-helical..., Grzyb [/bib_ref] [bib_ref] Engineering an artificial flavoprotein magnetosensor, Bialas [/bib_ref]. Future challenges include the creation of new biosynthetic pathways to synthesize novel cofactors in cells, and the design of specific binding sites with high affinity for the intended cofactor. To date, de novo proteins have been designed to mimic a variety of natural protein functions; advances in the last few years are discussed below, with perspectives on biocompatibility.
## Artificial enzymes
In Nature, protein catalytic function is often complex, with many factors working in concert to allow efficient chemistry and to ensure the reaction is thermodynamically favourable. These factors may include diffusion of the substrate and product in and out of the active site, quantum tunnelling effects, transition state stabilization, specificity and concerted protein dynamics [bib_ref] Catalytic efficiency of enzymes: a theoretical analysis, Hammes-Schiffer [/bib_ref] [bib_ref] Application of the theory of diffusion-controlled reactions to enzyme kinetics, Alberty [/bib_ref] [bib_ref] At the dawn of the 21st century: is dynamics the missing link..., Kamerlin [/bib_ref] [bib_ref] A perspective on enzyme catalysis, Benkovic [/bib_ref]. In many cases, the precise alignment and proximity of a substrate molecule and active-site amino acid side chains is important [bib_ref] Aldehyde dehydrogenasemaintaining critical active site geometry at motif 8 in the class..., Hempel [/bib_ref] [bib_ref] Functional evidence for a small and rigid active site in a high..., Kim [/bib_ref]. Imparting of catalytic function onto a de novo scaffold can therefore be a challenge. There are many examples of catalytic de novo proteins and peptides, and these are reviewed in Zozulia et al. [bib_ref] Catalytic peptide assemblies, Zozulia [/bib_ref] , some of which are shown in . A successful route to achieving catalysis has included the use of metal cofactors to perform chemistry, for example the aforementioned heme C-containing maquette, C45, which performs efficient catalysis without a highly specific substrate binding site [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref] [bib_ref] The ascent of man(made oxidoreductases), Grayson [/bib_ref] (figure 2b). However, in many cases it has been necessary to incorporate the cofactors in vitro, and further research and development is required to enable full functional assembly in vivo.
As most natural diiron-containing enzymes contain at their core a 4-helix bundle that binds the two iron ions necessary for their diverse and powerful enzymatic functions, these enzymes were early targets of de novo protein design [bib_ref] Retrostructural analysis of metalloproteins: application to the design of a minimal model..., Lombardi [/bib_ref]. The natural enzymes catalyse a variety of substrate oxidations and oxygenations, including the thermodynamically challenging hydroxylation of methane [bib_ref] Reactions of nonheme iron(ii) centers with dioxygen in biology and chemistry, Feig [/bib_ref]. Within the iron-binding core of the natural enzymes, the iron ions are generally coordinated by two histidine and four carboxylate residues [bib_ref] Artificial diiron enzymes with a de novo designed four-helix bundle structure, Chino [/bib_ref]. The DeGrado group has performed much work on its due ferri (DF) de novo proteins, whose simplicity has provided significant insight into the natural non-heme diiron enzymes; for a review of artificial diiron-oxo proteins of the DF family, see Chino et al. [bib_ref] Artificial diiron enzymes with a de novo designed four-helix bundle structure, Chino [/bib_ref]. Iterative design processes on the singlechain protein, DFsc, have produced variants with increased solvent and substrate accessibility, and modified reactivityfrom hydroquinone oxidation to selective N-hydroxylation of arylamines [bib_ref] Alteration of the oxygendependent reactivity of de novo Due Ferri proteins, Reig [/bib_ref]. The incorporation of a third ligating histidine residue to the variant G4DFsc altered the enzyme's function, mimicking the active site of p-aminobenzoate N-oxygenase, a natural diiron protein [bib_ref] Alteration of the oxygendependent reactivity of de novo Due Ferri proteins, Reig [/bib_ref]. The modified protein gains oxygenase activity towards p-anisidine, while 4-aminophenol oxidation is disfavoured [bib_ref] Alteration of the oxygendependent reactivity of de novo Due Ferri proteins, Reig [/bib_ref]. As the coordination of one iron site is saturated, a slower substrate binding rate is observed, inhibiting oxidase reactivity [bib_ref] Molecular-level insight into the differential oxidase and oxygenase reactivities of de novo..., Snyder [/bib_ref] [bib_ref] Systematic perturbations of binuclear non-heme iron sites: structure and dioxygen reactivity of..., Snyder [/bib_ref]. While both G4DFsc and 3His-G4DFsc bind p-anisidine near the biferrous site, the geometry of this site is differentially perturbed in such a way as to influence reactivity. While these proteins are expressed in E. coli, they are reconstituted with iron in vitro, and would probably require a significant increase in iron affinity to promote in vivo assembly and activity.
Other metal ions have been effectively used as catalytic cofactors in de novo designed proteins. The Pecoraro group has designed a carbonic anhydrase (CA) mimic, with a zinccontaining active site analogous to that of CA II, but in a 3-stranded a-helical coiled coil as opposed to the b-sheets found in CA II. While the efficiency of CO 2 hydration is within two orders of magnitude of CA II, it is a parallel assembly whose symmetry hinders improvements and is not manufacturable in vivo [bib_ref] Hydrolytic catalysis and structural stabilization in a designed metalloprotein, Zastrow [/bib_ref]. In a more recent study, the Pecoraro group modified an existing single-chain DeGrado scaffold, a3D [bib_ref] Solution structure and dynamics of a de novo designed three-helix bundle protein, Walsh [/bib_ref] [bib_ref] From coiled coils to small globular proteins: design of a native-like three-helix..., Bryson [/bib_ref] , to bind zinc and hydrate CO 2 (figure 2a) [bib_ref] A de novo designed metalloenzyme for the hydration of CO 2, Cangelosi [/bib_ref]. Although the catalytic efficiency is 1400-fold less than that of CA II and 11-fold less than that of CA III, the simple scaffold and iterative design strategy will facilitate the design of improved function. The protein can be expressed in E. coli, laying the groundwork for future design of in vivo activity [bib_ref] A de novo designed metalloenzyme for the hydration of CO 2, Cangelosi [/bib_ref]. a3D has been modified to bind various other metal ions to perform functions other than catalysis, such as copper [bib_ref] De novo design and characterization of copper metallopeptides inspired by native cupredoxins, Plegaria [/bib_ref] (see §4.3 Designed electron transport proteins) and heavy metals [bib_ref] Apoprotein structure and metal binding characterization of a de novo designed peptide,..., Plegaria [/bib_ref] (see §4.4 Artificial metal-sequestering proteins).
Many natural proteins contain manganese cofactors, which have a wide variety of functions, notably the evolution of molecular oxygen. Allen and colleagues have altered a DeGrado DF protein, Df2t, to produce a series of four helix bundle proteins, made up of two monomers, with dinuclear manganese (Mn) binding sites, analogous to that found in Mn-catalase (figure 2c) [bib_ref] Design of dinuclear manganese cofactors for bacterial reaction centers, Olson [/bib_ref] [bib_ref] Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins, Olson [/bib_ref]. These proteins exhibit catalase activity, converting hydrogen peroxide into molecular oxygen. Variants, 'P1' and 'P3', with three metal-binding sites exhibited higher activity than those with fewer binding sites, 'P0' (one site) and 'P2' (two sites). The apo-proteins were purified from E. coli, and the Mn cofactors were rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472 incorporated in vitro through incubation with MnCl 2 . These proteins can also transfer electrons to bacterial reaction centres (RCs), discussed in §4.2 Light-responsive artificial proteins.
Catalytic function has also been integrated into de novo designed scaffolds without the use of cofactors. Burton et al. [bib_ref] Installing hydrolytic activity into a completely de novo protein framework, Burton [/bib_ref] reported a de novo helical barrel with active sites that mimic those of natural cysteine/serine hydrolases, in which amino acid side chains alone catalyse the reaction (figure 2e). This structure comprises seven helices forming an 8 Å diameter channel, with each helix featuring a cysteine-histidine-glutamic acid catalytic triad in the barrel lumen, resulting in a total of seven catalytic triads in the fully assembled protein. A combination of rational design and iterative strategies was used in which the design was fully characterized with each additional mutation. Although there have been other de novo protein hydrolase designs containing Zn 2þ with higher catalytic efficiency [bib_ref] Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme..., Der [/bib_ref] [bib_ref] A designed supramolecular protein assembly with in vivo enzymatic activity, Song [/bib_ref] , this protein forms a unique structure with a highly mutatable de novo scaffold, and sequential rounds of directed evolution may aid further enhancement of its catalytic ability. While this protein was produced using peptide synthesis and is a parallel assembly and not a single-chain construct and therefore cannot be constructed in vivo, it is a good recent example of how catalysis may be performed in a de novo designed protein without the requirement for cofactors.
## Steps towards catalysis in de novo designed proteins
With the exception of a few notable examples, most de novo enzymes fall short of the catalytic efficiencies exhibited by natural enzymes. A richer understanding of how natural enzymes work and how to import these functions into de novo designed elements may enable us to perform more complex or tuneable reactions. This section briefly discusses some current ways in which de novo design is being used to understand some features of natural enzyme catalysis with the hope that we may be able to use this new knowledge to create improved artificial protein catalysts.
Enzymes can stabilize high-energy intermediates, and some make use of radicals, which must be controlled to avoid damage to the enzyme. It is not well understood how natural proteins can stabilize these unstable species, and Tommos and colleagues have used de novo proteins as models to study the stabilization of amino acid and mercaptophenol radicals [bib_ref] Exploring amino-acid radical chemistry: protein engineering and de novo design, Westerlund [/bib_ref] [bib_ref] Formal reduction potential of 3,5-difluorotyrosine in a structured protein: insight into multistep..., Ravichandran [/bib_ref] [bib_ref] Reversible phenol oxidation and reduction in the structurally well-defined 2-mercaptophenol-alpha C-3 protein, Tommos [/bib_ref]. The DeGrado group has used rational protein design to stabilize ortho-semiquinones, common radical intermediates found in natural catalysis and redox processes [bib_ref] Designed metalloprotein stabilizes a semiquinone radical, Ulas [/bib_ref]. In this study, a 4-helix bundle protein, DFsc, was used to bind Zn(II), to which the semiquinone was stabilized through binding (figure 1d). The location of the Zn(II) in the hydrophobic cleft of the protein excludes the bound semiquinone from the bulk water. At room temperature and in aqueous medium, binding of the semiquinone radical, SQ †, was favoured over binding of the more stable QH 2 and Q forms. The design principles used here, in which the radical is stabilized through metal-ligand interactions and burial of hydrophobic groups, may allow us to design artificial enzymes that can perform more complex chemistry [bib_ref] Designed metalloprotein stabilizes a semiquinone radical, Ulas [/bib_ref].
The rate of an enzymatic reaction in a de novo designed active site is often limited by imperfect geometry. Therefore, the ability to customize a ligand-binding pocket towards high specificity is an advantage. The Baker group investigated using beta sheets to custom-design backbones for binding a specific ligand. Furthermore, in natural proteins, amino acids far (10-20 Å ) from the active site can contribute to ligand-binding geometry, and DeGrado and colleagues [bib_ref] De novo design of a hyperstable non-natural protein-ligand complex with subangstrom accuracy, Polizzi [/bib_ref] have designed a de novo protein, PS1, with this in mind. PS1 was designed to mimic natural proteins with apolar folded cores which support cofactor-binding regions. Prior to cofactor binding, the binding region is flexible relative to the tightly packed core. On binding the cofactor, the entire protein is tightly packed and stabilized. PS1 is faithful to the design to sub-Å level, and binds a non-natural porphyrin with high thermostability. The success of this approach is promising for the design of improved catalytic de novo proteins, and their structural characterization [bib_ref] De novo design of a hyperstable non-natural protein-ligand complex with subangstrom accuracy, Polizzi [/bib_ref].
## Light-responsive artificial proteins
Natural photosynthesis is a highly organized process using pigment-protein complexes to harvest light energy, which is ultimately used to power ATP synthesis. There are many components in natural photosynthetic pathways working synergistically to ensure efficiency and productivity while maintaining the ability to quickly adapt to changing environmental conditions. Although complex, photosynthesis provides a rich source of natural engineering principles from which to draw inspiration for the design of functional de novo proteins. Ultimately, the de novo design of photosynthetic proteins may allow the construction of customizable, modular photosynthetic pathways that are adaptable to and stable within the desired conditions, to light-power the production of valuable products.
Many of the key proteins involved in photosynthesis are membrane proteins. Therefore, efforts have been made to design artificial transmembrane proteins that can emulate the functions of their natural counterparts. Artificial membrane proteins can bind a variety of cofactors such as heme, Znand Ni-bacteriochlorophylls, and synthetic tetrapyrroles [bib_ref] Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely..., Noy [/bib_ref] [bib_ref] Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions, Discher [/bib_ref]. Amphiphilic maquettes consist of two distinct continuous hydrophilic and lipophilic domains, the latter being of a suitable length to span a lipid bilayer [bib_ref] Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely..., Noy [/bib_ref] [bib_ref] Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions, Discher [/bib_ref]. Until recently, amphiphilic maquettes were constructed from self-associating, unlinked helical peptides; however, to break symmetry and therefore increase mutability, a single-chain amphiphilic maquette has been designed [bib_ref] First principles design of a core bioenergetic transmembrane electron-transfer protein, Goparaju [/bib_ref]. This maquette is expressed in E. coli, and, in vitro, can bind multiple b-type hemes and photoactive Zn protoporphyrin IXs, potentially capable of supporting a light-activatable intra-protein electron transfer (ET) chain. Although this protein forms inclusion bodies when expressed in E. coli, the authors ultimately aim to express and assemble these proteins in vivo with natural cofactors [bib_ref] First principles design of a core bioenergetic transmembrane electron-transfer protein, Goparaju [/bib_ref].
Despite advances in designing de novo membrane proteins, it remains simpler to design and work with soluble proteins. Membrane proteins require solubilization with detergents, particular design principles for folding and membrane insertion [bib_ref] The distribution of positively charged residues in bacterial inner membraneproteins correlates with..., Vonheijne [/bib_ref] and often more complex purification protocols than soluble proteins. There is therefore increasing interest in creating water-soluble proteins that perform the function of proteins found in natural photosynthesis, recent examples of which are detailed below.
Photosynthetic organisms tend to occupy particular spectral niches depending on the absorbance of their biosynthesized pigments, and therefore do not exploit the full range of the available solar spectrum [bib_ref] Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement, Blankenship [/bib_ref]. It would thus be exceptionally beneficial to design artificial light-harvesting pathways that could use a greater range of solar energy [bib_ref] Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter..., Grayson [/bib_ref] , rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472 and there has been a corresponding interest in the design of artificial proteins that selectively bind natural or artificial light-harvesting molecules. Maquettes have been designed to bind light-active chlorins [bib_ref] Zincbacteriochlorophyllide dimers in de novo designed four-helix bundle proteins. A model system..., Cohen-Ofri [/bib_ref] [bib_ref] Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions, Koder [/bib_ref] : these de novo proteins afford the protein engineer a greater freedom for design than natural light-harvesting proteins, and may be customized to absorb specific wavelengths of light. The hydrophobic nature of many photoactive tetrapyrroles, both natural and synthetic, can render binding to artificial proteins problematic. Natural proteins often use accessory proteins to obfuscate this problem, but a less complex approach would be beneficial to the assembly of de novo proteins. Successful methods in incorporating such hydrophobic molecules into soluble proteins have included the use of detergents [bib_ref] An unusual role for the phytyl chains in the photoprotection of the..., Agostini [/bib_ref] or water-in-oil emulsions [bib_ref] Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions, Bednarczyk [/bib_ref] , although other strategies would need to be developed to achieve this in vivo. One approach used in previous work regarding the incorporation of chlorophyll (Chl) and bacteriochlorophyll (BChl) into soluble de novo proteins involved the removal of their hydrophobic tails to improve solubility and prevent aggregation [bib_ref] Zincbacteriochlorophyllide dimers in de novo designed four-helix bundle proteins. A model system..., Cohen-Ofri [/bib_ref] [bib_ref] Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions, Koder [/bib_ref] [bib_ref] Design, synthesis and properties of synthetic chlorophyll proteins, Rau [/bib_ref]. However, to best make use of the wide range of synthetic and natural light-harvesting pigments (e.g. synthetic chorins and bacteriochlorins) available for use in synthetic systems and whose solubility is variable, we must understand how to effectively incorporate them into the desired protein scaffold without the need to modify the properties of the molecule. Kodali et al. [bib_ref] Design and engineering of water-soluble light-harvesting protein maquettes, Kodali [/bib_ref] have recently demonstrated how to strike a balance between the hydrophobic and hydrophilic nature of the cofactors by producing a soluble light-harvesting maquette which partitions the nonpolar region of amphiphilic tetrapyrroles into the interior of the 4-helix bundle, while the polar portion is exposed to the aqueous environment. In this work, the authors used Zn tetraphenyl porphyrins and Zn chlorins whose solubility in different environments was altered through substitutions at the meso-position of the tetrapyrrole ring. These porphyrins were then titrated into a maquette with ligating histidines to determine the optimum cofactor characteristics for binding. Furthermore, within the same maquette, it is possible to include ligating histidines with different binding affinities to incorporate more than one cofactor type into the same scaffold [bib_ref] Design and engineering of water-soluble light-harvesting protein maquettes, Kodali [/bib_ref].
To achieve biocompatibility, artificial components must work in symbiosis with natural proteins, and functional interactions between man-made and natural proteins may allow us to access functions beyond the current capabilities of de novo protein design alone. One approach has been to create natural-artificial protein chimeras. The Noy group fused a domain of a natural phycobiliprotein with a de novo 4-helix bundle binding a light-active zinc porphyrin or bacteriochlorin [bib_ref] Bimodal intramolecular excitation energy transfer in a multichromophore photosynthetic model system: hybrid..., Zeng [/bib_ref]. The direction of Förster resonance energy transfer (FRET) between the fusion domains could be altered depending on the particular pigments bound. Such directionality is highly important in natural light-harvesting proteins, ensuring light energy is captured efficiently. In recent research, Mancini et al. [bib_ref] Multi-step excitation energy transfer engineered in genetic fusions of natural and synthetic..., Mancini [/bib_ref] have created a light-harvesting/energy transfer fusion between a natural bilin-binding protein, CpcA and a 4-helix maquette featuring two photoactive tetrapyrrole-binding sites. The first cofactor-binding site of the maquette can ligate Zn-tetrapyrroles through a histidine residue in the hydrophobic interior of the maquette. The hydrophobic regions of the Zn-chlorin are buried, while maintaining close proximity to the phycobiliprotein for efficient FRET. The second binding site within the maquette is a cysteine residue which facilitates covalent attachment of a synthetic maleimide-functionalized bacteriochlorin, which buries itself in the maquette interior. The entire construct, fully assembled with cofactors, covers a large portion of the UV and visible absorbance spectrum. It can capture light energy and perform multistep excitation energy transfer from the natural bilin-binding protein to the tetrapyrroles of the synthetic protein. These studies demonstrate how natural and synthetic proteins may 'work together' for multistep excitation energy transfer, and present tailorable light-harvesting properties due to the option to incorporate different cofactors that absorb different wavelengths of light. Building on this work, de novo proteins with more complex systems of light-active cofactors may be created to more closely mimic natural light-harvesting proteins which can possess a higher number of bound pigments per polypeptide chain.
Though these artificial proteins can be genetically encoded, the functional photoactive complexes are currently assembled in vitro. To achieve full biocompatibility, these proteins must bind their photoactive prosthetic group in vivo; this can be achieved through the specific binding of photoactive molecules either endogenous to the cell, supplemented in culture or produced from a recombinantly expressed biosynthetic pathway in vivo. Mancini et al. [bib_ref] Multi-step excitation energy transfer engineered in genetic fusions of natural and synthetic..., Mancini [/bib_ref] report that chlorins native to the photosynthetic cyanobacterium Synechocystis sp. PCC603 bind maquettes expressed in vivo. Phycobilins can also be attached onto maquette cysteine residues in E. coli when co-expressed with bilin synthases and lyases [bib_ref] Multi-step excitation energy transfer engineered in genetic fusions of natural and synthetic..., Mancini [/bib_ref].
Beyond light harvesting, there are many downstream photosynthetic functions that could be imprinted onto artificial components, including, for example, de novo designed RCs for biomimetic, photoinduced charge separation. Here, the light-harvested electronic excitation energy is converted into the release of electrons into an electron transport chain. Photosystem II obtains these electrons through the photolysis of water, and a prototype PSII maquette is in development by Dutton and colleagues. De novo designed electron transport proteins are discussed in §4.3.
During photosynthesis photoprotection is required. In the presence of high or fluctuating light conditions, Chl and BChls can form triplet states capable of donating their energy to O 2 , producing singlet oxygen. Photosynthetic organisms have evolved various strategies to prevent singlet oxygen damage, including non-photochemical quenching (NPQ)the dissipation of excess energy as heat, and the involvement of pigments such as carotenoids which can directly quench the singlet oxygen state and the triplet B/Chl state, followed by NPQ [bib_ref] How carotenoids function in photosynthetic bacteria, Cogdell [/bib_ref]. An artificial light-harvesting system must be able to perform similar protective mechanisms. The maquette, HP7, bound with two zinc-substituted Chl derivatives, can efficiently undergo relaxation through NPQ when the pair is photoexcited [bib_ref] Zincbacteriochlorophyllide dimers in de novo designed four-helix bundle proteins. A model system..., Cohen-Ofri [/bib_ref]. The protein environment surrounding the BChl pair can control the fate of the excitation, so the maquette can be adapted for light harvesting, leading to energy transfer, for charge separation or for energy dissipation [bib_ref] The role of chargetransfer states in energy transfer and dissipation within natural..., Wahadoszamen [/bib_ref]. Although this designed protein is not a single chain and therefore probably cannot be constructed in vivo, the design principles learned here may be adapted for a biocompatible component.
In addition to roles in photosynthesis, light-sensing proteins can perform other functions. Cryptochrome proteins contain a flavin adenine dinucleotide (FAD) cofactor that forms a radical pair (RP) when exposed to blue light. These proteins are responsible for a variety of functions, including growth towards light in plants, the control of plant development and the regulation rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472 of circadian rhythms. Cryptochromes are also thought to be involved in magnetic sensing [bib_ref] The cryptochromes: blue light photoreceptors in plants and animals, Chaves [/bib_ref]. Interconversion of the singlet and triplet states of the RP is sensed in terms of its timing and extent to achieve magnetosensitivity [bib_ref] Chemical magnetoreception in birds: the radical pair rsif.royalsocietypublishing.org, Rodgers [/bib_ref]. A flavomaquette that is capable of light-mediated magnetic field sensing has been designed and constructed [bib_ref] Engineering an artificial flavoprotein magnetosensor, Bialas [/bib_ref]. This maquette was created to aid understanding of the properties required by natural proteins to sense magnetic fields. The maquette does not bear any resemblance to the natural chryptochrome fold but forms a chryptochrome-like light-active RP that is magnetically sensitive. As in natural chryptochromes, photoinduced ET from a nearby tryptophan to the flavin results in formation of the RP. The protein was expressed in and purified from E. coli, but the flavin cofactor, 8-bromo-riboflavin, was covalently incorporated in vitro. Natural cryptochromes contain a triad of tryptophans that act as a light-activated electron transport chain, which the authors aim to replicate in future designs.
## Designed electron transport proteins
An area that has been much explored in de novo protein design is the creation of artificial ET proteins. These proteins could be integrated with respiratory processes/complexes in vivo to divert electrons directly towards the production of useful products or improving respiratory energy conversion in humans in the case of disease or ageing [bib_ref] Design and engineering of a man-made diffusive electron-transport protein, Fry [/bib_ref] , or could redress redox imbalances caused as a result of metabolic engineering [bib_ref] Metabolic engineering of carbon and redox flow in the production of small..., Thakker [/bib_ref]. They may also have a role in photosynthesis; the previously discussed Mn-proteins designed by Allen and colleagues can transfer electrons to natural bacterial RCs, subsequent to illumination-which induces the RC charge-separated state. This process is analogous to the rapid reduction of RCs by natural secondary electron donors, such as cytochrome c 2 . Modelling results indicate that the artificial proteins bind the periplasmic face of the RC in a manner similar to cytochrome c 2 [bib_ref] Design of dinuclear manganese cofactors for bacterial reaction centers, Olson [/bib_ref] [bib_ref] Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins, Olson [/bib_ref].
In Nature, proteins containing iron-sulfur clusters are often involved in respiratory and photosynthetic electron transport chains. De novo ET proteins have been engineered to incorporate [4Fe-4S] clusters [bib_ref] Structural principles for computational and de novo design of 4Fe-4S metalloproteins, Nanda [/bib_ref]. In a recent example, a de novo ferredoxin mimic was designed, which incorporates two [4Fe-4S] clusters in a 3-helix scaffold [bib_ref] A de novo designed 2 4Fe-4S ferredoxin mimic mediates electron transfer, Roy [/bib_ref]. In a subsequent study mutants of this protein were produced to modulate redox potential and stabilize [3Fe-4S] [bib_ref] Modulation of cluster incorporation specificity in a de novo iron-sulfur cluster binding..., Sommer [/bib_ref]. For the construction of synthetic ET chains, or for accessing more sophisticated redox catalysis, this ability to tailor redox properties, such as potential and directionality, is an advantage. These proteins are produced by peptide synthesis and are not single chain; however, it is possible to incorporate an iron-sulfur cluster into a single-chain scaffold [bib_ref] De novo design of a nonnatural fold for an iron-sulfur protein: alpha-helical..., Grzyb [/bib_ref]. See also Dizicheh et al.for a review of the incorporation of FeS clusters into both natural and artificial scaffolds.
A rate-limiting step in inter-protein ET between the multiprotein complexes of the respiratory chain is the transient encounter between the redox partners so that they are within a suitable distance for efficient ET [bib_ref] Interprotein electrontransfer, Mclendon [/bib_ref]. Cytochrome c is a diffusible protein of the mitochondrial electron transport chain and it, along with many other natural proteins, uses complementary surface electrostatics to promote transient interactions between it and its redox partners [bib_ref] Electron transfer interactome of cytochrome c, Volkov [/bib_ref]. As maquettes are highly tolerant to extensive changes in their surface residues, they provide an ideal platform for investigating the effect of differing electrostatic surface characteristics. Fry et al. [bib_ref] Design and engineering of a man-made diffusive electron-transport protein, Fry [/bib_ref] have designed a genetically encodable heme-binding maquette that can reduce cytochrome c at physiologically relevant (millisecond) rates. In this way, cytochrome c may act in vivo as a mediator between the artificial maquette and the natural redox partners of cytochrome c, who either have a net negative charge or have negatively charged surface regions. It therefore followed that the maquette with a net negative charge was demonstrated to more rapidly reduce cytochrome c than that with a net positive charge. This work demonstrates that, for effective biological ET, a net complementary charge is sufficient as opposed to more specific protein-protein binding interactions. In this case, it was not necessary to design a specific docking site; however, it would be advantageous to design de novo proteins that specifically interact with natural proteins.
The Pecoraro group has sought to explore whether the characteristics and properties of metals in natural proteins can be retained when placing a metal-binding site in a de novo designed protein with a different topology. Natural ET cupredoxin, CuT1, proteins contain copper-binding centres that are often found within a beta-barrel framework. The de novo protein, a3D [bib_ref] Solution structure and dynamics of a de novo designed three-helix bundle protein, Walsh [/bib_ref] [bib_ref] From coiled coils to small globular proteins: design of a native-like three-helix..., Bryson [/bib_ref] (see §4.1 Artificial enzymes), was modified to incorporate a copper-binding centre [bib_ref] De novo design and characterization of copper metallopeptides inspired by native cupredoxins, Plegaria [/bib_ref] [bib_ref] Electron transfer activity of a de novo designed copper center in a..., Plegaria [/bib_ref]. A 2HisCys(Met) metal-binding site was modelled within the 3-helix bundle, and the resulting protein can be purified from E. coli. The spectroscopic properties of native cupredoxins were not fully replicated in the designed protein; however, the de novo protein is capable of intramolecular ET, and can perform ET with a photosensitizer. In the future, the authors aim to optimize the structure to improve its properties, including ET efficiency, and to incorporate both an ET and a catalytic site within the same scaffold to mimic natural enzymes such as copper nitrate reductase. In recent research, the same group has used the a3D scaffold to build in a rubredoxin site, containing one iron, using a CXXC motif, with the same spectroscopic characteristics as its natural counterpart despite the site being in a different fold [bib_ref] Development of a rubredoxintype center embedded in a de dovo-designed threehelix bundle, Tebo [/bib_ref].
The ET components described above have the potential to be integrated into systems in conjunction with man-made oxidoreductases or natural proteins. This could mimic the function of natural systems, or be used to create an assortment of biomimetic components that can interact in ways not observed in Nature.
## Artificial metal-sequestering proteins
Natural proteins may bind metal ions or compounds for purposes other than catalysis, photosynthesis and ET. In Nature, features such as teeth, bones and shells are created by biomineralization [bib_ref] An overview of biomineralization processes and the problem of the vital effect, Weiner [/bib_ref] ; in many cases, proteins and enzymes are involved in these processes [bib_ref] Computational design of a self-assembling symmetrical beta-propeller protein, Voet [/bib_ref]. These proteins are of interest in many fields as the materials they deposit may have desirable electric, optical and magnetic properties. To this end, a de novo designed protein, Pizza, has been engineered to synthesize cadmium chloride nanocrystals. One Pizza variant, Pizza6, is a computationally designed self-assembling de novo b-propeller protein with sixfold symmetry and high thermostability, and is expressed and purified from E. coli. [bib_ref] Computational design of a self-assembling symmetrical beta-propeller protein, Voet [/bib_ref]. A version of Pizza was designed with histidine residues at the trimeric interfaces (nvPizza2-S16H58; see figure 1a), with the intention that it would only assemble in the presence of metal ions [bib_ref] Biomineralization of a cadmium chloride nanocrystal by a designed symmetrical protein, Voet [/bib_ref]. This would inevitably increase stability of the protein complex but also aid disassembly by exposure to chelators. The designed subunits consist of two propeller domains per polypeptide which rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472 spontaneously trimerize. The presence of cadmium chloride induces a dimerization of these trimers through the coordination of a 19-atom cadmium chloride nanocrystal by the symmetrically positioned histidine residues. Trimerization is observed in the absence of cadmium chloride, probably through a water molecule forming hydrogen bonds with the three histidine residues. The authors envisage introducing catalytic activity to the structure, as a water-filled tunnel is formed on one face, reminiscent of the buried tunnels containing catalytic metal ions in the active sites of catalase and superoxide dismutase.
Metalloregulatory proteins regulate ion flux and delivery within the cell while limiting levels of potentially toxic heavy metals. Pecoraro and colleagues [bib_ref] Design of a three-helix bundle capable of binding heavy metals in a..., Chakraborty [/bib_ref] have created an artificial 3-helix bundle protein, a 3 DIV, that can bind Hg(II), Pb(II) and Cd(II). a 3 DIV is a redesigned variant of the DeGrado protein a 3 D and contains the triscysteine motif found in many metalloregulatory proteins. a 3 DIV is stable, with a more tightly packed core than a 3 D, and heavy metal binding induces further stability. The protein can be genetically encoded and expressed in E. coli and has been structurally characterized by nuclear magnetic resonance [bib_ref] Apoprotein structure and metal binding characterization of a de novo designed peptide,..., Plegaria [/bib_ref]. In a subsequent study, a fourth cysteine residue was introduced at one of two different sites in a 3 DIV to mimic the tetrathiolate binding site found in CadC, a transcriptional repressor protein that regulates the levels of intracellular Cd(II) [bib_ref] Variable primary coordination environments of Cd(II) binding to three helix bundles provide..., Tebo [/bib_ref]. As was identified with CadC, the tetrathiolate site in the artificial protein coordinates Cd(II) as a mixture of rapidly exchanging CdS 3 O and CdS 4 species. Thus, the artificial metal binder acts as a model for its natural counterparts.
## De novo designed membrane pores
Membranes provide a vital boundary between the cell and the outside world, strictly controlling what goes in and out of the cell. Natural membrane pores have many functions, including signalling and transport. Although providing selective transport across the cell membrane without compromising membrane integrity is a particular challenge, the capability of creating de novo designed membrane pores is highly advantageous, potentially facilitating the design of proteins tailored to specific 'cargo' molecules, through pore size and sequence. However, the design of de novo membrane protein scaffolds is hindered by the relatively small proportion of solved membrane protein structures compared with soluble proteins. Currently, de novo membrane nanopores come in many different forms, with examples constructed from proteins, DNA and organic materials [bib_ref] Building membrane nanopores, Howorka [/bib_ref].
A notable example of a de novo designed membrane pore is Rocker, a Zn 2þ /H þ antiporter, designed by the DeGrado laboratory [bib_ref] Design of self-assembling transmembrane helical bundles to elucidate principles required for membrane..., Joh [/bib_ref]. Rocker is a computationally designed transporter protein which can transport Zn 2þ or Co 2þ ions, but not Ca 2þ , across membranes, with the concurrent antiport of protons. The phospholipid bilayer of biological membranes is impermeable to metal ions, and transporters are required to transport metal ions such as Zn 2þ across the membrane. Rocker was designed with the aim of emulating the alternating access 'rocker-switch' model by which many natural transporter proteins are thought to operate, rocking between different states. It is a membrane-spanning de novo designed 4-helix protein that features two di-metal binding sites with negative cooperativity of binding. As more than 100 protons are transported per Zn 2þ ion, Rocker's efficiency does not match that of natural proteins; however, it represents an example of how function can be achieved through a protein scaffold that is simple in comparison with its natural counterparts, with potential to engineer additional features to improve the transport efficiency. Moreover, although Rocker was produced through solidphase peptide synthesis, production of a genetically encoded variant may be possible as the helices run antiparallel, facilitating a single-chain design. The fact that the protein can assemble in micelles and phospholipid bilayers is a promising indication that similar de novo proteins may readily insert into cell membranes.
Peptides that form pores inside membranes in acidic conditions were recently designed by the DeGrado group [bib_ref] Computational design and experimental characterization of peptides intended for pH-dependent membrane insertion..., Zhang [/bib_ref]. The computationally designed 28-residue peptides consisting of four 7-residue repeats are of sufficient length (42 Å ) to span the cell membrane. The in vivo extracellular environment is at a pH of 7.4, and at this pH the peptides are water soluble. As the pH is lowered, the peptides bind discretely to membrane. At pH 5.5, resembling the acidic conditions found within the endosome or lysosome, the peptides assemble to form transmembrane pores. When the peptides were added to red blood cells, miRNA and ATP were selectively released, but not haemoglobin, while preserving membrane integrity. With further research and understanding, the authors envisage creating 'selective membrane-permeating tools' for a variety of cargo.
Another approach to selectively breach the cell membrane is to mimic the function of a virus, in which the encapsulated DNA or RNA is delivered into a host cell. Noble et al. [bib_ref] A de novo virus-like topology for synthetic virions, Noble [/bib_ref] have created TecVir, a de novo design that forms a viruslike topology from coiled-coil peptide helices, which can transfer both RNA and DNA into human cells without cytotoxic effects. Each peptide helix was designed with one hydrophobic face and two polar interfaces, which allow it to interact with three identical neighbour helices; they pack to form a shell and can be co-assembled with DNA or RNA. Although TecVir is made in vitro through peptide synthesis, the assembly shows biocompatibility as it can transfect human dermal fibroblasts with a plasmid encoding eGFP without appearing to disrupt cell morphology as it is taken up by cells through endocytosis. Like the pore-forming peptides above, TecVir is pH responsive; at acidic pH, TecVir unfolds, allowing entry of the genetic cargo to the cytoplasm.
## Chemical mimics of protein function
It is not exclusively artificial proteins and peptides that can mimic protein function. Some designed chemical constructs have the potential to be biocompatible, and work with or in cells. This section delineates two recently reported studies in which molecules are used for transmitting signals across membranes.
Natural G-protein-coupled receptor (GPCR) signalling proteins are membrane bound. When a ligand signal is bound, a conformational change is induced in the GPCR, which initiates a signalling cascade within the cell. The Claydon group considered the minimal set of components required to create a GPCR mimic and subsequently designed a membrane-bound synthetic receptor (figure 4a) [bib_ref] Ligand-modulated conformational switching in a fully synthetic membrane-bound receptor, Lister [/bib_ref]. The receptor binding site contains a Cu(II) ion, to which a carboxylate ligand binds. This binding induces a conformational change across the receptor, consisting of a helical foldamer core derived from peptabiols, a fungal class of antibiotics.
rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472 Peptabiols can insert into the membrane bilayer and contain the quaternary amino acid, a-aminoisobutyric acid (aib). Oligomers of Aib have strong helical propensity, and decamers are long enough to span the membrane bilayer. In solution these oligomers switch between two different conformational states, left-or right-handed screws, and the binding of chiral ligands determines which of these states is favoured. At the other end of the molecule is a fluorophore consisting of a pair of pyrene molecules attached to a chiral diamine. This fluorophore is sensitive to conformational change: monomeric pyrene has an emission at 378 nm, but in a certain spatial arrangement a pair of pyrene fluorophores may emit at 450 nm. Thus, the binding of the cofactor affects the conformation of the whole receptor structure, which is reported by the fluorophore component.
Another study focused on the transmission of a signal using a switch localized in either the outer or inner leaves of the membrane (figure 4b) [bib_ref] Controlled membrane translocation provides a mechanism for signal transduction and amplification, Langton [/bib_ref]. Two head groups are coupled to a steroid spacer; one of the head groups, a protonated morpholine, is the external sensor and the other is a neutral pyridineoxime 'pro-catalyst'. When the sensor head group is polar, it sits in the aqueous phase awaiting the chemical signal, while the non-polar pro-catalyst sits in the membrane. On binding a signal, the sensor head group becomes nonpolar, inducing translocation across the membrane. Binding of a zinc cofactor from within the vesicle switches the pro-catalyst head group to polar, thus moving it into the aqueous vesicle interior. This activates the catalyst and allows the hydrolysis of the substrate to a fluorescent product within the vesicle. This signal transduction mechanism has been used to trigger the release of cargo from a vesicle, which may aid the development of drug delivery systems [bib_ref] Triggered release from lipid bilayer vesicles by an artificial transmembrane signal transduction..., Langton [/bib_ref].
These artificial signal transducers must be assembled in vitro; however, as they can function in vesicle lipid bilayers, they may well be able to function in vivo, acting as an interface between biological and synthetic systems.
## Making non-natural components biocompatible
While many chemical entities may have potential as biocompatible mimics of natural function, further research is required to effectively interface between biological and chemical elements to achieve full biocompatibility and functionality under physiological conditions. A step towards achieving full biocompatibility with or within cells is to optimize the element's function under physiological conditions, and to work in partnership with natural proteins. To take an example, while natural proteins do not make use of precious metals for catalysis, these metals are used in many industrial reactions. To function as part of an artificial metalloenzyme requires strategies to aid their biocompatibility; biotin/streptavidin technology has been exploited for this purpose. The Ward group described an artificial metalloenzyme which has a biotinylated organometallic iridium catalyst anchored to an engineered streptavidin protein scaffold. The streptavidin has a C-terminal artificial activating tripeptide ligand which, on proteolytic cleavage, then coordinates to the metal of the cofactor and activates the enzyme. In another study by the same group, an organometallic catalyst is integrated with natural proteins as part of an enzyme cascade to produce enantiopure amines [bib_ref] An NAD(P)Hdependent artificial transfer hydrogenase for multienzymatic cascades, Okamoto [/bib_ref]. It is difficult to fully assemble such metalloenzymes in vivo, in part due to the presence of inhibitors such as glutathione in cells [bib_ref] Neutralizing the detrimental effect of glutathione on precious metal catalysts, Wilson [/bib_ref]. The Ward group reported that compartmentalization within cells can be an approach to overcome this, as, for example, the periplasm contains a relative lack of glutathione. An artificial metalloenzyme for olefin metathesis (a reaction that does not occur in Nature) was expressed with a periplasmic export tag. The protein assembled in vivo in its functional form, and in vivo directed evolution was used to optimize the protein. In this case, the cofactor was biot-Ru, which is inactive until it is assembled as the mature form of the metalloenzyme [bib_ref] Directed evolution of artificial metalloenzymes for in vivo metathesis, Jeschek [/bib_ref]. . De novo transmembrane components for signalling. (a) A synthetic GPCR mimic [bib_ref] Ligand-modulated conformational switching in a fully synthetic membrane-bound receptor, Lister [/bib_ref]. The synthetic receptor consists of a ligand-binding pocket featuring a cationic metal complex (red), an Aib oligomer (grey) and a pair of pyrene molecules attached to a chiral diamine ( purple and blue). This complex adopts one of two mirror image conformational states on complexation with a chiral ligand. The binding of a chiral ligand (magenta) to one end of an Aib oligomer propagates its conformational influence along the entire length. The signal is output by the conformationally responsive fluorophore ( purple and blue). Thus, the binding of the cofactor perturbs the global conformation, which is reported by the fluorophore component. (b) A translocatable sensor [bib_ref] Controlled membrane translocation provides a mechanism for signal transduction and amplification, Langton [/bib_ref] in which two head groups are coupled to a steroid spacer (grey). The external sensor is a protonated morpholine (red or blue), while the second head group is a neutral pyridineoxime 'pro catalyst' (magenta or green). When the head groups are polar (red or green), they prefer to sit in the aqueous phase; when non-polar (blue or magenta), they prefer to sit in the membrane. Binding of a zinc cofactor from within the vesicle pulls the pro-catalyst head group into the aqueous phase on the interior of the vesicle. This allows the hydrolysis of the substrate within the vesicle, generating the output signal.
# Conclusion
There is still much progress to be made when it comes to fully biocompatible functional de novo proteins, although there are a few examples which fulfil these criteria [bib_ref] Construction and in vivo assembly of a catalytically proficient and hyperthermostable de..., Watkins [/bib_ref] [bib_ref] A de novo enzyme catalyzes a lifesustaining reaction in Escherichia coli, Donnelly [/bib_ref] [bib_ref] Massively parallel de novo protein design for targeted therapeutics, Chevalier [/bib_ref] [bib_ref] Evolution of a designed protein assembly encapsulating its own RNA genome, Butterfield [/bib_ref]. With advances in high-throughput techniques, and accessibility of these methods, we envisage successful designs becoming more commonplace in the coming years. In addition, new methods of making synthetic components biocompatible promise to unlock in vivo possibilities beyond those which Nature can provide us. These biocompatible de novo units may lead to improved and tailored medical benefits through the design of drug delivery systems and therapeutic molecules. In addition, we may learn more about how natural systems work, and therefore increase our knowledge of natural design principles in order to create improved de novo components. Ultimately, we may be able to create tailor-made life forms, such as bacteria with artificial genomes that can function in environments beyond their natural niches, to create, for example, useful industrial products.
Data accessibility. This article has no additional data. Authors' contributions. Both the authors contributed to the writing and editing of the manuscript. K.J.G. was the principal contributor and performed the literature review. J.L.R.A. provided advice and direction. Both the authors gave their final approval for publication.
[fig] & 2018: The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. [/fig]
[fig] Figure 1: The diversity of de novo designed protein structures. (a) Pizza variant, nvPizza2-S16H58, which coordinates a CdCl 2 nanocrystal [26]. PDB: 5CHB. (b) De novo designed reaction centre with heme B, synthetic Zn porphyrin and Zn(II) cations [27]. PDB: 5VJS. (c) Catalytic helical barrel, CC-Hept-I18C-L22H-I25E. Catalytic triad residues are shown [28]. PDB: 5EZC. (d ) DFsc-Zn(II) 2 used by Ulas et al. [29] for semiquinone radical stabilization. PDB: 2LFD. (e) Designed beta solenoid proteins, SynRFR24.1 (red, PDB: 4YC5) and SynRFR24.t1428 (blue, PDB: 5DNS) [/fig]
[fig] Figure 3: Crystal structures of de novo inhibitors binding to their targets. Crystal structures of inhibitor complexes. (a) Inhibitor peptide aMCL1 (red) binds the human BCL2 homologue, Mcl-1 (blue), with picomolar affinity [60]. PDB: 5JSB. (b) Inhibitor peptide HB1.6928.2.3 (red), which can bind influenza haemagglutinin [59]. PDB: 5VLI. rsif.royalsocietypublishing.org J. R. Soc. Interface 15: 20180472 [/fig]
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Where does DNA replication start in archaea?
Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins. Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.
Use of genome sequences is a powerful art that goes beyond finding protein homologs: it has changed how we can approach basic biological questions. This is particularly apparent for the enigmatic archaebacteria. Here, more than for other organisms, available genome data far exceed traditional biological study. A recent striking example of the insights that can be gained from archaeal genomics is provided by a report in Science from Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] showing the use of DNA strand compositional bias, or GC skew, to find the likely replication origin in three Pyrococcus species. One reason for widespread interest in archaeal replication origins is the similarity between the factors involved in DNA replication in archaea and eukaryotes. Archaeal homologs of eukaryotic replication factors and DNA polymerase suggest that archaebacteria could become an important model to aid understanding of eukaryotic DNA replication.
## Where do archaea fit?
Are archaea like humans or bacteria? This was the issue raised when archaeal genome sequencing revealed some areas of surprising similarity between these prokaryotes and eukaryotes. Though archaeal metabolism and operon gene organization is certainly most similar to prokaryotic eubacteria, the archaeal factors for transcription, translation and DNA replication seem more akin to those found in eukaryotes. Thus, the third kingdom, archaebacteria, might serve as a simple model for mechanisms of eukaryotic cell function. And we are left wondering just how much these prokaryotes resemble ourselves. (For more extensive reviews of this issue see [bib_ref] Ancient ciphers: translation in Archaea, Dennis [/bib_ref] [bib_ref] Archaea and the origin(s) of DNA replication proteins, Edgell [/bib_ref] [bib_ref] Another bridge between kingdoms: tRNA splicing in archaea and eukaryotes, Belfort [/bib_ref] [bib_ref] Archaeal histones, nucleosomes, and transcription initiation, Reeve [/bib_ref] [bib_ref] Archaeal genomics: an overview, Olsen [/bib_ref]. Archaea (as exemplified by Pyrococcus sp.) replicate their circular genome from a single DNA replication origin as do bacteria, even though they may use eukaryotic-like proteins to do so ; [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref]. This single-origin replication is definitely un-human, as our DNA replication depends on initiation at thousands of different origins. The multiple sites of initiation are essential for timely replication of large eukaryotic genomes. The archaebacterial Pyrococcus genomes by contrast are smaller even than that of Escherichia coli, so perhaps we should not be surprised that archaea can replicate like E. coli using a single origin.
Identifying a replication origin in archaea may be more important than finding whether they use one origin or many. In fact, E. coli lacking RNaseH start replication at many different sites, yet these multiple replication origins are not at all eukaryotic-like. The observation that a single origin is used fails to enlighten us as to the mechanism of initiation, but identification of an origin does provide one of the most powerful tools for future studies of DNA replication initiation. Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] reported genomic analyses that strongly suggest a well conserved 600 base-pair sequence is the replication origin in three related archaea [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref].
## The importance of gc skew
How can nucleotide sequence be used to find replication origins? In most eubacteria, there is a statistical overrepresentation of guanine (G) in DNA of the leading strand and more cytosine (C) in DNA of the lagging strand. This GC skew changes sign at the replication origin and terminus [fig_ref] Figure 2: A bi-directional replication fork [/fig_ref] , though this change is most notable at the origin as termination may occur in a wider region. What creates GC skew is poorly understood but could include differences in errors, damage, and/or repair for lagging versus leading strand synthesis. Skewed distribution of short sequences may be even more predictive of bacterial origins than GC skew alone [bib_ref] Skewed oligomers and origins of replication, Salzberg [/bib_ref]. Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] measured strand distribution of the tetramer GGGT across several archaea genomes to look for a singularity where GGGT skew changed sign. This occured at the same place in genomes of three related Pyrococcus species analyzed, suggesting this region may be the replication origin for these organisms.
Several archaeal genomes lack obvious GC skew that would indicate a single DNA replication origin [bib_ref] Strand compositional asymmetry in bacterial and large viral genomes, Mrazek [/bib_ref] [bib_ref] Base composition skews, replication orientation, and gene orientation in 12 prokaryote genomes, Mclean [/bib_ref]. This has fueled speculation that archaea use multiple origins for DNA replication and could provide clues to the selection and use of many replication origins in eukaryotes. But GC skew is not clear for all eubacteria and may be obscured by biological constraints of factor binding sites and gene coding sequences (and see McLean et al. [bib_ref] Base composition skews, replication orientation, and gene orientation in 12 prokaryote genomes, Mclean [/bib_ref] for a discussion of these issues and a clear, thoughtful skew analysis of 12 prokaryotes including three archaea). By practicing on bacteria with known origins, scientists and mathematicians are finding countless ways to count Gs and Cs and have predicted single origins for some archaea [bib_ref] Skewed oligomers and origins of replication, Salzberg [/bib_ref] [bib_ref] Strand compositional asymmetry in bacterial and large viral genomes, Mrazek [/bib_ref] [bib_ref] Base composition skews, replication orientation, and gene orientation in 12 prokaryote genomes, Mclean [/bib_ref] [bib_ref] Bacterial DNA strand compositional asymmetry, Karlin [/bib_ref] [bib_ref] Asymmetric substitution patterns in the two DNA strands of bacteria, Lobry [/bib_ref]. So what is special about the report by Myllykallio et al.?
Myllykallio et al. did three important things. First, they obtained a signal revealing skewed strand distribution of nucleotides and the skew changed sign abruptly at one position in the genome (the putative origin). Use of a tetramer (GGGT) and mathematics to smooth out local variations were required to see a signal at all. Since it is not understood exactly what causes nucleotide skew and is even less clear what causes skewed distribution of short sequences, finding a signal was only the first step.
Second, they exploited the awesome power of comparative genomics. They compared three fully sequenced Pyrococcus species. Nucleotide skew as well as other properties predict origin location in the same place in all three genomes. For instance, a prediction for bacteria-like replication is that genes encoding replication factors will be clustered around the replication origin. Most notably, the gene for the bacterial replication initiator dnaA is so consistently linked to the origin that it is predictive of origin location. Several of the Pyrococcus replication factors cluster around the predicted origin, including Orc1/Cdc6, which resembles the putative eukaryotic initiator origin recognition complex and, therefore, is analogous to DnaA. Finally, the bacterial replication terminus is a hot spot for
## G g g t c c c a g g g t c c c a oric
## 5' 5' 3' 3'
Leading strand synthesis Lagging strand synthesis
## Figure 1
Evolutionary relationships between bacteria, archaea and eukaryotes takes into account the similarities between archaea replication, transcription and translation factors with eukaryotic factors. The report by Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] shows that the archaea share chromosome organization and replication pattern with prokaryotes although they use many eukaryotic-like factors to duplicate their chromosomes. rearrangement [bib_ref] When replication forks stop, Bierne [/bib_ref] [bib_ref] Analysis and possible role of hyperrecombination in the termination region of the..., Louarn [/bib_ref] and comparative genomics reveals this to be true for the three Pyrococcus species studied by Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref].
Third and most importantly, they tested the hypothesis derived by computer or in silico experiments using old-fashioned laboratory experimentation. They grew these third kingdom creatures keeping them warm at 95°C labeled newly synthesized DNA in vivo, and then determined which genome regions replicate first and last. As with any good story, all the pieces fit. Tetramer skew analysis predicted origins in the same place for all three species: the region identified has a highly conserved intergenic sequence that might bind replication factors, and, finally, DNA replication was shown to begin in this putative origin region.
## Towards a mechanism
The species studied by Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] performs the incredible feat of replicating its genome at 95°C a temperature hot enough to melt DNA duplexes. It is amazing that replication under extreme conditions, using many eukaryotic-like factors that differ considerably from bacterial replication machinery, could result in the bacterial GC skew. Perhaps the same skew will be detectable in eukaryotes once we better understand how to filter out biological noise and focus on chromosomal regions that are replicated most often by a fork passing in a single direction.
Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] used their information to calculate replication fork movement to be at a rate of 20 kilobases per minute. This is slower than DNA replication in E. coli, but is still ten times faster than fork movement in eukaryotes. Archaea have a DNA polymerase resembling eukaryotic DNA polymerases, but they also have their own unique DNA polymerase [bib_ref] Archaeal DNA replication: identifying the pieces to solve a puzzle, Cann [/bib_ref] ; perhaps this unique archael polymerase is required for the faster movement of the replication fork. Nevertheless, many factors for initiation and replication fork function in archaebacteria have clear homologs in eukaryotes.
Many scientists trying to decipher eukaryotic DNA replication are leaping at the chance to study something a bit simpler. Archaeal replication proteins look far more like eukaryotic replication proteins than those of eubacteria and there are fewer of them. For example, eukaryotic DNA replication requires six related mini-chromosome maintenance (MCM) proteins named MCM2-7, but the archaeal bacterium Methanobacter thermoautotrophicum has only one MCM homolog [bib_ref] Insights into DNA replication from the third domain of life, Tye [/bib_ref] ; eukaryotic DNA replication requires the replication factor C complex, comprised of five proteins, while M. thermoautotrophicum has only two subunits for replication factor C [bib_ref] A unique organization of the protein subunits of the DNA polymerase clamp..., Kelman [/bib_ref] ; eukaryotic DNA replication initiation requires the origin recognition complex (ORC) and Cdc6 (three ORC subunits and Cdc6 share sequence similarity [bib_ref] The metazoan origin recognition complex, Quintana [/bib_ref] , whereas the M. thermoautotrophicum has only two ORC/Cdc6-like subunits [bib_ref] Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics, Smith [/bib_ref].
Archaeal replication is thought to be the evolutionary precursor of eukaryotic replication, so archaea may use homooligomers evolved from an ancestral factor, where eukaryotes use hetero-oligomeric complexes of related proteins evolved from this same ancestral factor. Biochemical studies are already yielding valuable data from studying the simpler archaeal systems. For example, archaebacterial MCM has helicase function in vitro [bib_ref] The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance..., Shechter [/bib_ref] [bib_ref] The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains DNA helicase..., Kelman [/bib_ref] [bib_ref] A doublehexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase, Chong [/bib_ref] , and this lends strong support to the hypothesis that MCMs in eukaryotes function as a replicative helicase [bib_ref] A DNA helicase activity is associated with an MCM4, -6, and -7..., Ishimi [/bib_ref] , an idea that is reviewed elsewhere [bib_ref] Insights into DNA replication from the third domain of life, Tye [/bib_ref] [bib_ref] Emerging mechanisms of eukaryotic DNA replication initiation, Leatherwood [/bib_ref]. In another example, the archaeal homolog of DNA polymerase alpha subunit p50 has primase activity in vitro, strongly supporting the long-held, but never proven, hypothesis that p50 is the catalytic subunit of eukaryotic DNA polymerase alpha primase [bib_ref] Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus..., Desogus [/bib_ref]. Information on archaeal replication origins will be critical in reconstituting an in vitro archaebacterial replication system.
Myllykallio et al. [bib_ref] Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon, Myllykallio [/bib_ref] provide the best evidence to date that archaebacteria replicate DNA from a single origin. If this is indeed so, these organisms have no need to coordinate replication initiations at various sites. But they must still couple replication with growth and division; and how they do so is an interesting puzzle. E. coli achieves such regulation via SeqA, a negative regulator of replication initiation [bib_ref] SeqA: a negative modulator of replication initiation in E. coli, Lu [/bib_ref] [bib_ref] Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation..., Torheim [/bib_ref] [bib_ref] Effects of purified SeqA protein on oriC-dependent DNA replication in vitro, Wold [/bib_ref]. Eukaryotes do so via the master cell-cycle regulators, the cyclin-dependent kinases (Cdks) and cyclins, and eukaryotic replication is also dependent on the replication specific kinase Cdc7 [bib_ref] Emerging mechanisms of eukaryotic DNA replication initiation, Leatherwood [/bib_ref] [bib_ref] Cdc7p-Dbf4p becomes famous in the cell cycle, Sclafani [/bib_ref]. Archaea lack recognizable homologs of SeqA or cyclin dependent kinases, cyclins, or the kinase Cdc7. Perhaps archaebacteria have their own ways to couple replication, growth, and division, which may be achieved by some of the proteins encoded by the 50% of archaeal coding sequences specific to archaea.
[fig] Figure 2: A bi-directional replication fork: DNA replicated by laggingstrand synthesis on one side of the origin will be replicated by leading-strand synthesis on the other side. In bacteria, there is a switch in the strand bias of guanine content at the origin. Myllykallio et al.[1] measured the strand bias resulting from GGGT as illustrated. [/fig]
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Prevalence and Profile of High-Impact Chronic Pain in the United States
The multidimensional nature of chronic pain is not reflected by definitions based solely on pain duration, resulting in high prevalence estimates limiting effective policy development. The newly proposed concept of high-impact chronic pain (HICP) incorporates both disability and pain duration to identify a more severely impacted portion of the chronic pain population yet remains uncharacterized at the population level. As such, we used the 2011 National Health Interview Survey (N = 15,670) to 1) assess the likelihood of disability in the overall chronic pain population, 2) estimate the prevalence of HICP, and 3) characterize the disability, health status, and health care use profile of this population in the United States. Overall, chronic pain, defined as pain experienced on most days or every day in the previous 3 months, was strongly associated with an increased risk of disability after controlling for other chronic health conditions (odds ratio = 4.43; 95% confidence interval = 3.73−5.26), where disability was more likely in those with chronic pain than in those with stroke or kidney failure, among others. HICP affected 4.8% of the U.S. adult population, or approximately 10.6 million individuals, in 2011. The HICP population reported more severe pain and more mental health and cognitive impairments than persons with chronic pain without disability, and was also more likely to report worsening health, more difficulty with self-care, and greater health care use. HICP clearly represents a more severely impacted portion of the chronic pain population. Understanding this heterogeneity will contribute to developing more effective legislation promoting safe and cost-effective approaches to the prevention and treatment of chronic pain.
Adult Functioning and Disability Supplement (AFD), for a total of 15,670 individuals included in this analysis [fig_ref] Figure 1: Flowchart representing the NHIS sampling procedure for the 2011 survey [/fig_ref]. Full eligibility criteria, sources, and methods of selection of participants for the 2006−2015 NHIS are available at https://www.cdc.gov/nchs/data/nhis/ 2006var.pdf. The AFD assessed functioning and disability in domains including sensory, motor, communication, cognitive, emotional, pain, and fatigue. Data from the 2011 NHIS were used because 2011 the most recent year where the AFD contained items referring to specific activity limitations/participation restrictions.
## Demographic variable recoding
In some cases, NHIS variables were recoded to identify specific subgroups. Age and body mass index (BMI), both coded as continuous variables (AGE_P and BMI, respectively), were recoded into discrete categories. Ethnicity was recoded from 2 separate NHIS ethnicity items (MRACRP12 and HISPAN_I) to include 7 ethnicities: white, black/African American, Hispanic, American Indian/Inuit, Asian Indian, Asian, or multiple/unspecified. Marital status (R_MARITL) was recoded such that individuals who were married and living with a partner or unmarried but living with a partner were categorized as married/living with partner. Individuals who identified as divorced, separated, or married but not living with a partner were categorized as divorced/separated. All other marital status subcategories were not recoded. includes all NHIS questions used in this analysis.
## Operational definitions of chronic pain groups
The operational definition of chronic pain in the HICP and the CPWL groups involved the PAIN_2 question in the AFD: "In the past 3 months, how often did you have pain? Would you say never, some days, most days, or every day?" . HICP was defined as pain on most days or every day in the previous 3 months accompanied by ≥ 1 activity limitation/ participation restriction from among 8 relevant questions from the AFD, whereas CPWL was operationally defined as pain experienced on most days or every day in the previous 3 months without activity limitations/participation restrictions. The 8 items assessed the respondents' capacity (ie, do the activity, don't do the activity, unable to do the activity) to engage in the following activities: 1) working outside the home to earn an income, 2) going to school or achieving your education goals, 3) participating in leisure or social activities, 4) getting out with friends or family, 5) doing household chores such as cooking and cleaning, 6) using transportation to get to places you want to go, 7) participating in religious activities, and 8) participating in community gatherings . Individuals were considered to have an activity limitations/participation restriction if they indicated that they were unable to do ≥ 1 of the activities. The total population incorporates those with CPWL, HICP, and those without pain on most days/every day in the previous 3 months.
# Statistical analysis
All analyses were performed using the integrated complex samples analysis procedures in the SPSS software package (SPSS v. 22; IBM, Armonk, NY). Responses to questions from the Sample Adult core and the AFD were merged using the sample weighting variable WTFA-AFD (from the 2011 AFD data file) as well as the STRAT_P and PSU_P variables (from the 2011 Sample Adult core), accounting for stratification and clustering, respectively. The merged datasets represent a weighted population size of approximately 220.3 million noninstitutionalized adults. Descriptive statistics (mean ± standard error) were used to characterize groups according to demographic and psychosocial variables. In most cases, data are presented as a percent of the specified group representing the variable of interest, extracted from cross-tabulation tables. Given that odds ratios (ORs) reflect the effect of a putative predictor on the likelihood that a specified outcome will occur, binomial logistic regression was used to determine ORs for various outcomes. To adjust for potential confounding, we controlled for demographic variables such as age, sex, ethnicity, marital status, region, and BMI in all cases. In addition, we also controlled for chronic health conditions where indicated. Owing to the categorical nature of NHIS items addressing sex, ethnicity, marital status, and region, they were included into the logistic regression analysis as categorical covariates. Given that age and BMI were reported in the NHIS as continuous variables (ie, whole numbers between a specified range), these factors were included into the logistic regression analysis as continuous factors, with the exception of the demographic analysis illustrated in , where age and BMI were treated as categorical factors. The reference categories for sex, ethnicity, marital status, and region were male, white, married/ living with partner, and Northeast, respectively. For the chronic health condition analyses indicated in , each health condition−related OR also controlled for the remaining 14 chronic health conditions. In these cases, the reference category was set to no (ie, never been told by a doctor or health professional that I have the condition). Occasionally, a survey respondent failed to answer a given question, accounting for no more than 1 or 2 individuals or less per category (ie, total population/HICP/CPWL). As such, these responses were removed from the analysis of that question.
# Results
## Prevalence of chronic pain and hicp in the u.s. adult population
In 2011, 4.8% of the United States adult population (10.6 million persons) met our criteria for HICP defined as pain present on most days or every day over the previous 3 months and having ≥ 1 major activity limitation/participation restriction . Almost 3 times as many individuals-29.9 million individuals or 13.6% of the adult population-experienced chronic pain without activity limitations/participation restrictions (CPWL). Taken together, approximately 18.4% of the adult population, or >40 million adults, reported experiencing pain on most days or every day in the previous 3 months.
## Demographic profile
The demographic profile of the CPWL and HICP populations is shown in . Compared with those without pain, individuals with chronic pain (ie, CPWL and HICP together) were more likely to be female (OR = 1.16, 95% confidence interval [CI] = 1.03−1.30). Those with HICP, however, were no more likely to be female than those with CPWL (OR = 0.98, 95% CI = 0.80−1.22). Advancing age increased the likelihood of having chronic pain (compared with no pain) and HICP (compared with CPWL). This effect is particularly evident in individuals >45 years of age. Although individuals of white ethnicity comprised the majority of the total population as well as the chronic pain population, those of African American (OR = 1.76, 95% CI = 1.29−2.39), native American (OR = 2.86, 95% CI = 1.39−5.90), and Asian Indian (OR = 3.61, 95% CI = 0.85−15.31) descent had an increased likelihood of HICP compared with CPWL. In terms of education, those with chronic pain (vs no pain) and HICP (vs CPWL) were more likely to have achieved no more than a high school diploma. Although the majority of those in the total population, as well as those with CPWL and HICP, were married or living with a partner, those who were divorced/separated, widowed, or never married had an increased likelihood of HICP. Obesity increased the likelihood of experiencing chronic pain (OR = 1.67, 95% CI = 1.45−1.92); however, obesity did not increase the likelihood of HICP compared with CPWL (OR = 1.14, 95% CI = 0.87−1.51). Compared with those living in the North east, those from other regions were no more likely to have chronic pain or HICP. Overall, the HICP population was more likely > 45 years of age, predominantly white (but with a larger proportion of African Americans, native Americans, and Asian Indians vs the CPWL population), of lower educational level, and have higher rates of divorce/separation.
## Comorbid chronic health conditions
The HICP population bore a substantially greater illness burden than the CPWL population. For each of 15 chronic health conditions , a greater proportion of the HICP population reported having been told by a doctor or other health professional that they had the health condition compared with the CPWL population. Moreover, individuals with HICP were more likely than the CPWL population to have emphysema, a liver condition, weak/ failing kidneys, chronic bronchitis, arthritis, diabetes, or asthma, or to have had a stroke . Compared with the CPWL population, those with HICP were about as likely to report having had cancer, a heart condition, coronary heart disease, hypertension, a heart attack, or angina, or to be obese .
Considering that the defining characteristics of the HICP population were chronic pain with activity limitations/participation restrictions, we assessed whether activity limitations/ participation restrictions were explained by comorbid chronic health conditions. Controlling for demographic variables as well as other chronic health conditions, the OR for the presence of limitations in those with no pain/occasional pain versus those with pain on most days or every day was 4.23 (95% CI = 3.55−5.03), indicating that regardless of the presence of other chronic health conditions, individuals experiencing frequent pain were much more likely to have limitations than those with no/occasional pain. For added perspective, the OR for activity limitations/participation restrictions in those reporting frequent pain was compared with those reporting chronic health problems; the OR for the presence of activity limitations/participation restrictions in those with chronic pain (OR = 4.23, 95% CI = 3.55−5.03) was greater than the likelihood of activity limitations/participation restrictions in all other chronic health conditions, including those reporting weak/failing kidneys (OR = 3.64, 95%CI = 2.46−5.39) or a stroke(OR = 3.04, 95% CI = 2.17−4.26), among others . Taken together, those with chronic pain were more likely to have activity limitations/participation restrictions than those with other chronic health conditions.
## Pain characteristics
The operational definitions of both CPWL and HICP are based on pain frequency over the previous 3 months. However, considering the multidimensional nature of chronic pain, we also explored pain intensity and the number of bodily locations of pain. [fig_ref] Table 4: SamAdultThe following questions are about pain you may have experienced in the... [/fig_ref] indicates the proportion of the HICP and CPWL populations in the 3 categories defined by pain intensity (ie, a little pain, in between a little and a lot, a lot of pain). The HICP population was much more likely to report moderate and severe pain than the CPWL population (OR = 2.19, 95% CI = 1.59−3.01 and OR = 4.72, 95% CI = 3.40−6.55, respectively). Respondents were also asked about pain in 6 bodily regions including joint(s), low back, legs, neck, jaw/ear, and migraine/severe headache. The HICP population was more likely than the CPWL population to report severe headache/migraine and pain in the legs, low back, and joints but was equally likely to report pain in the neck or face. Those with HICP were about as likely as the CPWL population to report pain in 1 to 3 locations (OR = 0.97, 95% CI = 0.50−1.88) but somewhat more likely to report pain in 4 to 6 locations (OR = 1.79, 95% CI = 0.96−3.33; [fig_ref] Table 4: SamAdultThe following questions are about pain you may have experienced in the... [/fig_ref]. A proportion of both the CPWL and the HICP populations reported pain that did not refer to any of the 6 listed locations, likely owing to the limited number of possible pain locations available in the survey (ie, no item referencing pain of cutaneous or visceral origin). These individuals were classified as having 0 pain locations (ie, pain location not specified). Overall, the HICP population carries a more severe pain burden than those with CPWL.
# Activity limitations/participation restrictions
Activity limitations/participation restrictions were based on the respondent's engaging in 8 major life activities . Most individuals in the HICP population reported being unable to work outside the home (83.2 ± 1.7%), whereas substantial proportions of the HICP population also reported being unable to go to school or to attain educational goals (42.0 ± 2.1%), engage in leisure or social activities (27.1 ± 1.9%), and perform household chores (25.3 ± 1.9%), among others. In terms of the number of limitations/restrictions per individual, 32.1 ± 2.0% reported having 2 or 3 limitations/restrictions and 22.8 ± 1.8% reported having ≥ 4 limitations/restrictions . Some of the limitations/restrictions may not be mutually exclusive (ie, leisure/social activities, friend/family outings, and community activities), potentially increasing the estimated number of limitations/restrictions per individual. Taken together, almost 85% of the HICP population was unable to work, and approximately 55% of the HICP population reported having ≥ 2 major activity limitations/ participation restrictions in their daily life.
## Mental health, fatigue, and cognitive impairment
Individuals with HICP had a much greater likelihood to report daily or weekly depression than those with CPWL (43.7 ± 2.2% vs 18.0 ± 1.0%, respectively; OR = 3.85, 95% CI = 3.01−4.93). Only 8.9% of the total population experienced daily/weekly depression . Likewise, the HICP population was also much more likely to be taking antidepressant medication than those with CPWL (33.3 ± 1.7% vs 14.8 ± 1.0%, respectively; OR = 3.13, 95% CI = 2.48−3.95). Similarly, the HICP population was more likely to experience daily or weekly anxiety (54.7 ± 2.1% vs 30.6 ± 1.2%, respectively; OR = 3.30, 95% CI = 2.62−4.15) and to take anxiety medications (36.8 ± 2.0% vs 15.7 ± 1.0%, respectively; OR = 3.41, 95% CI = 2.69−4.33) compared with the CPWL population. Those with HICP were also more likely than the CPWL to report fatigue on most days or every day (59.1 ± 2.1% vs 31.4 ± 1.2%, respectively; OR = 3.46, 95% CI = 2.76−4.33) and to have difficulty remembering/ concentrating (47.6 ± 2.2% vs 20.8 ± 1.1%, respectively; OR = 3.18, 95% CI = 2.51−4.02; .
## Health status
Compared with the CPWL population, the HICP population had a greater likelihood of reporting worsening health over the previous 12 months (OR = 2.92, 95% CI = 2.30−3.70; . Moreover, the HICP population was less likely to report their current health status as very good or excellent, and much more likely to report it as fair or poor . Although most of the CPWL and the total populations reported no difficulties with self-care, approximately 33% of the HICP population reported at least some difficulty with self-care . Indeed, the HICP population was much more likely to report being unable to maintain self-care than the CPWL population (OR = 11.99, 95% CI = 2.74−52.46). The HICP population had a much greater likelihood of spending ≥ 11 days in bed over the previous 12 months compared with the CPWL population (64.9% vs 24.6%, respectively; OR = 5.03, 95% CI = 3.96−6.38; .
## Health care use
Health care use includes services for all health conditions, including pain. The HICP population was more likely to consult health professionals including general doctors, specialists, physical/occupational therapists, and mental health professionals . Although the majority of all 3 populations reported no surgical procedures and no emergency room visits in the previous 12 months , a greater portion of the HICP population reported ≥ 2 surgical procedures compared with the CPWL group (10.7% vs 3.9%, respectively; OR = 2.93, 95% CI = 1.71−5.04) and ≥ 2 emergency room visits over the previous 12 months (30.3% vs 11.9%, respectively; OR = 2.77, 95% CI = 2.01−3.81). Finally, whereas only 2.3% and 2.9% of the total adult and CPWL populations, respectively, received home care in the previous 12 months, >15% of the HICP population required home care . As such, the HICP population was much more likely than the CPWL population to have received home care from a health professional in the previous year (OR = 5.14, 95% CI = 3.30−7.99).
# Discussion
As evidenced by its nomenclature, chronic pain has traditionally been defined by pain duration. [bib_ref] Report of the American College of Rheumatology Pain Management Task Force, Borenstein [/bib_ref] [bib_ref] The prevalence of chronic pain in United States adults: Results of an..., Johannes [/bib_ref] [bib_ref] Chronic pain, psychopathology, and DSM-5 somatic symptom disorder, Katz [/bib_ref] [bib_ref] Prevalence of persistent pain in the US adult population: New data from..., Kennedy [/bib_ref] [bib_ref] A classification of chronic pain for ICD-11, Treede [/bib_ref] [bib_ref] Common chronic pain conditions in developed and developing countries: Gender and age..., Tsang [/bib_ref] Using this approach, our prevalence estimate of the overall chronic pain population is approximately 18.4% of the adult population, or >40 million people. Although this estimate is similar to some estimates, [bib_ref] The prevalence of chronic pain in United States adults: Results of an..., Johannes [/bib_ref] [bib_ref] Prevalence of persistent pain in the US adult population: New data from..., Kennedy [/bib_ref] [bib_ref] Estimates of pain prevalence and severity in adults: United States, Nahin [/bib_ref] it is much lower than others, 10,26 likely owing to methodological aspects of data collection in those studies as well as how chronic pain was operationalized. More important, whereas pain enduring ≥ 3 months certainly constitutes a significant burden to the sufferer, not everyone is impacted equally. [bib_ref] Von Korff M: A prognostic approach to defining chronic pain: Replication in..., Dunn [/bib_ref] [bib_ref] Assessment of chronic pain in epidemiological and health services research, Korff [/bib_ref] [bib_ref] Chronic pain reconsidered, Korff [/bib_ref] [bib_ref] Graded chronic pain status-An epidemiologic evaluation, Korff [/bib_ref] [bib_ref] Assessing global pain severity by self-report in clinical and health services research, Korff [/bib_ref] [bib_ref] A prognostic approach to defining chronic pain, Korff [/bib_ref] [bib_ref] Grading the severity of chronic pain, Korff [/bib_ref] [bib_ref] United States National Pain Strategy for population research: Concepts, definitions, and pilot..., Korff [/bib_ref] To address this variability in outcomes, some studies have further stratified the chronic pain population according to pain severity, where greater severity is generally associated with poorer outcomes. [bib_ref] Chronic back pain and major depression in the general Canadian population, Currie [/bib_ref] [bib_ref] Characterizing the course of low back pain: A latent class analysis, Dunn [/bib_ref] [bib_ref] The relation between pain intensity, disability, and the episodic nature of chronic..., Mcgorry [/bib_ref] [bib_ref] Estimates of pain prevalence and severity in adults: United States, Nahin [/bib_ref] [bib_ref] Categorizing the severity of pain using questions from the 2012 National Health..., Nahin [/bib_ref] However, it has been long agreed that several other factors interact with pain characteristics to produce negative outcomes, including affective distress, life control, and, crucially, functional disability. [bib_ref] Behavioral concepts in the analysis of chronic pain syndromes, Keefe [/bib_ref] [bib_ref] Towards a comprehensive assessment of chronic pain patients, Turk [/bib_ref] [bib_ref] Toward an empirically derived taxonomy of chronic pain patients: Integration of psychological..., Turk [/bib_ref] [bib_ref] Chronic pain reconsidered, Korff [/bib_ref] [bib_ref] A prognostic approach to defining chronic pain, Korff [/bib_ref] [bib_ref] Grading the severity of chronic pain, Korff [/bib_ref] As proposed in the U.S. National Pain Strategy, 24 the inclusion of disability (ie, activity limitations/ participation restrictions) into the standard chronological definition of chronic pain is meant to differentiate those with debilitating chronic pain from those with less impactful chronic pain. Responding to the National Pain Strategy's call for better information on the prevalence and impact of chronic pain in the U.S. population, we used nationally representative data to show that chronic pain (ie, pain on most days or every day over the previous 3 months) is strongly associated with an elevated risk of developing activity limitations/participation restrictions such as the inability to work for a living; go to school; or engage in social, community or religious activities. Our use of general measures of disability not related to pain rather than pain-related interference constitutes an important advancement over previous work. Here, respondents were simply asked about their capacity to engage in certain activities rather than the degree to which pain interferes with these tasks. By not requiring an individual with severe pain to distinguish the increased effort required to carry out important life activities from the actual incapacity to participate in these activities, our findings reflect a more clinically relevant assessment of impact. Moreover, this approach also allows us to directly compare the impact of chronic pain with the impacts of several other chronic health conditions, including stroke, kidney failure, cancer, and heart disease. We not only demonstrate that individuals with chronic pain are much more likely to have disabilities than those without pain on most days or every day but also show that disability is more likely in the chronic pain population than in any other chronic health condition assessed, including stroke, kidney failure, cancer, diabetes, and heart disease. A potential limitation of using general disability questions rather than more specific pain-related interference questions may be that the disability is owing to other chronic health conditions rather than pain. However, our analyses controlled for the presence of >15 chronic health conditions, strongly suggesting that general disability questions may indeed reflect the impact of chronic pain.
In 2011, approximately one-quarter of the overall chronic pain population in the United States had HICP, or chronic pain with ≥ 1 activity limitation/participation restriction. The overall prevalence of HICP was 4.8%, or approximately 10.6 million adults. That this estimate is considerably lower than estimates based on pain interference [bib_ref] Racial and socioeconomic disparities in disabling chronic pain: Findings from the Health..., Janevic [/bib_ref] [bib_ref] United States National Pain Strategy for population research: Concepts, definitions, and pilot..., Korff [/bib_ref] is not unexpected. Indeed, considering the stringent criteria for social role disability used in this assessment, our estimate reflects the most severely impacted portion of the chronic pain population. The application of more inclusive disability criteria would likely result in considerably higher prevalence estimates. Indeed, the CPWL population likely includes a sizable portion of individuals with varying degrees of social role disability who do not meet the stringent requirements used in this study. Although it is possible that some people with CPWL may progress to HICP at some point in the future, our approach seems to identify a smaller but more heavily burdened portion of the population. Compared with those with CPWL, the HICP population experienced poorer overall health outcomes. The most prevalent limitation in the HICP population was incapacity to work outside the home. The impacts of lost work are apparent both for the individual (ie, loss of self-worth, income, and lower quality of life), as well as for society (ie, lost productivity, higher health care expenditures). Accordingly, the HICP population also exhibited higher levels of anxiety, depression, fatigue, and cognitive difficulty than those with CPWL. The HICP population tended to report more severe pain, markedly poorer health outcomes, and dramatically more health care use than those with CPWL. Importantly, as discussed else-where in this article, the disabilities characterizing the HICP population seem to be more closely related to the frequent pain than the presence of other chronic health conditions. Nonetheless, a substantial proportion of the CPWL population also reported important pain and mental health impacts that should not be ignored; as discussed, CPWL may progress to HICP owing to ongoing pain duration or other factors.
Although this study expands our understanding of the population health impact of CPWL and HICP, the cross-sectional nature of the survey precludes any assessment of potential contributory and/or causal factors to the development of CPWL or HICP. Furthermore, it is possible that alternative stratifications of some variables (ie, demographics, health-related outcomes) may have altered certain logistic regression outcomes. However, considering that our analytical strategy yielded prevalence estimates and other measures mirroring previous studies also using NHIS data, we are confident in the relevance of our findings. Other potential limitations include the external validity of our results. The results described in this study are based on a large-scale, nationally representative health survey. As such, they can be expected to accurately represent the U.S. noninstitutionalized, adult population. However, individuals such as veterans, the incarcerated, and those in residential care facilities are not represented. Although the prevalence estimates described herein do not include these populations, there is no reason to expect that the health-related impact of chronic pain would be any less burdensome. Although the survey response rates were high (81.6%), there is potential for selection bias. Indeed, prior research has found survey respondents to be somewhat healthier than nonrespondents. [bib_ref] A selective follow-up study on a public health survey, Linden-Boström [/bib_ref] [bib_ref] Survey non-response in the Netherlands: Effects on prevalence estimates and associations, Van Loon [/bib_ref] However, nonresponse bias, whereby respondents fail to respond to some or all questions, is generally understood to be modest in face-to-face interview paradigms compared with other types of surveys. [bib_ref] Effect of survey mode on response patterns: Comparison of face-to-face and self-administered..., Christensen [/bib_ref] Moreover, adjustments for nonresponse were made before the NHIS data were released and is reflected in the sample weights provided by NHIS.Overall, our findings demonstrate that defining HICP with binary questions addressing social role limitations without reference to pain identifies the most severely disabled segment of the chronic pain population, a segment representing >10 million American adults. As such, preventing the development of disability in this population should be a public health priority. Considering the heterogeneity in health-related and quality of life−related outcomes within the chronic pain population, stratification of the CPWL and HICP populations in clinical research and practice will lead to an improved understanding of the risk factors, causes, and consequences of chronic pain. Of significant interest would be risk factors associated with the transition from CPWL to HICP. In addition, considering the ongoing opioid epidemic, improved understanding of opioid use and analgesic efficacy in these populations is required. From a policy perspective, the incorporation of World Health Organization recommendations 37 (ie, activity limitations/participation restrictions) into the stratification of chronic pain will permit greater cogency and relevance to potential legislative outcomes. In short, this article evaluates an assessment of HICP that does not refer to pain-related interference with life activities and highlights the role of disability as a key indicator of pain impact. This knowledge may not only serve to refine clinical research and streamline treatment, it also provides much-needed information to policymakers.
[fig] Figure 1: Flowchart representing the NHIS sampling procedure for the 2011 survey. [/fig]
[table] Table 4: SamAdultThe following questions are about pain you may have experienced in the past 3 months. Please refer to pain that lasted a whole day or more. Do not report aches and pains that are fleeting or minor. During the past 3 months, did you have...Facial ache or pain in the jaw muscles or the joint in front of the ear?Sociodemographic Distribution of the CPWL and Populations Compared With the Total Adult U.S. Population Prevalence of Comorbid Health Conditions and ORs for Activity Limitations/Participation Restrictions Obese (BMI ≥ 30) 1.23 (1.05−1.44) Abbreviations: SE, standard error; N/A, not applicable. Complex samples logistic regression model included sex, age, ethnicity, education, marital status, BMI, and region, as well as all other chronic health conditions.Pain Characteristics in the CPWL and HICP Populations [/table]
[table] Table 5 Table 6: Activity Limitations/Participation Restrictions in the HICP PopulationACTIVITY LIMITATIONS (UNABLE TO DO THE ACTIVITY) HICP ESTIMATED NUMBER (MILLIONS) PREVALENCE, %(SE) Abbreviation: SE, standard error. Mental Health Status, General Health Status, and Health Care Use in the CPWL and HICP Populations [/table]
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Tobramycin exposure from active calcium sulfate bone graft substitute
Background: Bone graft substitute such as calcium sulfate are frequently used as carrier material for local antimicrobial therapy in orthopedic surgery. This study aimed to assess the systemic absorption and disposition of tobramycin in patients treated with a tobramycin-laden bone graft substitute (Osteoset® T). Methods: Nine blood samples were taken from 12 patients over 10 days after Osteoset® T surgical implantation. Tobramycin concentration was measured by fluorescence polarization. Population pharmacokinetic analysis was performed using NONMEM to assess the average value and variability (CV) of pharmacokinetic parameters. Bioavailability (F) was assessed by equating clearance (CL) with creatinine clearance (Cockcroft CLCr). Based on the final model, simulations with various doses and renal function levels were performed. (ClinicalTrials.gov number, NCT01938417).
# Background
Since the end of the sixties, bone cement such as polymethyl methacrylate (PMMA) and bone graft substitute such as calcium sulfate are frequently used as carrier material for local antimicrobial therapy in orthopedic surgery for osteomyelitis, infected arthroplasty, soft tissue infections or prophylaxis. Unlike PMMA, calcium sulfate is resorbable, thus obviating the need for surgical removal. Osteoset® T (Wright Medical Technology Inc, Arlington, TN, USA) is one of these products; it contains 4% tobramycin sulfate in calcium sulfate pellets. Most data on Osteoset® T come from animal studies, where high local and low systemic tobramycin concentrations have been observed [bib_ref] The treatment of experimental osteomyelitis by surgical debridement and the implantation of..., Nelson [/bib_ref]. No pharmacokinetic (PK) observations with patients treated with Osteoset® T have been published so far, despite well known concentration-related potential toxicity of aminoglycosides. Only concentrations have been described in patients implanted with a tobramycinladen polymer carrier material (Simplex™ P Bone Cement with Tobramycin) showing high tobramycin levels at the operative site but low systemic absorption [bib_ref] The Pharmacokinetics of Simplex-tobramycin bone cement, Sterling [/bib_ref].
The aim of this study was to develop a population PK model for tobramycin in patients treated with an active calcium sulfate bone substitute and to predict tobramycin systemic exposure under various dose and renal function levels.
# Method
## Patients and sampling
The data were collected prospectively between October 2006 and March 2008 from all adult patients treated with Osteoset® T in our orthopedic surgery department. Osteo-set® T was implanted whenever estimated a useful adjunct to standard therapy for bone, soft tissues and prosthetic joint infections requiring surgical debridement. Either 10 g or 20 g of Osteoset® T with 4% tobramycin sulfate were implanted, representing 262 or 524 mg tobramycin repectively taking into account the salt factor (tobramycin/tobramycin sulfate = 0.655). Additional intravenous aminoglycosides were not used in these patients. No wound drains were used.
Blood samples were taken at 3 h, 6 h, 12 h, 24 h, 48 h and on days 3, 5, 7 and 10 after implantation. Whenever a tourniquet had been used during the operation, its release-time was taken into account instead of implantation time. Blood was sampled in BD Vacutainer® SST II Advance (Becton Dickinson AG, Allschwil, Switzerland) serum separator tubes.
The study protocol was approved by the Institutional Review Board of Fribourg Cantonal Hospital and all subjects gave their written informed consent. This study was registered in ClinicalTrials.gov under code number NCT01938417.
## Drug assay
Tobramycin concentrations were measured on a COBAS INTEGRA 800 (Roche Diagnostics GmbH, Mannheim, Germany) fluorescence polarization detector, using a standard tobramycin measurement kit (ref. 20737925). The lower limit of quantification (LOQ) of the assay was 0.1 mg/L. Coefficient of variation (CV) within runs was 1.7% at 2.2 mg/L, while CV between runs were 6.2%, 2.6% and 2.5% at 0.9 mg/L, 2.8 mg/L and 4.5 mg/L, respectively. Accuracy was monthly checked by participating to the external quality control scheme provided by LGC Standards for antibiotics (LGC Standards Proficiency Testing, Bury, UK). Assays were processed within 6 hours after sampling.
# Population pharmacokinetic analysis
The pharmacokinetic analysis was performed using nonlinear mixed-effects modeling with the NONMEM computer program (version VI). Models were fitted to the data with the first-order conditional estimation with interaction (FOCE INTER) method. One and two compartment models with first-order absorption were compared. The first tobramycin concentration below the LOQ was set to half of the LOQ value and subsequent points below the LOQ were dropped out. Average value and variability of clearance (CL), volume of distribution (V) and absorption rate constant (k a ) were assessed. As aminoglycosides are known to be completely and exclusively eliminated by glomerular filtration, tobramycin CL was equated to the creatinine clearance (CL cr ) value, thus enabling the estimation of absolute bioavailability (F) in the absence of intravenous injection. CL cr was estimated with the Cockcroft formula, based on patient's serum creatinine, body weight, and sex [bib_ref] Prediction of creatinine clearance from serum creatinine, Cockcroft [/bib_ref] , with linear interpolation for days without creatinine measurement. The residual variability was adequately modeled using an additive plus proportional error model. The final model was then developed by testing in a stepwise fashion the potential influence of sex, age, body weight, Osteoset® T quantity and implantation site on the pharmacokinetic parameters.
Based on the final average parameters and variability, simulations using various tobramycin doses (262 mg; 786 mg; 1310 mg) at various CL cr levels (120, 90, 60, 30, 20 and 10 mL/min) were performed, using 2000 virtual patients each to predict the doses associated with concentrations exceeding the 2 mg/L threshold over a prolonged period of time (5 days or more).
# Results
Twelve patients (7 males, 5 females) were included in this observational study. Their mean age was 52 years (range 19-82, SD ± 20), body weight 73 kg (53-116, ± 17), and CL cr 119 mL/min (34-288, ± 55).
Osteoset® T implantation sites were tibia/fibula (6 patients), hip (2), calcaneum (2), femur (1) and lumbar spine (1), either for established infections (9) or prophylaxis (3). Eight patients were implanted 10 g Osteoset® T containing 262 mg tobramycin, while 4 were implanted 20 g Osteo-set® T containing 524 mg tobramycin.
Osteoset® T was well tolerated; further clinical details have been presented elsewhere [bib_ref] Systemic exposure to tobramycin after local antibiotic treatment with calcium sulfate as..., Wahl [/bib_ref]. Systemic tobramycin concentrations measured remained low (<2 mg/L at 24 h) in all patients. Concentration profiles were consistent with a one compartement model with absorption rate-limited first-order release, while showing important variability. A two compartment model was also tested but did not improve the fit. With CL equated to CL Cr , mean estimated k a was 0.0603 h −1 , volume of distribution 16.6 L (CV 89%) and F was 63% and 32% (CV 74%) in the 8 patients having received 10 g Osteoset® T and in the 4 patients having received 20 g, respectively. Sex, age, body weight or implantation site did not seem to affect tobramycin absorption or disposition when introduced as covariates. Population pharmacokinetic parameter estimates and variability are summarized in [fig_ref] Table 1: Tobramycin population pharmacokinetic parameter estimates CL, mean apparent clearance [/fig_ref]. The concentration values observed are represented on [fig_ref] Figure 1: Concentrations versus time plot of tobramycin with mean population prediction [/fig_ref] , along with mean population prediction and 90% prediction intervals for CL Cr 120 mL/min.
The simulations of concentration profiles (using an average F estimate of 47%) showed that more than 5% patients would maintain concentrations over 2 mg/L during at least five days post implantation with tobramycin 262, 786 and 1310 mg when CL cr was respectively set at 10, 20 and 30 mL/min.
# Discussion and conclusions
This is the first tobramycin pharmacokinetic study after Osteoset® T implantation in a clinical setting. Tobramycin systemic concentration values were measured well below the traditional toxicity threshold of 2 mg/L from 24 h and later on, which was not unexpected considering the low tobramycin dose implanted and the good renal function of the study subjects. Whether continuous systemic exposure to low concentrations below minimal inhibitory concentration may favor antimicrobial resistance remains an issue [bib_ref] Once-daily dosing of aminoglycosides: review and recommendations for clinical practice, Freeman [/bib_ref].
Prominent inter-individual variability was observed, probably due to heterogeneity of patients, indications, surgical techniques and implantation sites. There have probably been too few patients included for reliably testing the influence of all covariates. Loss through wound oozing has been neglected (not measured) but could represent another source of variability. Imprecision on low tobramycin levels measurement, very close to the LOQ, could also have increased residual variability.
The significant difference in bioavailability between 10 g and 20 g Osteoset® T (63% versus 32% respectively) could indicate that higher amounts of bone graft substitute either limit tobramycin release to some extent, or slow it down sufficiently for the resulting concentrations to fall below the LOQ. Unbalanced loss through wound oozing could also partly account for this difference. Implantation site and tissue perfusion could also play a role, although this could not be demonstrated in our analysis, due to limited data.
Our model gained in credibility when tobramycin CL was equated to CL cr ; indeed there is no argument to think of a different elimination route for the drug absorbed from Osteoset® T compared to intravenous delivery. The Cockcroft equation that we used has yet its limitations, in particular in obese or bedridden patients. Our model found a distribution volume of 0.22 L/kg body weight, consistent with previously published values. The large inter-patient variability found in both V and F is likely to incorporate some amount of variability in CL and k a .
High sustained potentially toxic concentrations were predicted by simulations with standard doses Osteoset® T at renal failure stages 4 and 5. Considering the limited study power, this simulation should be taken as a rough estimate of the potential for toxicity of this product. Our model is probably too imprecise to deduce precise dose adjustment guidelines. However, it indicates that caution is warranted when Osteoset® T implant is considered for patients with severe renal failure, as its benefit/risk ratio could turn out unfavourable [bib_ref] A case of an acute kidney injury secondary to an implanted aminoglycoside, Wu [/bib_ref].
[fig] Figure 1: Concentrations versus time plot of tobramycin with mean population prediction (solid line) and 90% prediction interval (dashed lines) after Osteoset® 10 g (left) or 20 g (right) in patients with normal renal function. Dose difference is almost fully compensated by different bioavailability, giving similar predictions. Superimposed points show the concentrations observed in 8 patients after 10 g and 4 patients after 20 g. v : first concentration below the limit of detection. [/fig]
[table] Table 1: Tobramycin population pharmacokinetic parameter estimates CL, mean apparent clearance; V, mean apparent volume of distribution; k a , mean absorption rate constant; F, mean bioavailability. estimates of variability expressed as coefficient of variation (CV%). s.e = standard error of the estimates (S.E), defined as S.E/estimate and expressed as percentage.c s.e = standard error of the coefficient of variations, taken as ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi s:e estimate =estimate p , expressed as a percentage. [/table]
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Hydrogenated vacancies lock dislocations in aluminium
Supplementary Figure 1. Process to achieve individual-dislocation configuration through 2 small-strain-amplitude cyclic compression. (a) As-fabricated pillar contains high density of 3 dislocations. (b) After 339 cycles of cyclic compression with peak stress of 330 MPa, the 4 dislocation density dramatically decreased. (c) After another 355 cycles of cyclic compression 5 with peak stress of 250 MPa, only a few long dislocations remained inside the pillar. (d) The 6 diffraction pattern of the single crystal aluminum pillar, for which the loading axis is inclined to 7 the [11 0] direction by 7°. (e) Schematic of the mobile dislocations in (c) and one of the possible 8 slip system, for which the Schmidt's factor M is calculated to be 0.070. All scale bars represent 9 200 nm. 10 11 2 12 Supplementary Figure 2. The threshold loads for each dislocation determined in the first 13 loading cycle in vacuum. (a) Load vs. time curve of the slow leading cycle. The triangular 14 markers correspond to the threshold loads of each dislocation to be activated. (b) The original 15 configuration of the dislocations in the pillar before loading. (c), (d), and (e) show the abrupt jump 16 of dislocations #2, #3 and #5 at critical loading stresses of 99, 125, and 213 MPa respectively. The 17 red lines and white lines are the dislocation profiles before and after the jump. For more details see 18 supplementary Movie 1. All scale bars represent 200 nm. 19 20 109 Critical loading stress for dislocation #1, #3, and #5 are 99 MPa, 125 MPa, and 213 110 MPa respectively. 111 112 Supplementary Note 2: Monotonic compression 113 Four typical engineering stress-strain curves from monotonic compression tests are 114 shown in Supplementary Fig. 3. Before the stress reaches 200 MPa, the curve looks 115 linear without obvious yielding or strain burst. But when correlated with the movie 116 frames, discrete movements of preexisting dislocations can be observed. These 117 preexisting dislocations in the as-fabricated pillars mainly result from FIB damage in 118 the milling process, and are pinned by obstacles such as surface junctions, point 119 defects and other dislocations. As the applied load increases, dislocations start 120 breaking away from pinning points, exhibiting abrupt movements. After depinning, 121 186 the two maps match relatively well. The tensile region corresponds to a much lower 187 energy state, where most of the hydrogen atoms distribute. The most energy-favored 188 sites for hydrogen are right in the dislocation core region. The hydrogen concentration 189 around dislocation line is characterized with N H /L, where N H is the number of 190 hydrogen atoms and L is the length of the dislocation line 8 . After adding hydrogen 191 atoms, the system was further relaxed at 300 K for 100 ps using a Nosé-Hoover 192 thermostat 9, 10 . Finally shear strain was applied in the xz-plane to drive dislocation 193 glide along the x direction. The strain rate was on the order of 10 8 s -1 . The whole 194 calculations were carried out using the LAMMPS code 11 and the atomic 195 configurations were displayed via AtomEye 12 .196
aluminum and (b) total energy difference of hydrogen (ΔE H,i ) in interstitial sites around the 54 dislocation core. Generally, interstitial hydrogen in the region with minus hydrostatic stress 55
(tension) has a smaller value of ΔE H,i and that in the region with positive hydrostatic stress 56
(compression) has a larger value of ΔE H,i . The tensile region around dislocation core corresponds 57
to the most energy-favored sites for hydrogen atoms. , i.e. for each following cycle, the bow out extent of each dislocation remained 106 the same at each peak stress.
## 107
In
[fig] 21 Supplementary, Figure 3, Figure 4, Figure 5, Figure 6: Monotonic compression of aluminum pillars in vacuum and 22 hydrogen gas. (a) Engineering stress-displacement curves showing similar yield stress and flow 23 stress for tests in vacuum and in hydrogen. The inset illustrates the experimental setup. (b) The 24 critical stress (σ c ) for dislocation depinning events in various samples, showing that a higher stress 25 is required to set off dislocation motion in hydrogenated samples. Scale bar in inset in (Hydrogen effect on dislocation motion during bending tests. (a) 30 Bright field TEM image of a typical bending cantilever containing dislocations and other defects. 31 (b) The corresponding diffraction pattern showing the single crystal nature of the cantilever. (c) 32 and (d) are the magnified area in (a) showing two typical dislocation profile at the given moments 33 before and after sudden movements. (e) Force vs. displacement curves of two typical samples with 34 similar dimensions but tested in vacuum and hydrogen environment, respectively. The first 5 35 dislocation events of each test are labeled on the curve with triangle marks. All scale bars 36 represent 100 nm. Destruction of dislocation configuration due to bucking during the 41 test in 2 Pa H 2 . (a) The dislocation configuration inside the pillar remained unchanged after 85 42 cycles. (b) Due to the tip drift, the pillar was buckled. The dislocation configuration underwent a 43 sudden change under the coupling of loading stress and buckling stress. Note pillar contour 44 change before drift (red dashed line) and after drift (red solid line), as well as the change of black 45 contrast inside the pillar due to buckling. (c) When the tip is detached from the pillar, the pillar 46 contour recovered but the dislocation configuration can't be resumed. All scale bars represent 200 47nm. The distribution maps of (a) hydrostatic stress of edge dislocation in 53 [/fig]
[fig] Figure 7, Figure 8, Figure 9: Atomistic simulations based on the model with hydrogen atoms in 61 a Boltzmann distribution. (a) Stress-strain curves at different hydrogen concentration levels. The 62 critical shear stress (τ c ) for dislocation motion shows a linear increase with the increase of 63 hydrogen concentration (N H /L), as shown in the inset. (b) Initial configuration of dislocation 64 decorated with hydrogen atoms. (c) The configuration at the depinning state. Atoms with golden 65 and black colors refer to aluminum and hydrogen. The distribution maps of (a) hydrostatic stress of edge dislocation in 69 aluminum and (b) total energy difference of VaH complex ( MD simulations of dislocation pinning effect by hydrogen-vacancy 74 complex. The different colored lines show the stress-strain curves at different concentrations of 75 hydrogen-vacancy complex. The critical shear stress (τ c ) for dislocation motion shows a linear 76 increase with the increase of hydrogen-vacancy complex concentration (N VaH /L), as shown in the 77 inset. 78 79 Supplementary [/fig]
[fig] Supplementary Note 5 Supplementary Note 7: three dislocations show abrupt position change during the 108 loading, whereas the two other dislocations only show smooth position change. they either vanish at surfaces or get stopped at a stronger pinning point. We have 122 tracked all the observed depinning events and their corresponding critical stresses, and 123 found that the lowest depinning stress increases from ~40 MPa to ~90 MPa when 124 changing the testing environment from vacuum to the 2 Pa hydrogen gas, as 125 demonstrated in Supplementary Fig. 3b. 126 From the engineering stress-displacement curves, monotonic compressions in 127 hydrogen environment show similar stiffness and strain hardening behavior when 128 compared to compressions in vacuum. In these tests, the profile of stress-strain curve 129 is mainly governed by sample-size dependent dislocation nucleation and 130 multiplication. In Supplementary Fig. 3a, H 2 #2 specimen show lower stiffness than 131 the other three pillars due to the slight misalignment and initial point contact at the 132 beginning of the compression test.133 134 Supplementary Note 3: Hydrogen effect on dislocation motion in bending tests 135 8 smooth cantilevers with width ranging from 222 nm to 292 nm and thickness from 136 600 nm to 642 nm were bent inside the ETEM. All the cantilevers (e.g. 137 Supplementary Fig. 4a) contained preexisted dislocations resulting mainly from FIB 138 milling. The dislocation density was estimated to be between 10 13 m -2 and 10 14 m -2 . 139 Monotonic bending loads were applied to the cantilevers at loading rate of 5 nm/s 140 under displacement control mode. Total 8 samples were tested, 4 in vacuum and 4 in 141 hydrogen environment. Similar to that observed in monotonic compression tests 142 (Supplementary Fig. 3), the threshold loads for dislocation activation in the hydrogen 143 atmosphere are always higher than those tested in vacuum. One typical comparison is 144 shown in Supplementary Figure 4e. It again demonstrates that hydrogen can improve 145 the threshold loads significantly for those preexisted dislocations. Supplementary Note 4: Reconfiguration of dislocations due to buckling/bending 148 induced by probe drift 149 Since a typical cyclic loading test lasts 40 to 100 seconds, the diamond punch may 150 undergo obvious drift in all three space coordinate directions. Under the load control 151 system, drift in the indentation direction can be automatically compensated by the 152 feedback control system so as to ensure a correct loading force applied to the pillar.153 However, drifts perpendicular to the indentation axis can buckle the samples 154 obviously due to the friction confinement at the contact interface between the 155 diamond tip and the samples. This will apply addition stress to the dislocations. One 156 typical example is shown in Supplementary Fig. 5. The sample was bended/buckled 157 obviously (Supplementary Fig. 5b) during the cyclic loading, as suggested by the 158 profile deviation (red dashed lines) and the significant contrast change. New 159 dislocations coming out from the bottom disrupted the dislocation configuration 160 inside the pillar. Interestingly, the sample can go back to its original position after the 161 diamond probe was retracted despite that the dislocation configurations had been Evaluation of Al-H angular dependent potential 165 To provide more details about the empirical angular-dependent potential (ADP) 6 we 166 used in MD simulations, here we show various properties predicted by this potential 167 in comparison to the experiments and ab initio calculations, especially some 168 energetics related to the point defects (H, Va and VaH), as shown in the following 169 three Tables 1, 2 and 3. Among them, most has a relatively good match with the 170 referenced data, but unfortunately, the binding energy of VaH is very small. More 171 information for this potential is shown in refs 6, 7. Supplementary Note 6: Molecular dynamics simulation of the influence of 174 hydrogen atmosphere on dislocation motion 175 We first created the a/2<110>-type edge dislocations in Al. The simulation box was 176 oriented along x-[10 1], y-[111], z-[121] , with dimensions of 23nm × 28nm × 16nm. 177 Periodic boundary conditions were applied in the x and z directions. The hydrogen 178 atoms were then introduced in the interstitial tetrahedral sites around the dislocation the total energy difference of hydrogen in site i ( H,i E ) 182 with reference to that in bulk tetrahedral interstitial site ( Bulk H,i E ), k B is Boltzmann 183 constant and T is absolute temperature. Supplementary Fig. 6a shows the distribution 184 of hydrostatic stress of the edge dislocation and Supplementary Fig. tetrahedral site around the dislocation core. We can see that value of critical shear stress for edge dislocation motion increases in hydrogen 199 environment. The dependence of locking strength on hydrogen concentration is 200 studied by conducting a serial of simulations with different N H /L. The inset in 201 Supplementary Fig. 7a shows the variation of critical shear stress (τ c ) with different 202 amount of N H /L. We find that the value of τ c increases almost linearly with hydrogen τ 0 (= 15 MPa) is the critical shear stress for dislocation motion without 206 hydrogen and the coefficient α is 11.7 MPa·nm in the present Al-H system. It is 207 interesting to observe that even hydrogen with a density of N H /L = 1 nm -1 could 208 double the τ c . Supplementary Fig. 7b shows the initial configuration of dislocation 209 decorated with hydrogen atoms at the level of N H /L = 3 nm -1 . When the shear stress 210 reaches the critical value, the dislocation suddenly escapes from the hydrogen 211 atmosphere, as illustrated in Supplementary Fig. 7c. 212 Our MD simulations reveal in a straightforward manner that hydrogen atoms could 213 enhance the critical stress for dislocation motion. The interaction between dislocation 214 and diffusing solute hydrogen atoms can lead to locking effect, similar to that in strain Molecular dynamics simulation of the influence of 218 hydrogen-vacancy complexes on dislocation motion 219 [/fig]
[table] Table 1: Energetics of isolated H atom in Al. ΔE s refers to the solution energy of 80 H in Al. T and O refer to the tetrahedral (T) interstitial and octahedral (O) interstitial sites. E m 81 refers to the migration barrier of one H atom from a T-site to the O-site. The units are in eV. 82 Supplementary Table 3. Energetics of VaH. E f (T-Va) refers to the formation energy of vacancy 91 with H in T-site. E f (O-Va) refers to the formation energy of vacancy with H in O-site. E b (T-Va) 92 refers to the binding energy of vacancy with H in T-site. The units are in eV. 93Supplementary Note 1: Mechanical annealing by cyclic compressionThe loading function consists of a slow leading cycle and tens of fast trailing cycles in each group of test. In the loading half-cycle, each dislocation marches forward, like a bending bow, with both ends pinned on surface; upon unloading, each swings back to the original location (see supplementary Movie 1).The initial compression tests were conducted with peak stress of ~330 MPa. By the 101 end of 339 th cycle (N339), only a few isolated mobile dislocations were left, with their 102 both ends pinned by the surface of the sample(Supplementary Fig. 1b). In order to [/table]
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Management of tumor rupture and abdominal compartment syndrome in an infant with bilateral high risk stage 4 neuroblastoma
Rationale: Tumor rupture and bleeding at initial presentation of infants with neuroblastoma (NBL) is a rare, but life threatening condition and challenge in pediatric oncology. Here, we report successful multidisciplinary management of an abdominal compartment syndrome as a result of tumor rupture and bleeding in an infant with bilateral high risk stage 4 NBL.Patient concerns: The patient was admitted to a cooperating hospital with vomiting, failure to thrive and a large mass in the abdomen and was then referred to our center.Diagnoses: Stage 4 NBL with MYC-N amplification and 1p36 deletion was diagnosed in an 11 months old girl. Due to rapid and massive tumor growth she developed abdominal compression with renal failure, severe bleeding, and tumor lysis syndrome (TLS).Interventions: Surgical decompression by enterostomy, local, and systemic bleeding control with platelets and coagulation factors, antiinfective and TLS therapy were effective in stabilizing the patient's condition. This allowed initiation of the multimodal antineoplastic treatment according to protocol NB 2004.Outcomes: Mechanical ventilation was stopped after 11 days, the abdominal wall was closed 3 months after the start of therapy, and treatment according to the protocol be started and successfully completed.Lessons: Only the immediate, coordinated multidisciplinary intervention managed to overcome the life-threatening abdominal compartment syndrome and its associated problems, eventually enabling successful curative treatment.Abbreviations: ACS = abdominal compartment syndrome, ASCT = autologous stem cell transplantation, GD2 = disialoganglioside GD2, IAP = intraabdominal pressure, MIBG = meta-iodo-benzylguanidine, MRI = magnetic resonance imaging, MYC-N = V-myc myelocytomatosis related oncogene, neuroblastoma derived, NB = neuroblastoma, NB2004 = German neuroblastoma protocol, VGPR = very good partial remission.
# Introduction
Neuroblastoma (NB) is the most common malignant solid tumors of infancy and childhood. The overall survival rate of stage 4 patients still remains poor despite intensive multimodal treatment regimens.In 40% to 50% of patients NB arise in the adrenal glands.Spontaneous abdominal hemorrhage within the adrenal glands may be a leading sign for NB and occurs particular during the neonatal period,less frequently in older children.Massive intraabdominal hemorrhage resulting in an abdominal compartment syndrome due to tumor rupture is rare and life-threatening.Here we report on an infant with bilateral high-risk stage 4 neuroblastoma (1p36 deletion, MYC-N amplification) presenting with tumor rupture and abdominal hemorrhage. Only by immediate, coordinated interdisciplinary action the life-threatening abdominal compartment syndrome and its associated problems could successfully be managed, eventually enabling curative treatment.
## Case
Parents provided written consent to report the case of their child. A Caucasian girl (11 months) was admitted to a local hospital with vomiting, failure to thrive (development of body weight, age, weight, percentile: 10 days, 3320 g, P50; 1 month, 4050 g, P50; 3 months, 5090 g, P25; 6 months, 5970 g, P3; 11 months, 6320 g, <<P3) and loss of milestones (stopped walking). Physical examination revealed a solid resistance in the right upper abdomen, and 2 massive bilateral solid tumors were diagnosed by ultrasound. Initial laboratory findings were as follows: Hemoglobin 7.8 g/dl, serum lactate dehydrogenase 3250 IU/L (<300 IU/L), uric acid 8.3 mg/dl (<5.0 mg/dl), neuron specific enolase 1084 mg/l (<16 mg/L), vanillylmandelic acid/creatinine 50 mmol/mmol (<10.7 mmol/mmol), homovanillylmandelic acid 75 mmol/mmol (<20.1 mmol/mmol). Bone marrow infiltration was not detectable by microscopy and anti-GD 2 immunocytochemistry (aspirates and biopsies from 4 locations; 2Â tibia, 2Â iliac crest). Magnetic resonance imaging (MRI) and scintigraphy with 123 I meta-iodo-benzylguanidine revealed bilateral NBoriginating from both adrenal glands with metastases to retroperitoneal lymph nodes and lesions of the skeleton at the left scapula and the right 4th rib.
During staging procedures, the child developed progressive tachypnea (>40/minute), tachycardia (>150 bpm), and a distended abdomen with bluish discoloration of the skin. At the same time urine output decreased (<0.5 ml/ kg/hour), and a gradual increase of the intragastric pressure (max. 17 cmH 2 O) established the diagnosis of an abdominal compartment syndrome (ACS). On ultrasound, the abdominal blood vessels (vena cava, abdominal aorta) were compressed, and free abdominal fluid was detectable. Based on the deteriorating clinical condition, the sustained rise of the intraabdominal pressure (IAP) together with the newly developed organ dysfunction it was decided to perform an enterostomy in order to decompress the abdominal cavity Steinau. Laparotomy revealed a tumor rupture with extensive bleeding located at the right abdominal component of the primary tumorwhich infiltrated the entire right hepatic lobe. A tumor biopsy was taken from the smaller left sided primary tumor, and a central line was implanted (dual lumen 6,6fr broviac catheter). Closure of the abdominal cavity was not possible due to the huge ruptured tumor infiltrating the liver.
Small fragments of the biopsy were cultured in RPMI containing 10% human serum in a standard incubator (5% CO2, 100% H 2 O saturation) yielding a stable and rapidly growing cell line. The cells expressed ganglioside GD 2 and were MYC-N amplified, which are typical features of neuroblastoma cells. Histology of the tumor revealed an undifferentiated stroma-poor NB Sanowith MYC-N amplification (20-40 copies) and 1p36 deletion establishing the diagnosis of a high risk stage 4 NB Monclairin an infant less than twelve months of age.
During the first postoperative days the bleeding of the ruptured right sided tumor continuedrequiring immediate interdisciplinary action.
First, hematologists and blood bank were required to substitute blood loss by continuous infusion of red blood cells (40 ml/hour) to maintain hemoglobin levels above 8 g/dl, fresh frozen plasma (10 ml/kg q8 hour) to keep fibrinogen above 1 g/L and platelets to keep thrombocytes above 50,000/ml with focus on homeostasis of electrolytes, blood glucose, and fluid balance. In addition, coagulation factors had to be substituted (activated factor VII, prothrombin complex, factor XIII, anti-thrombin III, and, at the same time fibrinolysis was inhibited with tranexamic acid.
In parallel, pediatric surgeons performed surgery to control bleeding in ruptured tumor areas through electrocoagulation and the use of TachoSil, a topical fibrin-based hemostatic agent.
Chemotherapy according to the German neuroblastoma protocol (NB2004) was started by pediatric oncologists, at first with chemotherapy (element N5) consisting of vindesin (0.1 mg/ kg, day 1), cisplatinum (1.3 mg/kg, day 1-4) and etoposide 4.2 mg/kg, day 1-4) followed by GCSF support.
At the same time, radiotherapists began irradiating the rightsided primary tumor at low dose (5 Â 1 Gy) to accelerate and assist cytoreductive therapy, thereby reducing intratumoral pressure.
In parallel, the infection team delivered a preventive antiinfective therapy with broad spectrum antibiotics and antimycotics. During the entire course of the treatment no evidence of bacterial, fungal, or viral infection was found.
Tumor lysis syndrome with hyperuricaemia was treated with fluids (3l/m 2 body surface area), electrolytes, and furosemide in combination with rasburicase.
This multidisciplinary management was finally effective and successful. After 6 days, the bleeding was stopped, and the enterostomy could be closed by inserting a gore-tex patch accompanied by vacuum dressing. Intensive care management also included cardiovascular therapy (noradrenaline and dobutamine) and thyroxine against low T 3 syndrome. Respirator therapy was discontinued after 11 days and the tumor lysis syndrome resolved gradually over a period of 2 weeks.
Chemotherapy was continued according to protocol NB2004 for high risk stage 4 neuroblastoma patientswith alternating N5/N6 chemotherapy cycles (N5 see above, N6: vincristine 0.05 mg/kg day 1 and 8, dacarbacine 6.7 mg/kg day 1-5, ifosfamide 50 mg/kg day 1-5, adriamycin 1 mg/kg/day 6 and 7). Between the courses peripheral blood stem cells were collected for the planned high-dose chemotherapy. Surgically, 6 successive reductions of the gore-tex patch were performed in weekly intervals followed by sequential resections of the left and right primary tumor. Finally, 3 months after the start of therapy the abdominal wall could be closed. Magnetic resonance imaging and mIBG scans indicated a very good partial remission (VGPR). The little girl was in a good general condition, on oral feeding and gradually gaining weight and could be prepared for high dose chemotherapy followed by autologous stem cell rescue.
# Discussion
The typical manifestation of NB in newborns and infants is stage 4s which is associated with a favorable outcome. The outcome related to survival of the neuroblastoma remains favorable even if laparostomy has been required in patients with rapidly growing tumors due to liver metastases resulting in an abdominal compartment syndrome (ACS).Nevertheless, mortality from ACS ranges from 50% to 60%, and early decompression by enterostomy has been described to improve outcome.Another specific and in part lethal problem in newborns and young infants with NB may be disseminated intravascular coagulation.In contrast, true high-risk metastatic NB with MYC-N amplification, 1p36 deletion, and unfavorable histology in infants is rare. The aggressiveness of the NB of patient reported here is also documented by the rapid growth of tumor cells in vitro. With these cells, a new neuroblastoma cell line (HGW-CM) could be established expressing ganglioside GD 2 and sharing myc-n amplification as well as 1p36 deletion like the parental tumor.
In a report from the Neuroblastoma Group of the International Society of Paediatric Oncology Europe (SIOPEN) MYC-N amplified NB accounted for only 10% of affected infants.Here, infants with MYC-N amplified NB had a poor outcome Published case reports are scarce on infants with high-risk NB and abdominal compartment syndrome. The patient reported here was complex with 4 life threatening conditions including high-risk NB, tumor rupture, and abdominal compartment syndrome followed by tumor lysis syndrome and severe bleeding problems.
Immediate interdisciplinary well coordinated parallel interventions of a variety of pediatric subspecialties including pediatric surgery, pediatric oncology, radiotherapy, transfusion medicine, infectiology, and supportive therapy was key to successful management of this complex condition.
Very important was an early decompression to reinstitute renal function as it is essential for the timely clearance of cytotoxic agents as well as for the management of the tumor lysis syndrome. It is speculative, which further intervention was most important, but the outcome demonstrates impressively that such conditions require the full commitment of all disciplines involved.
In conclusion, timely enterostomy is capable of effectively bridging a life threatening abdominal compartment syndrome in neuroblastoma to make subsequent curative multimodal treatment possible.
Author contributions |
Association between diabetic eye disease and other complications of diabetes: Implications for care. A systematic review
Funding informationSupport for this project was provided by the Vision Academy, a Bayer-sponsored initiative comprising over 70 leading retinal specialists.The aim of this systematic review was to examine the associations between diabetic retinopathy (DR) and the common micro-and macrovascular complications of diabetes mellitus, and how these could potentially affect clinical practice. A structured search of the PubMed database identified studies of patients with diabetes that assessed the presence or development of DR in conjunction with other vascular complications of diabetes. From 70 included studies, we found that DR is consistently associated with other complications of diabetes, with the severity of DR linked to a higher risk of the presence of, or of developing, other micro-and macrovascular complications. In particular, DR increases the likelihood of having or developing nephropathy and is also a strong predictor of stroke and cardiovascular disease, and progression of DR significantly increases this risk. Proliferative DR is a strong risk factor for peripheral arterial disease, which carries a risk of lower extremity ulceration and amputation.Additionally, our findings suggest that a patient with DR has an overall worse prognosis than a patient without DR. In conclusion, this analysis highlights the need for a coordinated and collaborative approach to patient management. Given the widespread use of DR screening programmes that can be performed outside of an ophthalmology office, and the overall cost-effectiveness of DR screening, the presence and severity of DR can be a means of identifying patients at increased risk for microand macrovascular complications, enabling earlier detection, referral and intervention with the aim of reducing morbidity and mortality among patients with diabetes. Healthcare professionals involved in the management of diabetes should encourage regular DR screening.K E Y W O R D Sdiabetes complications, diabetic retinopathy
# | introduction
Diabetic retinopathy (DR) is the most common complication of diabetes and remains the leading cause of blindness among working-age individuals in most developed countries. 1,2 DR has long been considered a microvascular complication of diabetes; however, growing evidence suggests that abnormalities in retinal function can be detected in patients without any evidence of microvascular abnormalities, and the American Diabetes Association defined DR as a highly specific neurovascular complication. [bib_ref] European Consortium for the Early Treatment of Diabetic Retinopathy (EUROCONDOR), Simó [/bib_ref] [bib_ref] Diabetic retinopathy: a position statement by the American Diabetes Association, Solomon [/bib_ref] DR is characterized by degeneration of endothelial cells and pericytes in retinal capillaries, which leads to ischaemia and the formation of microaneurysms. [bib_ref] Diabetic retinopathy: targeting vasoregression, Hammes [/bib_ref] In the advanced stage of the disease, pathologic proliferation of retinal vessels occurs via upregulation of proangiogenic mediators, particularly vascular endothelial growth factor (VEGF). [bib_ref] Diabetic retinopathy: targeting vasoregression, Hammes [/bib_ref] The resulting alterations of the retinal microvasculature and increased leakage from the retinal vessels may lead to sight-threatening vision loss. [bib_ref] Diabetic retinopathy: targeting vasoregression, Hammes [/bib_ref] As the number of adults with diabetes worldwide is projected to increase from 415 million to 642 million by 2040, 6 DR represents a significant global health threat. According to recent estimates, DR accounts for 2.6 million cases of moderate to severe vision impairment worldwide, and this figure is expected to rise to 3.2 million by 2020. [bib_ref] Global causes of blindness and distance vision impairment 1990-2020: a systematic review..., Flaxman [/bib_ref] A pooled analysis of data from 35 studies conducted in the United States, Europe, Asia and Australia estimated that in 2010, worldwide, the number of patients with diabetes who had DR or vision-threatening DR (VTDR) was 92.6 and 28.4 million, corresponding to prevalence rates of 34.6% and 10.2%, respectively. [bib_ref] Global prevalence and major risk factors of diabetic retinopathy, Yau [/bib_ref] The global number of patients with diabetes who have retinopathy in the general population is projected to reach 191 million by 2030. [bib_ref] The worldwide epidemic of diabetic retinopathy, Zheng [/bib_ref] Longer duration of diabetes is a major risk factor for DR. [bib_ref] The Wisconsin epidemiologic study of diabetic retinopathy. II. Prevalence and risk of..., Klein [/bib_ref] One study estimated that individuals who have type [bib_ref] Global prevalence and major risk factors of diabetic retinopathy, Yau [/bib_ref] 96% CI, 7.10-10.63), compared with individuals who have type 2 diabetes mellitus (T2DM) for less than 10 years. [bib_ref] Global prevalence and major risk factors of diabetic retinopathy, Yau [/bib_ref] However, more recently, DR has also been reported with various prevalence rates in patients with newly diagnosed diabetes mellitus. [bib_ref] Prevalence and risk factors for diabetic retinopathy: the Korea National Health and..., Jee [/bib_ref] Treatment of DR significantly improved after the introduction of pharmacological agents with anti-VEGF activity. In phase III clinical trials of patients with diabetic macular oedema (DMO), a complication of DR, anti-VEGF therapy improved visual acuity from baseline and lowered the risk of progression to proliferative DR (PDR), indicating that VEGF inhibitors can interfere with the underlying pathogenesis of retinopathy. [bib_ref] Ranibizumab for diabetic macular edema: results from 2 phase III randomized trials:..., Nguyen [/bib_ref] [bib_ref] Intravitreal aflibercept for diabetic macular edema, Korobelnik [/bib_ref] However, delayed DR screening and detection can hamper treatment. In one UK study, there was a significant trend (P = 0.0004) relating time from diagnosis of T2DM to detection of worsening retinopathy at screening. [bib_ref] Delay in diabetic retinopathy screening increases the rate of detection of referable..., Scanlon [/bib_ref] The consequences of delayed detection of DR extend beyond suboptimal visual acuity outcomes; increasing evidence links retinopathy to other microvascular complications of diabetes, most notably nephropathy and cardiac autonomic neuropathy (CAN). [bib_ref] Determinants of development of microalbuminuria in normotensive patients with type 1 and..., Villar [/bib_ref] [bib_ref] Retinopathy as a predictor of other diabetic complications, El-Asrar [/bib_ref] [bib_ref] Risk factors for development of incipient and overt diabetic nephropathy in type..., Rossing [/bib_ref] [bib_ref] Diabetic retinopathy is strongly predictive of cardiovascular autonomic neuropathy in type 2..., Huang [/bib_ref] Retinopathy has also been linked to macrovascular comorbidities, including stroke, myocardial infarction and, potentially, heart failure. [bib_ref] Type 2 diabetes-associated carotid plaque burden is increased in patients with retinopathy..., Alonso [/bib_ref] [bib_ref] Diabetic retinopathy, its progression, and incident cardiovascular events in the ACCORD trial, Gerstein [/bib_ref] Additionally, a recent meta-analysis of epidemiological observational studies found that patients with diabetes who have DR are at increased risk of all-cause mortality, compared with patients with diabetes who do not have retinopathy (risk ratio [RR], 2.33; 95% CI, 1.92-2.81).With regard to the link between DR and cardiovascular events, Alonso et al. conducted a cross-sectional study of patients with T2DM who did not have renal deficits or prior history of cardiovascular disease (CVD). They found that the percentage of patients with carotid plaques, a risk factor for ischaemic stroke, 20 was higher among those with DR than among those without DR (68.0% vs 52.2%; P = 0.0045). [bib_ref] Type 2 diabetes-associated carotid plaque burden is increased in patients with retinopathy..., Alonso [/bib_ref] Other research has demonstrated that, not only is DR associated with future cardiovascular events, but also that the risk of these events increases with increasing severity of DR (hazard ratio
[HR], 1.49; 95% CI, 1.12-1.97 for mild retinopathy and HR, 2.35; 95% CI, 1.47-3.76 for severe retinopathy). [bib_ref] Diabetic retinopathy, its progression, and incident cardiovascular events in the ACCORD trial, Gerstein [/bib_ref] Similar associations have been observed between DR and cerebrovascular events. [bib_ref] Different risk factor profiles for ischemic and hemorrhagic stroke in type 1..., Hägg [/bib_ref] [bib_ref] Incidence of stroke according to presence of diabetic nephropathy and severe diabetic..., Hägg [/bib_ref] [bib_ref] Rates and predictors of risk of stroke and its subtypes in diabetes:..., Hankey [/bib_ref] Our analysis aimed to further elucidate the relationships between DR and micro-and macrovascular complications, and to identify any examples where the presence of DR increases the risk of having or developing other complications of diabetes. Irrespective of the directionality of any such relationships, that is, whether DR is antecedent to or develops after other complications, an increased awareness and understanding of these relationships can help promote timely referral and discussion between ophthalmologists and the other healthcare professionals involved in the management of these co-morbities, including diabetologists, cardiologists, vascular surgeons and nephrologists. There is currently a lack of communication between the diverse healthcare disciplines involved in the treatment of patients with diabetes, which can lead to a breakdown in the care pathway and contributes to suboptimal patient outcomes. DR screening can be performed effectively outside the ophthalmologist's office by trained personnel (eg, primary care physician's office, optometrist, mobile unit). Indeed, this approach is generally deemed more practical and efficient than screening at ophthalmology offices.The identification of patients with DR and a greater understanding of the wider relevance of DR may provide an opportunity to identify patients who are at increased risk of the presence or development of other diabetic complications, thus forming the basis for enhanced inter-disciplinary communication with the aim of improving patient outcomes.
## | materials and methods
## | research questions
The aim of this study was to answer the following research questions:
- Is there a link between DR and the micro-and macrovascular complications of diabetes?
- Does DR increase the risk of other complications of diabetes?
- What is the clinical significance of the associations between DR and other complications of diabetes?
## | literature search strategy
A literature search of the PubMed database was performed using a structured approach to identify relevant studies. The search term combination used was: ((((((diabetes) OR diabetic) OR diabetes mellitus)) AND (((retinopathy) OR eye disease) OR macular disease))) AND (((((((((neuropathy) OR neurological complication*)) OR ((((nephropathy) OR renal disease) OR microalbuminuria) OR kidney disease)) OR ((((((coronary heart disease) OR myocardial ischemia) OR cardiovascular disease) OR cardiovascular event*) OR myocardial infarction) OR atherosclerosis)) OR (((((((cerebrovascular accident) OR cerebral infarction) OR cerebral ischemia) OR cerebrovascular disease) OR stroke) OR cerebrovascular event*) OR transient ischemic attack)) OR ((((((((intermittent claudication) OR vasculopathy) OR peripheral artery disease) OR ulcer) OR gangrene) OR peripheral vascular disease) OR necrosis) OR amputation))) AND ((((((((((risk) OR risk factor) OR association) OR correlation) OR logistic regression) OR predictive) OR predictor) OR predictive factor) OR link) OR relationship)).
Diabetes search terms were restricted to title content, and the remaining search terms were limited to title and abstract. Only peerreviewed original research studies and reviews were included, and no geographical, date or language restrictions were applied.
## | study selection
## | data extraction
For the first stage of the selection process, identified titles were screened for relevance. If the publication passed this stage, the abstract was read to verify relevance, and if this was confirmed, the full text of the paper was assessed against the inclusion and exclusion criteria. Extracted information included study design, population characteristics, conclusions and limitations. Precedence was given to studies specifically designed to investigate the relationship between DR and other complications of diabetes, and to studies in which statistical analyses were adjusted for confounding factors.
# | results
## | study selection
Of 3028 potentially relevant papers initially identified through the PubMed search, 2418 were excluded because they were not relevant to the research questions. The abstracts of the remaining 610 papers were read, which led to the exclusion of an additional 330 papers. This resulted in a total of 280 papers of which the full text was assessed for relevance. Of these papers, 71 met the criteria for inclusion in the structured review.
## | dr and other microvascular complications of diabetes
A correlation was found between DR and other microvascular complications of diabetes, specifically, nephropathy and neuropathy. A summary of the findings of the reviewed studies that examined the relationship between DR and these microvascular complications is presented in Tables 1 and 2.
## | nephropathy
Diabetic nephropathy affects approximately 40% of patients with diabetes and is characterized by increased urinary albumin excretion (UAE). It is categorized into stages, microalbuminuria (UAE >20 μg/ min and ≤199 μg/min) and macroalbuminuria (UAE ≥200 μg/min),
and it is associated with increased mortality. [bib_ref] Diabetic nephropathy: diagnosis, prevention, and treatment, Gross [/bib_ref] Glomerular filtration rate (GFR), a measure of renal function, is also used to assess the presence of diabetic nephropathy. [bib_ref] Diabetic nephropathy: diagnosis, prevention, and treatment, Gross [/bib_ref] Overall, risk estimates from the reviewed studies suggest a correlation between DR and nephropathy, and between DR and declining renal function [fig_ref] TABLE 1: Studies describing the association between DR and nephropathy and renal decline [/fig_ref].
## Dr and albuminuria
In a cross-sectional study of patients with T1DM or T2DM, the presence and severity of DR were significantly predictive of nephropathy. [bib_ref] Retinopathy as a predictor of other diabetic complications, El-Asrar [/bib_ref] Multivariate logistic regression showed that nephropathy was the only complication of diabetes independently associated with DR, and that retinopathy increased the likelihood of developing nephropathy by 4.37 times. However, the predictive value of DR was strongest for T1DM. [bib_ref] Retinopathy as a predictor of other diabetic complications, El-Asrar [/bib_ref] These findings have been confirmed in other studies that identified DR as a significant independent predictor of progression to micro-or macroalbuminuria in patients with or T2DM [bib_ref] Prevalence and risk factors for microalbuminuria in a referred cohort of type..., Parving [/bib_ref]. Risk of nephropathy has been shown to increase with DR severity, [bib_ref] Retinopathy as a predictor of other diabetic complications, El-Asrar [/bib_ref] and PDR was identified as a significant independent marker of incident nephropathy in a 25-year follow-up study in T1DM. [bib_ref] Proliferative retinopathy predicts nephropathy: a 25-year follow-up study of type 1 diabetic..., Karlberg [/bib_ref] Interestingly, data from the Diabetes Control and Complications Trial (DCCT) in T1DM demonstrated that progression of DR independently increases the risk of development of nephropathy, and also showed that the development of nephropathy increases the risk of DR progression. [bib_ref] Concordance of retinopathy and nephropathy over time in type 1 diabetes: an..., Kramer [/bib_ref] Although a previous study demonstrated that these two complications influence the incidence of each other, 36 a retrospective study in T2DM demonstrated that the association between DR and diabetic nephropathy is unidirectional and that renal injury precedes retinal damage. [bib_ref] Link between retinopathy and nephropathy caused by complications of diabetes mellitus type..., Kotlarsky [/bib_ref] Accordingly, several studies have shown that nephropathy is a risk factor for DR, including severe forms of retinopathy. [bib_ref] Relationship between diabetic retinopathy, microalbuminuria and overt nephropathy, and twenty-year incidence follow-up..., Romero-Aroca [/bib_ref] [bib_ref] Risk factors for retinopathy and DME in type 2 diabetes-results from the..., Hammes [/bib_ref] [bib_ref] Diabetic retinopathy in patients with diabetic nephropathy: development and progression, Jeng [/bib_ref] [bib_ref] Urinary albumin excretion as a predictor of diabetic retinopathy, neuropathy, and cardiovascular..., Savage [/bib_ref] Concerning T1DM, a 20-year prospective study by Romero-Aroca et al. found that both micro-and macroalbuminuria correlated with the development of VTDR but not with the development of any DR. [bib_ref] Relationship between diabetic retinopathy, microalbuminuria and overt nephropathy, and twenty-year incidence follow-up..., Romero-Aroca [/bib_ref] In the same study, DR was a significant risk factor for nephropathy. [bib_ref] Relationship between diabetic retinopathy, microalbuminuria and overt nephropathy, and twenty-year incidence follow-up..., Romero-Aroca [/bib_ref] Notably, in a 10-year prospective analysis of patients with T1DM, macroalbuminuria was identified as a significant independent risk factor for the development of DMO, but not of DR. [bib_ref] Tenyear incidence of diabetic retinopathy and macular edema, Romero-Aroca [/bib_ref] Concerning T2DM, Hammes et al. analysed data from 6478 patients. The presence of microalbuminuria was found to independently increase the risk of DR, severe DR and DMO, and the risk further increased in the presence of macroalbuminuria. [bib_ref] Risk factors for retinopathy and DME in type 2 diabetes-results from the..., Hammes [/bib_ref] However, research has also shown that nephropathy did not affect development or progression of DMO, although it was an independent risk factor for DR, PDR and the progression from non-proliferative DR (NPDR) to PDR. [bib_ref] Diabetic retinopathy in patients with diabetic nephropathy: development and progression, Jeng [/bib_ref] Previously, macroalbuminuria, but not microalbuminuria, had been found to be significantly associated with increasing severity of DR and with progression from NPDR to PDR in T2DM. 32 Diabetic symmetric polyneuropathy is also a major risk factor for foot ulcers and amputations. [bib_ref] Diabetic neuropathy: clinical manifestations and current treatments, Callaghan [/bib_ref] Concerning CAN, reported prevalence rates among individuals with diabetes vary from 17% to 66% in T1DM, and from 31% to 73% in T2DM. [bib_ref] Cardiac autonomic neuropathy in patients with diabetes mellitus: current perspectives, Fisher [/bib_ref] Nerve injury occurs in both the autonomic and peripheral nervous systems, increasing the likelihood of experiencing heart rate abnormalities that progress to resting tachycardia, as well as orthostatic hypotension, ischaemia, CVD, chronic kidney disease and anaemia. [bib_ref] Cardiac autonomic neuropathy in patients with diabetes mellitus: current perspectives, Fisher [/bib_ref]
## | cerebrovascular complications
An early case-control study identified DR as a risk factor for nonembolic ischaemic stroke in individuals with diabetes, independent of smoking, blood pressure and other complications of diabetes. [bib_ref] Retinopathy as a risk factor for nonembolic stroke in diabetic subjects, Petitti [/bib_ref] The findings were later supported by a population-based, prospective study of patients with diabetes that found DR to be significantly correlated with incident stroke, although no association was identified between DR grade and increasing risk of ischaemic stroke. [bib_ref] Is diabetic retinopathy an independent risk factor for ischemic stroke?, Cheung [/bib_ref] This differs from the results of the Wisconsin Epidemiologic Study of Diabetic
Retinopathy in T1DM, which demonstrated an association between DR severity and increased odds of stroke. [bib_ref] Cardiovascular disease, mortality, and retinal microvascular characteristics in type 1 diabetes: Wisconsin..., Klein [/bib_ref] Similarly, in the Finnish Diabetic Nephropathy (FinnDiane) Study, severe DR independently increased the risk of stroke, cerebral infarction and cerebral haemorrhage in patients with T1DM who were followed for 9 years, and the risk increased if patients had concomitant diabetic nephropathy. [bib_ref] Incidence of stroke according to presence of diabetic nephropathy and severe diabetic..., Hägg [/bib_ref] Further analysis of this data set revealed severe DR to be independently associated with haemorrhagic stroke but not with ischaemic stroke, [bib_ref] Different risk factor profiles for ischemic and hemorrhagic stroke in type 1..., Hägg [/bib_ref] whereas a large prospective study in patients with T2DM who were followed for 5 years found DR to be correlated with smallartery ischaemic stroke, but not with large-artery ischaemic stroke or haemorrhagic stroke. [bib_ref] Rates and predictors of risk of stroke and its subtypes in diabetes:..., Hankey [/bib_ref] An analysis of data from the Japan Diabetes Complications Study revealed that even patients with mild to moderate NPDR had an increased risk of stroke after adjusting for traditional cardiovascular risk factors. [bib_ref] Risk of cardiovascular diseases is increased even with mild diabetic retinopathy: the..., Kawasaki [/bib_ref] Furthermore, in the Atherosclerosis Risk in Communities (ARIC) Brain MRI Study, DR was associated with the emergence of subclinical brain infarcts. [bib_ref] Retinal microvascular abnormalities and subclinical magnetic resonance imaging brain infarct: a prospective..., Cheung [/bib_ref]
## | cardiovascular complications
Increased carotid intima-media thickness (cIMT) is a broadly accepted marker for early or subclinical atherosclerosis, [bib_ref] Carotid intima-media thickness (cIMT) and plaque from risk assessment and clinical use..., Bartels [/bib_ref] [bib_ref] Proliferative diabetic retinopathy in type 2 diabetes is related to coronary artery..., Reaven [/bib_ref] It is worth noting, however, that, in a prospective study of individuals with T2DM who were followed for up to 8 years, even mild to moderate DR increased the risk of CHD and stroke. [bib_ref] Risk of cardiovascular diseases is increased even with mild diabetic retinopathy: the..., Kawasaki [/bib_ref] In addition, in a case-controlled study, the odds ratio of myocardial perfusion defects in an asymptomatic patient who
## Nr
Abbreviations: ABI, ankle-brachial index; β, β coefficient; CI, confidence interval; cIMT, carotid intima-media thickness; CAC, coronary artery calcium; CHD, coronary heart disease; CVD, cardiovascular disease; DR, diabetic retinopathy; HR, hazard ratio; ICA-IMT, internal carotid artery intima-media thickness; NPDR, non-proliferative diabetic retinopathy; NR, not reported; OR, odds ratio; PDR, proliferative diabetic retinopathy; RR, relative risk; STDR, sight-threatening diabetic retinopathy (defined as clinically significant macular oedema and/or severe non-proliferative DR, or clinically significant macular oedema and/or PDR); T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; VTDR, vision-threatening diabetic retinopathy (defined as clinically significant macular oedema or PDR). a Adjusted for nephropathy.
had diabetes with DR in comparison to patients who had diabetes without DR was 11.7 (95% CI, 3.7-37). [bib_ref] Prevalence and risk factors accounting for true silent myocardial ischemia: a pilot..., Hernández [/bib_ref] Overall, the above findings are consistent with those from other prospective studies, indicating that DR, particularly advanced DR, is an independent risk factor for CVD in patients with diabetes. [bib_ref] Coronary computed tomographic angiographic findings in asymptomatic patients with type 2 diabetes..., Park [/bib_ref] [bib_ref] Nephropathy, but not retinopathy, is associated with the development of heart disease..., Torffvit [/bib_ref] Notably, the association did not remain after adjusting for the presence of macroalbuminuria. [bib_ref] Nephropathy, but not retinopathy, is associated with the development of heart disease..., Torffvit [/bib_ref] However, this contrasts with results from the Atherosclerosis Risk in Communities (ARIC) Study in T2DM, in which adjusting for nephropathy had minimal impact on the association between DR and CHD. [bib_ref] Diabetic retinopathy and the risk of coronary heart disease: the Atherosclerosis Risk..., Cheung [/bib_ref] In this study, the risk of incident CHD increased with DR severity, and severe DR was also a significant independent risk factor for fatal CHD. [bib_ref] Diabetic retinopathy and the risk of coronary heart disease: the Atherosclerosis Risk..., Cheung [/bib_ref] In the ACCORD Eye Study, DR severity and progression were associated with the composite outcome of first occurrence of nonfatal myocardial infarction, non-fatal stroke or cardiovascular death in patients with T2DM. [bib_ref] Diabetic retinopathy, its progression, and incident cardiovascular events in the ACCORD trial, Gerstein [/bib_ref] Analyses revealed that the risk of this primary outcome increased by 38% for every category of change in DR severity. [bib_ref] Diabetic retinopathy, its progression, and incident cardiovascular events in the ACCORD trial, Gerstein [/bib_ref] Elsewhere, retinopathy and PDR have been shown to increase the risk of cardiovascular or all-cause mortality in patients with T1DM and patients with T2DM. 74-76 PDR. [bib_ref] Predicting development of proliferative diabetic retinopathy, Nwanyanwu [/bib_ref] A number of papers have also identified DR as a key risk factor for lower extremity amputation in both patients with T1DM and patients with T2DM. [bib_ref] Amputations and foot ulcers in patients newly diagnosed with type 2 diabetes..., Bruun [/bib_ref] [bib_ref] Risk factors, ethnic differences and mortality associated with lower-extremity gangrene and amputation..., Chaturvedi [/bib_ref] [bib_ref] Factors predicting lower extremity amputations in patients with type 1 or type..., Hämäläinen [/bib_ref] [bib_ref] Incidence and risk factors of lower extremity amputations in people with type..., Lai [/bib_ref] [bib_ref] Prevalence and risk factors for diabetic lower limb amputation: a clinic-based case..., Rodrigues [/bib_ref] [bib_ref] Predictors for limb loss among patient with diabetic foot infections: an observational..., Saltoglu [/bib_ref] Notably, patients who have T2DM and undergo lower extremity amputation (LEAs) have been found to be at higher risk of developing DR than those without LEAs. [bib_ref] Subsequent ischemic events associated with lower extremity amputations in patients with type..., Tsai [/bib_ref] This was also observed in the ADVANCE-ON post-trial observational study in T2DM, which demonstrated that lower extremity ulceration or amputation, as a major presentation of PAD, increased the risk of retinal photocoagulation or blindness. [bib_ref] Presentations of major peripheral arterial disease and risk of major outcomes in..., Mohammedi [/bib_ref] PAD is diagnosed using the ABI, with values less than 0.9
## | peripheral complications
indicative of the disease. [bib_ref] Peripheral arterial disease in diabetes-a review, Jude [/bib_ref] [bib_ref] Incidence of stroke according to presence of diabetic nephropathy and severe diabetic..., Hägg [/bib_ref] and was associated with small-artery ischaemic stroke in patients with T2DM. [bib_ref] Rates and predictors of risk of stroke and its subtypes in diabetes:..., Hankey [/bib_ref] Evidence presented here also demonstrates that DR is associated with subclinical atherosclerosis, [bib_ref] Rural Epidemiology Study-2. Rural Epidemiology Study-2. Association of carotid intima-media thickness and..., Rema [/bib_ref] [bib_ref] Association between diabetic retinopathy and subclinical atherosclerosis in China: results from a..., Liu [/bib_ref] [bib_ref] Retinopathy is a strong determinant of atherosclerosis in type 2 diabetes: correlation..., Saif [/bib_ref] and that the presence of this complication is independently correlated with cIMT and carotid plaques in T2DM populations. [bib_ref] Type 2 diabetes-associated carotid plaque burden is increased in patients with retinopathy..., Alonso [/bib_ref] [bib_ref] Rural Epidemiology Study-2. Rural Epidemiology Study-2. Association of carotid intima-media thickness and..., Rema [/bib_ref] [bib_ref] Coronary computed tomographic angiographic findings in asymptomatic patients with type 2 diabetes..., Park [/bib_ref] Two studies showed that DR is associated with increased risk of CHD-related events and mortality [bib_ref] Is diabetic retinopathy an independent risk factor for ischemic stroke?, Cheung [/bib_ref] [bib_ref] Risk of cardiovascular diseases is increased even with mild diabetic retinopathy: the..., Kawasaki [/bib_ref] and, overall, DR was found to be an independent risk factor for CVD. [bib_ref] Coronary computed tomographic angiographic findings in asymptomatic patients with type 2 diabetes..., Park [/bib_ref] [bib_ref] Ability of retinopathy to predict cardiovascular disease in patients with type 2..., Gimeno-Orna [/bib_ref] [bib_ref] Risk factors for coronary disease and stroke in previously hospitalized African-Americans with..., Roy [/bib_ref] [bib_ref] Diabetic retinopathy is associated with an increased incidence of cardiovascular events in..., Targher [/bib_ref] Similarly, outcomes from the reviewed studies demonstrate that DR, and particularly PDR, is a strong risk factor for PAD, which carries a risk of lower extremity ulceration and amputation. [bib_ref] Prevalence of and risk factors for abnormal ankle-brachial index in patients with..., Li [/bib_ref] [bib_ref] Abnormally low or high anklebrachial index is associated with proliferative diabetic retinopathy..., Chen [/bib_ref] [bib_ref] High prevalence of lower extremity peripheral artery disease in type 2 diabetes..., Chen [/bib_ref] Therefore, worsening DR could prove useful as a marker for individuals at increased risk of these serious macrovascular complications. Specifically, the evidence indicates that patients with diabetes who have PDR are more likely than those with NPDR to have an abnormal ABI. [bib_ref] Abnormally low or high anklebrachial index is associated with proliferative diabetic retinopathy..., Chen [/bib_ref] Furthermore, DR was identified as an independent risk factor for foot ulceration and a key risk factor for lower extremity amputation. [bib_ref] A longitudinal study of foot ulceration and its risk factors in community-based..., Baba [/bib_ref] [bib_ref] Factors associated with diabetic patients at high risk for foot ulceration, Leymarie [/bib_ref] [bib_ref] Amputations and foot ulcers in patients newly diagnosed with type 2 diabetes..., Bruun [/bib_ref] Interestingly, two studies found that patients with diabetes who have non-healing foot ulcers or who have undergone lower extremity amputation are at risk of DR development and progression. [bib_ref] Predicting development of proliferative diabetic retinopathy, Nwanyanwu [/bib_ref] [bib_ref] Presentations of major peripheral arterial disease and risk of major outcomes in..., Mohammedi [/bib_ref] We performed a comprehensive search of the literature that resulted in the inclusion of 71 studies, with a total of over 400 000 patients, and the approach taken to review was objective and systematic, complying with several of the key items included in the PRISMA checklist for systematic reviews and meta-analyses. One limitation of our study was that only the PubMed literature database was searched.
Despite this, our findings are an important contribution to the understanding of the relationship between DR and the micro-and macrovascular complications of diabetes and, as such, provide valuable information to support the wider multidisciplinary team that is involved in the care of individuals with diabetes. In particular, the findings underscore the need for prompt screening and referral for DR, which can both reduce vision loss and, potentially, identify patients at increased risk of other complications of diabetes. This highlights the need for a coordinated and collaborative approach to patient management on the part of all the healthcare professionals involved in diabetes care in order to optimize clinical outcomes. A risk assessment model that incorporates DR presence as an input factor, rather than an outcome, may provide a powerful tool for identifying individuals at high risk of potentially life-threatening complications of diabetes.
Indeed, a study has demonstrated that incorporation of microvascular complications into cardiovascular risk algorithms improves CVD risk prediction in T2DM. [bib_ref] Microvascular disease and risk of cardiovascular events among individuals with type 2..., Brownrigg [/bib_ref] The ability to identify the individuals most at risk of developing complications could improve the targeting of preventive treatments, potentially reducing morbidity and mortality among patients with diabetes. Further exploration in this area is warranted.
# Acknowledgments
# Author contributions
All authors contributed to the concept development, determining the search strategy and evaluating the results for inclusion, and provided critical review of the manuscript.
## Orcid
## Rafael simó
https://orcid.org/0000-0003-0475-3096
Marc Evans https://orcid.org/0000-0003-0671-0778
[table] TABLE 1: Studies describing the association between DR and nephropathy and renal decline [/table]
[table] TABLE 2: Studies describing the association between DR and diabetic neuropathy Abbreviations: CAN, cardiac autonomic neuropathy; CI, confidence interval; DPN, diabetic peripheral neuropathy; DR, diabetic retinopathy; HR, hazard ratio; NPDR, non-proliferative diabetic retinopathy; NR, not reported; OR, odds ratio; PDR, proliferative diabetic retinopathy; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.In a single-centre observational cohort study by Takagi et al., the presence of DR was a common predictor of both albuminuria onset and decreased estimated GFR (eGFR) in patients with T2DM.33 In earlier research, also involving patients with T2DM, Rossing et al. found a significant correlation between degree and presence of DR at baseline and increased rate of eGFR decline.48 Interestingly, in the Japan Diabetes Complications Study, patients with T2DM who had both microalbuminuria and DR at baseline had the highest risk of developing macroalbuminuria and displayed significantly faster eGFR decline (−1.92 mL min −1 1.73 m −2 y −1 ; P < 0.01) compared with those with one of the two complications (−0.69 mL min −1 1.73 m −2 year −1 ;DR was found to be associated with two neuropathies: diabetic peripheral neuropathy (DPN) and CAN (Table 2). DPN is estimated to affect 30% to 50% of individuals with diabetes; it is characterized by peripheral nerve injury and manifests most commonly as distal symmetric polyneuropathy (DSP). Symptoms of DSP include pain, numbness and weakness in the lower limbs, which may lead to falls.49 [/table]
[table] TABLE 3: Studies describing the association between DR and cerebrovascular complications [/table]
[table] TABLE 4: Studies describing the association between DR and cardiovascular complications [/table]
[table] TABLE 5: Studies describing the association between DR and peripheral complications [/table]
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Impact of grafting using thin upper pole artery ligation on living-donor adult kidney transplantation
This study aimed to investigate the impact of grafting using thin upper pole artery ligation for living-donor adult kidney transplantation. Few reports have examined the safety of thin upper pole artery ligation.Between January 2008 and May 2015, 613 consecutive living-donor adult kidney transplantations were performed. We excluded 21 recipients who experienced graft loss due to factors that were unrelated to surgical complications and 3 recipients with grafts treated with arterial reconstruction and thin upper pole artery ligation for 3 arteries. We included 439 kidney grafts with single arteries (Single Artery Group), 123 with reconstructed arteries (Arterial Reconstruction Group) and 27 with ligated thin upper pole arteries (Arterial Ligation Group) in this retrospective cohort study. To evaluate the safety of thin upper pole artery ligation, we compared the Arterial Ligation Group with the Single Artery and Arterial Reconstruction groups. We evaluated the characteristics of the enrolled donors, recipients, and their grafts. Thereafter, we investigated recipients' perioperative and postoperative estimated glomerular filtration rate (eGFR) and complication rates.Significant differences among the 3 groups were identified for donor sex and endoscopic nephrectomy rates. Recipient eGFR and the complication rates were adjusted according to these factors. The perioperative and postoperative eGFR of recipients did not differ significantly in the Arterial Reconstruction and Single Artery groups with low complication rates.Thin upper pole artery ligation is a safe procedure for living-donor adult kidney transplantation and may prevent unnecessary arterial reconstruction and associated complications.Abbreviations: 3D-CT = 3-dimensional computed tomography, BMI = body mass index, CI = confidence interval, eGFR = estimated glomerular filtration rate, TIT = total ischemia time, WIT = warm ischemia time.
# Introduction
Reportedly, 18% to 30% of potential kidney donors require grafting of >2 arteries unilaterally and 15% require grafting of >2 arteries bilaterally. [bib_ref] Living-related donors with multiple renal arteries. A twenty-year experience, Roza [/bib_ref] Grafting with >2 arteries is sometimes required after endoscopic donor nephrectomy. [bib_ref] Increased transplantation of kidneys with multiple renal arteries in the laparoscopic live..., Troppmann [/bib_ref] The safety and efficacy of arterial reconstruction has been the topic of several reports. [bib_ref] Impact of arterial reconstruction with recipient's own internal iliac artery for multiple..., Hiramitsu [/bib_ref] [bib_ref] Short and long term outcomes of kidney transplants with multiple renal arteries, Benedetti [/bib_ref] [bib_ref] Successful renovascular reconstruction for renal allografts with multiple renal arteries, Makiyama [/bib_ref] [bib_ref] Long-term graft outcome after arterial reconstruction during living related kidney transplantation, Sagban [/bib_ref] Several reports have recommend the reconstruction of the lower pole arterial branches to prevent ureteral complications. [bib_ref] Successful renovascular reconstruction for renal allografts with multiple renal arteries, Makiyama [/bib_ref] [bib_ref] Complex vascular anatomy in live kidney donation: imaging and consequences for clinical..., Kok [/bib_ref] On the other hand, there have not been any reports on the importance of preserving the thin upper pole arteries. Some grafts have very thin upper pole arteries that feed small areas and that are very thin to reconstruct. Most arterial reconstructions are performed using the conjoined, end-to-side, and interposition methods. For grafts of very thin upper pole arteries, arterial reconstruction presents a risk of arterial complications. To prevent arterial complications, thin upper pole artery ligations are performed instead. However, very few reports have discussed the safety of thin upper pole artery ligation. [bib_ref] Laparoscopic procurement of kidneys with multiple renal arteries is associated with increased..., Carter [/bib_ref] Thus, we investigated the safety of grafting using thin upper pole artery ligation in living-donor adult kidney transplantation.
# Methods
## Ethics review
This study was approved by the Institutional Review Board of Nagoya Daini Red Cross Hospital, Aichi, Japan, and was conducted according to the Declaration of Helsinki.
## Study design
To investigate the safety of thin upper pole arterial ligation, adult recipients of living-donor kidney transplantation were divided into 3 groups: the Arterial Ligation, Arterial Reconstruction, and Single Artery groups. The perioperative and postoperative estimated glomerular filtration rate (eGFR) and complication rates in the Arterial Ligation Group were compared with those of the 2 other groups. This retrospective cohort study was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.
## Participants
Between January 2008 and May 2015, 613 consecutive livingdonor adult kidney transplantations were performed at our hospital and included in this study. The recipients were observed every month between January 2008 and May 2015 (mean observation period: 43.3 ± 24.9 months). Twenty-one recipients dropped out of the study with graft failure unrelated to surgical complications [fig_ref] Figure 1: Patient [/fig_ref].
In total, 592 recipients were followed up. However, 439 of 592 kidney grafts were single artery (Single Artery Group), and 123 kidney grafts underwent arterial reconstruction (Arterial Reconstruction Group). Thin upper pole arteries (<2 mm) were ligated in 27 kidney grafts (Arterial Ligation Group). Three kidney grafts were excluded because they required arterial reconstruction and thin upper pole graft artery ligation for a total of 3 arteries (2 arteries and 1 thin upper pole artery) [fig_ref] Figure 1: Patient [/fig_ref]. The outcomes were evaluated by examining the perioperative and postoperative eGFR and complication rates of the recipients. Arterial thrombosis, urine leakage, ureterial stricture, delayed graft function, postoperative bleeding, lymphocele, acute cellular rejection, and antibody-mediated rejection were investigated. Patient data were retrospectively collected from patients' charts, and there were no missing data.
# Statistical analysis
Statistical analyses were performed using the analysis of variance for continuous data and the chi-square exact test for categorical variables. Bonferroni correction was used for multiple comparison analyses. The generalized linear model analysis (gamma with log link) was used to make comparisons between surgical procedures and operative results. Generalized estimating equation (GEE) analysiswas usedtomakecomparisons betweensurgicalprocedures and longitudinal data on eGFR. The risk ratio was used to make comparisons between surgical procedures and incidence of complication rates. The analyses were performed using donor sex and laparoscopic donor nephrectomy as adjustment factors. P values of <0.05 were considered statistically significant. Analyses were performed using SAS package 9.0 (SAS Institute, Cary, NC).
## Preoperative evaluation of graft arteries and indications for thin upper pole artery ligation
To select the reconstruction method, the number and size of graft arteries were preoperatively evaluated using 3-dimensional computed tomography (3D-CT). Ligation was indicated for very thin upper pole arteries that posed a risk of arterial complications (<2 mm). Lower pole graft arteries evident in 3D-CT images were always reconstructed to avoid ureteral complications.
## Perfusion area of each ligated upper pole artery
The perfusion area of each ligated upper pole artery was estimated during cold perfusion. In all kidney grafts, the perfusion area of each ligated upper pole artery was <5%.
# Arterial reconstruction methods
In general, 3 types of arterial construction methods were used for grafts with >2 arteries in the Arterial Reconstruction Group: the conjoined, end-to-side, and interposition methods.
## Definition of time to initial urination
The time to initial urination was defined as the interval between blood reperfusion and the initial urination from the "graft" ureter.
# Results
## Participants
We observed 589 recipients every month between January 2008 and May 2015 (mean observation period: 43.3 ± 24.9 months) at our hospital; no patients dropped out during this period. Four hundred thirty-nine of the 589 kidney grafts had a single artery, and arterial reconstructions were performed in 123 kidney grafts. Thin upper pole artery ligation was performed in 27 kidney grafts.
## Excluded recipients
A total of 24 recipients were excluded from this study, 21 due to graft failure and 3 due to a combination of arterial reconstruction and thin upper pole artery ligation. Among the 21 recipients, no thin upper pole artery ligation was identified. One graft underwent arterial reconstruction for 2 arteries, and 20 grafts were with a single artery. One recipient with arterial reconstruction dropped out from following death from pneumonia.
## Descriptive data
We compared the characteristics of recipients and donors among the Arterial Ligation Group, the Arterial Reconstruction Group, and Single Artery Group [fig_ref] Table 1: Characteristics of recipients and donors [/fig_ref]. We did not identify any significant differences in the characteristics of the recipients. The donor sex and endoscopic donor nephrectomy rates significantly differed among the groups. The body mass index (BMI) of the recipients and donors was similar among the groups. The mean eGFR of donors was also similar. The multiple comparison analysis in [fig_ref] Table 2: Comparison of characteristics [/fig_ref] shows a significant difference in donor sex and endoscopic donor nephrectomy rates between the Arterial Ligation Group and the Single Artery Group. All patient data collected were complete and accurate.
## Characteristics of kidney grafts and results of surgeries
The attributes of all kidney grafts are shown in [fig_ref] Table 3: Characteristics of the kidney grafts [/fig_ref]. Kidney weights were similar among the groups. In the 3D-CT images, the mean diameter of the ligated arteries in the Arterial Ligation Group was 1.82 ± 0.36 mm, which was significantly thinner than that of the reconstructed arterial branches in the Arterial Reconstruction Group.
Operative results are shown in . Donor sex and endoscopic donor nephrectomy rates were significantly different according to the characteristics of recipients and donors. Operative results were adjusted with these factors in mind. There were no differences in the operative duration, blood loss of donor nephrectomy, and initial urination among the groups. The warm ischemia time (WIT; interval between arterial clamping and the beginning of cold perfusion) and total ischemia time (TIT; interval between arterial clamping and blood reperfusion) were significantly longer in the Arterial Reconstruction Group.
Complications that occurred in each group are shown in [fig_ref] Table 5: Complications in each group [/fig_ref]. Incidences of each complication (adjusted for donor sex and endoscopic operation rates) were not significantly different among the Arterial Ligation Group and the other groups.
The unadjusted eGFR of 3 groups are shown in [fig_ref] Figure 2: Mean estimated glomerular filtration rate of the recipients during the observation period [/fig_ref]. The eGFR and eGFR differences adjusted for donor sex and endoscopic operation rates are shown in [fig_ref] Figure 3: Mean and differences in the estimated glomerular filtration rate of the recipients... [/fig_ref]. Differences in eGFR were not significantly different among the Arterial Ligation The results of the operation. <0.001 --AL = arterial ligation, ANOVA = analysis of variance, AR = arterial reconstruction, CI = confidence interval, SA = single artery, SD = standard deviation. Difference: "AR group -AL group" or "SA group -AL group," P-value: difference estimates with Bonferroni correction. Group and the other groups throughout the entire observation period [fig_ref] Table 6: Perioperative and postoperative graft function [/fig_ref].
# Discussion
Using 3D-CT, it is easy to evaluate the number and size of kidney graft arteries preoperatively. [bib_ref] Prospective comparison of 3-dimensional volume rendered computerized tomography and conventional renal arteriography..., Fettouh [/bib_ref] [bib_ref] Multidetector CT angiography for preoperative evaluation of living laparoscopic kidney donors, Kawamoto [/bib_ref] [bib_ref] Multidetector CT angiography in live donor renal transplantation: experience from 156 concecutive..., Laugharne [/bib_ref] [bib_ref] Assessment of 100 live potential renal donors for laparoscopic nephrectomy with multi-Detector..., Holden [/bib_ref] [bib_ref] Role of multidetector-row computed tomography in evaluation of living renal donors, Zhang [/bib_ref] [bib_ref] Living donor kidneys: usefulness of multi-detector row CT for comprehensive evaluation, Kim [/bib_ref] [bib_ref] Preoperative evaluation of living kidney donors using multirow deterctor computed tomography: comparison..., Hänninen [/bib_ref] Studies have reported that the preservation of the arterial branches that feed the lower pole of the kidney graft is important to prevent ureteral complications such as ureteral leakage and stricture. [bib_ref] Complex vascular anatomy in live kidney donation: imaging and consequences for clinical..., Kok [/bib_ref] The safety of arterial branch reconstruction has been established. [bib_ref] Impact of arterial reconstruction with recipient's own internal iliac artery for multiple..., Hiramitsu [/bib_ref] [bib_ref] Short and long term outcomes of kidney transplants with multiple renal arteries, Benedetti [/bib_ref] [bib_ref] Successful renovascular reconstruction for renal allografts with multiple renal arteries, Makiyama [/bib_ref] [bib_ref] Long-term graft outcome after arterial reconstruction during living related kidney transplantation, Sagban [/bib_ref] However, very thin upper pole arteries are sometimes present, [bib_ref] Preoperative evaluation of living kidney donors using multirow deterctor computed tomography: comparison..., Hänninen [/bib_ref] and these are often ligated to shorten the TIT and prevent complications related to reconstruction. However, the safety of ligating these thin upper pole arteries is unclear. In this study, the mean diameter of 27 thin upper pole arteries was 1.82 mm, which was significantly thinner than that of the reconstructed arterial branches. We identified significant differences according to the donor sex and endoscopic nephrectomy rates. For accurate analysis, operative results, recipients' eGFRs, and complication rates were adjusted according to these factors. No significant differences in operative results were demonstrated, except for WIT and TIT. The significantly longer WIT and TIT evident in the Arterial Reconstruction Group were reasonable because dealing with >2 arteries during donor nephrectomy and their reconstruction was more time consuming. Although the WIT was significantly longer in the Arterial Reconstruction Group, the mean WIT was 140 s in the Arterial Ligation Group and 171.9 s in the Arterial Reconstruction Group. This difference in WIT of only 31.9 s may have been too small to yield specific complications. [bib_ref] Effect of warm ischemia on graft outcome in laparoscopic donor nephrectomy, Simforoosh [/bib_ref] Although the ligation of thin upper pole arteries significantly shortened the mean TIT in the Arterial Ligation Group compared to that in the Arterial Reconstruction Group, the perioperative eGFR, the time to initial urination, the incidence of delayed graft function, and the incidence of acute cellular rejection were similar between the Arterial Ligation and Arterial Reconstruction groups. The mean TIT was 100.9 min in the Arterial Ligation Group and 136.8 min in the Arterial Reconstruction Group. This difference was statistically significant, but the difference in TIT of only 35.9 min may have been too small to constitute a clinical difference. [bib_ref] Cold ischemia time and allograft outcomes in live donor renal transplantation: Is..., Simpkins [/bib_ref] With regard to other adjusted complications, arterial thrombosis did not occur in the Arterial Ligation Group, and occurred in only 1 recipient in the Arterial Reconstruction Group. This implies that the ligation of thin upper pole arteries minimized the risk of arterial thrombosis in the Arterial Reconstruction Group. The mean diameter of the 27 thin upper pole arteries examined in this study was only 1.82 mm, which may have been a risk factor for complications related to reconstruction. Although the safety of arterial reconstruction was established, further assessments of the criteria that indicate arteries for reconstruction and optimum techniques for this procedure are required. [bib_ref] Impact of arterial reconstruction with recipient's own internal iliac artery for multiple..., Hiramitsu [/bib_ref] Incidences of other complications in the Arterial Ligation Group compared with those in the Arterial Reconstruction Group were not significantly different. Furthermore complication rates were similar for the Arterial Ligation Group and Single Artery Group. These results demonstrated low complication rates in the Arterial Ligation Group.
Infarction of a small area of the upper pole (<5%) can occur during ligation of thin upper pole arteries. Although this might influence the postoperative kidney function of recipients, we did not observe significant differences in eGFRs in the Arterial Ligation Group and the other groups in this study. This suggests that the areas fed by thin upper pole arteries were too small to have an impact on graft function. These facts demonstrate the safety of the ligation of thin upper pole arteries.
One limitation of this study is its retrospective nature; prospective randomized studies on the impact of the ligation of thin upper pole kidney graft arteries are required in the future.
In conclusion, the ligation of thin upper pole arteries (<2 mm) is a safe procedure with a low incidence of complications when performed on selected thin upper pole arteries.
[fig] Figure 1: Patient [/fig]
[fig] 95%CI: = 95% confidence interval, AL = arterial ligation, AR = arterial reconstruction, RR = risk ratio, SA = single artery. P-value: Risk effect estimates with Bonferroni correction. * Donors gender-, Endoscopic donor nephrectomy-adjusted. [/fig]
[fig] Figure 2: Mean estimated glomerular filtration rate of the recipients during the observation period (unadjusted). [/fig]
[fig] Figure 3: Mean and differences in the estimated glomerular filtration rate of the recipients during the observation period. There was no significant difference among the Arterial Ligation Group and the Single Artery and Arterial Reconstruction groups. eGFR = estimated glomerular filtration rate, POD = postoperative day. [/fig]
[table] Table 1: Characteristics of recipients and donors. BMI = body mass index, CI = confidence interval, eGFR = estimated glomerular filtration rate. [/table]
[table] Table 2: Comparison of characteristics.AL = arterial ligation, AR = arterial reconstruction, CI = confidence interval, SA = single artery. Difference: "AR group -AL group" or "SA group -AL group," P: difference estimates with Bonferroni correction. [/table]
[table] Table 3: Characteristics of the kidney grafts. [/table]
[table] Table 5: Complications in each group. [/table]
[table] Table 6: Perioperative and postoperative graft function. P value >0.999 >0.999 >0.999 0.708 >0.999 >0.999 >0.999 >0.999 0.980 >0.999 >0.999 >0.999 >0.999 >0.999 95% CI À5.396 À8.662 À6.508 À3.949 À4.881 À4.731 À4.503 À6.426 À3.786 À5.486 À5.137 À6.646 À7.95% CI À1.286 À2.073 À0.816 À0.260 À0.156 À0.758 À2.074 À3.448 À0.007 À1.684 À2.392 À4.409 À4.101 À6.065 CI = confidence interval, POD = postoperative day. [/table]
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Oral arsenic trioxide for treating acute promyelocytic leukaemia: Implications for its worldwide epidemiology and beyond
## How oral ato treatment has influenced apl epidemiology
Acute promyelocytic leukaemia (APL) is a relatively uncommon type of highly aggressive acute myeloid leukemia, first characterised in 1957 [bib_ref] Acute promyelocytic leukemia and chronic lymphocytic leukemia diagnosed concurrently, Coombs [/bib_ref] [bib_ref] Acute promyelocytic leukemia: A history over 60 years-from the most malignant to..., Thomas [/bib_ref]. However, even till recently − the medical literature contained no authentic estimates for its prevalence, although incidences applicable to various parts of the world were readily available [fig_ref] TABLE 1: Incidence estimates [/fig_ref] [bib_ref] Acute promyelocytic leukemia: a population-based study on incidence and survival in the..., Chen [/bib_ref] [bib_ref] Incidence of acute promyelocytic leukemia across Europe: results of RARECAREnet-a population-based study, Dinmohamed [/bib_ref] [bib_ref] Characteristics and predictors of early hospital deaths in newly diagnosed APL: a..., Gill [/bib_ref] [bib_ref] Epidemiology and outcomes of acute promyelocytic leukaemia in the era of alltrans..., Gill [/bib_ref]. This seemingly perverse anomaly is very likely due to the very high early mortality of APL treated with conventional chemotherapy, 1 * which has now been superseded by targeted therapy with alltrans retinoic acid (ATRA) and arsenic trioxide (ATO). Paradoxically therefore, prior to this paradigm shift in management, the prevalence of APL could have been lower than its annual incidence.
Even for highly efficacious new treatments with acceptable safety, to become globally established and preferred over alternative more traditional therapeutic strategies ─ they should also be well tolerated, user friendly, convenient/nondisruptive for quality of life, and affordable. In all these respects, treatment of APL patients with oral ATO (prepared in accordance with Good Manufacturing Practice standards) confers distinct advantages when compared with intravenous (IV) delivery, which may also be somewhat more cardiotoxic [bib_ref] Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with..., Kumana [/bib_ref] [bib_ref] Effects of oral arsenic trioxide therapy on QT intervals in patients with..., Kumana [/bib_ref]. Based on these considerations, for more than two decades ─ increasing numbers of newly-diagnosed APL patients in Hong Kong have been treated with oral ATO. Predominantly, such treatment has been incorporated into a regimen comprising ATO, ATRA, and ascorbic acid (AAA) [bib_ref] Oral arsenic trioxide incorporation into frontline treatment with all-trans retinoic acid and..., Gill [/bib_ref]. In Hong Kong, patients with APL receiving oral AAA had reasonable quality-of-life with the out-patient regimen and had favourable relapse-free and overall survivals [bib_ref] Epidemiology and outcomes of acute promyelocytic leukaemia in the era of alltrans..., Gill [/bib_ref] [bib_ref] Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with..., Kumana [/bib_ref] [bib_ref] Effects of oral arsenic trioxide therapy on QT intervals in patients with..., Kumana [/bib_ref] [bib_ref] Oral arsenic trioxide incorporation into frontline treatment with all-trans retinoic acid and..., Gill [/bib_ref]. Nevertheless, there is lack of prospective data comparing the efficacies and surivals of patients treated with oral ATO-based regimens versus IV ATO/ATRA-based approach.
As to how the prognosis of APL has been transformed, ATO induces dose-dependent apoptosis and differentiation of abnormal promyelocytes via its effect on an abnormally generated PML-RARA anti-apoptotic fusion protein, which is involved in a complex series of oxidative processes (14-18). Moreover, its combination with ATRA and ascorbic acid has been shown to produce synergistic degradation of PML in APL cells, whilst also being able to induce apoptosis in myeloma cells [bib_ref] Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic..., Rego [/bib_ref] [bib_ref] Synergy against PML-RARa: targeting transcription, proteolysis, differentiation, and self-renewal in acute promyelocytic..., Santos [/bib_ref] [bib_ref] Malignant cells can be sensitized to undergo growth inhibition and apoptosis by..., Dai [/bib_ref] [bib_ref] Feasibility and correlates of arsenic trioxide combined with ascorbic acid-mediated depletion of..., Bahlis [/bib_ref].
Thus, assuming that annual incidence rates of APL continue to remain steady, and coinciding with the adoption of highly efficacious treatment with ATRA and ATO (starting around the year 2000) ─ year on year improved outcomes and verifiable increases in prevalence should be expected. This prediction has been amply borne-out, as reported in population based studies of the evolving epidemiology of APL in Hong Kong [bib_ref] Oral arsenic trioxide-based maintenance regimens for first complete remission of acute promyelocytic..., Au [/bib_ref] [bib_ref] Epidemiology and outcomes of acute promyelocytic leukaemia in the era of all-trans..., Gill [/bib_ref]. and illustrated in [fig_ref] FIGURE 1 APL: Incidence and prevalence in Hong Kong from 1991-2020 [/fig_ref] , which was constructed from available data as illustrated in Supplemental [fig_ref] TABLE 1: Incidence estimates [/fig_ref]. The overriding ensuing message is that since around the turn of this century, there has been a marked increase in APL prevalence, related to a dramatic increase in long-term patient survival presumably consequent upon the introduction of treatment with ATRA and ATO. Interestingly, some of the epidemiological publications on APL cited in [fig_ref] TABLE 1: Incidence estimates [/fig_ref] alluded to increasing year on year incidences [bib_ref] Epidemiology and outcomes of acute promyelocytic leukaemia in the era of alltrans..., Gill [/bib_ref] , whatever the reason. 2 # However, such reported incremental increases were minimal and insufficient to explain the magnitude of prevailing prevalence rate changes. Although it 2022: Estimates derived from data available from a Hong Kong APL epidemiology study and yielded a slightly increasing trend (see [fig_ref] FIGURE 1 APL: Incidence and prevalence in Hong Kong from 1991-2020 [/fig_ref].
➔ indicates trend over the years; APL: acute promyelocytic leukaemia.
1 * Median survival from diagnosis in untreated patients was reported to be less than a week. (3) 2 #Possible speculative explanation being the availability of better
[formula] 'diagnostic tools'. [/formula]
is reasonable to infer that both oral and IV dosing can give rise to equivalent improvements in overall survival and hence increased APL prevalence, the advantages of oral versus IV ATO are nevertheless highly pertinent. This is because intermittent long-term oral ATO administration is easily achieved. In contrast, long-term intermittent IV ATO is much more challenging to implement efficiently as it is less acceptable and burdensome for patients and health care delivery systems in many ways. The impediments specific to IV ATO dosing [bib_ref] Systemic availability of arsenic from oral arsenic-trioxide used to treat patients with..., Kumana [/bib_ref] [bib_ref] Arsenic trioxide for treating acute promyelocytic leukaemia: an evidence review group perspective..., Ramaekers [/bib_ref] include being: much more patient unfriendly and restrictive (entailing hospitalisations and absence from work), expensive, 3 ‡ highly time consuming, and very demanding on health-care resources. [bib_ref] Acute promyelocytic leukemia: A history over 60 years-from the most malignant to..., Thomas [/bib_ref] - In contrast, oral ATO can be taken at home under outpatient supervision. In most countries however, reliance on IV dosing still continues to be the norm. This means that APL patients in the poorest parts of the world are needlessly dying, simply because they lack access to oral ATO, an affordable, simple to imbibe, convenient, and quality of life preserving lifesaving remedy.
How experience from treating APL patients with oral ATO could help to expand its indications for the management of patients with non-APL diseases/disorders
Appreciating the favourable impact of a treatment on disease epidemiology, and unravelling the mechanisms for such outcomes, can also have important implications on the potential to expand the indications for such an intervention. The success of ATO therapy, and particularly its oral formulation for APL patients seems to be a case in point, because as of late, there has been a renewed interest in invitro, animal, and even clinical studies exploring a possible role for ATO for many other conditions. Thus, there is now a wide array of publications about such non-APL haematological and non-haematological diseases/conditions for which possible ATO-induced treatment benefits are being postulated (28-44). The experimental studies targeted nucleophosmin-1 (NPM1)mutated acute myeloid leukaemia, multiple myeloma, certain lymphomas, lung cancers, several autoimmune diseases, graft versus host disease, certain neurological diseases, and idiopathic pulmonary fibrosis. Preliminary clinical reports also suggest that treatment with ATO may even result in outcome benefits for mantle cell lymphoma and systemic lupus erythematosus (SLE). Compared to APL, moreover, several of the aforementioned non-haematological diseases are much more prevalent and thus clinically important (see [fig_ref] TABLE 2: The epidemiology of acute promyelocytic leukaemia and other possible high profile arsenic... [/fig_ref] (43-50). Provided the possible putative clinical outcome benefits of ATO for at least some of these conditions are borne out, even for small proportions of such patients, exciting new treatment strategies may well emerge.
These considerations are particularly pertinent to the use of oral ATO treatment, as many non-APL diseases entail long-term disability, for which intermittent IV delivery would pose considerable burdens and expense. Thus, the known safety and other advantages of oral ATO 5 *, gleaned from experience treating Hong Kong APL patients, indicate that further clinical studies warrant exploring ATO treatment via the same route for patients having such non-APL disorders and not surprisingly such investigations are being planned. The known likely systemic equivalence of IV and oral ATO means that the latter route offers yet another crucial advantage. Notably, investigations entailing oral dosing offer a means of rejecting or affirming such claims affordably and expeditiously, without imposing quality of life disruption on patients being studied. It also follows that for patients with the latter high prevalence non-APL diseases, robust clinical studies validating worthwhile benefits following oral ATO dosing could confer important benefits for large numbers of chronically symptomatic patients. Improved outcomes for selected patients with such non-APL conditions could also generate heightened interest and support for oral ATO dosing in the medical community and pharmaceutical industry. For diseases that potentially respond to alternative IV arsenicals, 6 † efforts should also be made to determine whether oral ATO may also be effective.
If the findings of such diverse investigations confirm that clinically significant benefits can accrue, very large patient numbers may stand to benefit and thus provide additional clinical and commercial interest in oral ATO.
## Conclusions and future perspectives
Adoption of oral-ATO based treatment for patients with APL in Hong Kong as part of the oral AAA regimen has been associated with vastly improved overall survival and a dramatic increase in estimated prevalence, from about 0.1 to 6.0 per 100,000 persons during the last 2 decades. In contrast to regimens entailing IV ATO, this has been achieved with far less patient inconvenience and quality of life disruption, with greatly reduced direct and indirect costs. In underprivileged parts of the world however, patients with APL are needlessly dying because they lack access to oral ATO. Even in more affluent countries, most patients still continue to receive their ATO intravenously, which imposes unnecessary inconvenience and quality of life disruption, as well as logistical and financial burdens on their health care delivery. For patients with a number of diverse diseases/disorders other than APL, many experimental and a few clinical studies suggest that ATO may also have a therapeutic benefit. Several such diseases are highly prevalent worldwide and give rise to prolonged distress and disability. Hence, robust clinical studies entailing oral ATO in patients with such diseases are obviously warranted. If the expected clinical, logistic and financial benefits for patients with such non-APL diseases can also be validated, very large numbers of patients could stand to benefit. This would very likely rekindle greater interest in this form of ancient therapy within the medical profession and the pharmaceutical industry.
# Author contributions
CRK: Conceived the study, wrote and approved the manuscript. Y-LK: Conceived the study, wrote and approved the manuscript. HG: Conceived the study, wrote and approved the manuscript. All authors contributed to the article and approved the submitted version.
## Conflict of interest
Authors HG, CRK and Y-LK are employed by or associated with the University of Hong Kong.
The University of Hong Kong currently holds two United States US patents 7,521,071 B2 and 8,906,422 B2, one Japanese patent 4786341 and one European patent EP 1562616 B1 for the use of arsenic trioxide oral solution in the treatment of leukaemias and lymphomas.
## Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
[fig] FIGURE 1 APL: Incidence and prevalence in Hong Kong from 1991-2020. For the 18 Hong Kong government hospitals with specialised haematology services, annual incidence and prevalence rates were derived, assuming an average HK population of 7.5 million throughout those years. These estimates were based on available details in the computerised Clinical Data Analysis and Reporting System (CDARS) of the Hospital Authority, Hong Kong. (9) Prevalence refers to numbers of surviving patients diagnosed with APL on December 31 of each year, even if deemed to be 'cured'. The trend line shows that the point prevalence increased from 0.1 to 6.0 per 100,000 persons between 1991 and 2020. 3 ‡ One month's typical IV ATO treatment for an adult with APL retails for around US$10,000; equivalent dosages of oral treatment (Arsenol ® ) used in Hong Kong do so for <40% of this figure, whilst also avoiding the costs and inconvenience of recurring life-long hospitalisations and IV infusion paraphernalia. 4 ∞Repeated time commitments from medical staff and recourse to necessary equipment for IV infusions. [/fig]
[fig] Funding: The work was supported by the Health and Medical Research Fund (HMRF) (ref.:08191946), Food and Health Bureau, the Government of the Hong Kong Special Administrative Region, China. [/fig]
[table] TABLE 1: Incidence estimates (number of new patients with APL encountered annually, -expressed as a proportion of a given region's inferred population as cited in relevant publications). [/table]
[table] TABLE 2: The epidemiology of acute promyelocytic leukaemia and other possible high profile arsenic trioxide responsive diseases. Unrealistic estimates based on very small samples; ‡Very variable depending on ethnicity & location (extremely high in Afro-Caribbeans, and high in Chinese & S Asians) making accurate comparisons challenging. Special circumstances may need consideration; e.g. in chloroquine tre ate d Lup us pa tie nts , c o-t reat ment wit h ATO may ris k electrocardiographic QT prolongation related cardiotoxicity. 6 † For example, phase I studies in patients with relapsed/refractory Peripheral T-Cell Lymphoma indicate that IV treatment with darinaparsin (an organo-arsenical that leads to cancer cell apoptosis) may confer clinical benefits.(48) [/table]
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Characterization of Membrane-Type Dissolution Profiles of Clinically Available Orally Inhaled Products Using a Weibull Fit and a Mechanistic Model
## S.1 dissolution of polydisperse powders
In order to evaluate the expression that results from Eqs. (6) and (7) in the main text, we change variable to = ln( 1 ⁄ ) with inverse = 1 . Letting = 1/( √2), the lognormal particle-size distribution takes the form
[formula] = 1 √ −( ) 2 −(S1) [/formula]
Noting that d = 1 d and letting = − / 1 , the integrand in the numerator of Eq. (7), here denoted , can be expressed as
[formula] = 1 3 √ ( + ) 3 −( ) 2 = 1 3 √ � � 3 � 3− 3 =0 −( ) 2 + (S2) [/formula]
where binomial coefficients (with values 1, 3, 3 and 1 for = 0, 1, 2 and 3) have been used to expand the product. To evaluate the integral, first use the identity
[formula] − � − 2 � 2 + � 2 � 2 = −( ) 2 + (S3) [/formula]
and the definitionof the complementary error function erfc, to obtain
[formula] � −( ) 2 + d ∞ ℎ = √ 2 exp �� 2 � 2 � erfc � ℎ − 2 � (S4) [/formula]
where ℎ is the lower limit of integration. Each term in Eq. (S2) is proportional to the integrand in Eq. (S4), with = . Hence the integral becomes
[formula] Φ = � d ∞ = 1 2 1 3 � � 3 � 3− 3 =0 exp �� 2 � 2 � erfc � ℎ − 2 � (S5) where ℎ = ln( 1 ⁄ ); c.f. Eq. (7) [/formula]
in the main text. Moreover, the denominator of Eq. (7)
represents the 3 rd moment of the lognormal distribution, which can be expressed as 1 3 9 2 /2 = sink poly = 1
[formula] 2 −9/(4 2 ) � � 3 � 3− 3 =0 exp �� 2 � 2 � erfc � ℎ − 2 � . (S6) [/formula]
The time dependence comes from = − / 1 and ℎ = ln( 1 ⁄ ); hence sink poly can be expressed in non-dimensional form as a function of / 1 . As in our previous work, we introduce a characteristic time for dissolution, denoted diss , from the requirement that the magnitude of the initial dissolution rate equals 0 diss ⁄ , where 0 is the initial value of . In other words, we demand that
[formula] 1 0 � d d � =0 = � d sink poly d � =0 = − 1 diss .( [/formula]
The factor 2 originates from the limit of the complementary error function obtained when its argument tends to −∞. Upon using using ′(0) = − / 1 and substituting = 1/( √2) we finally obtain
[formula] � d sink poly d � =0 = −3 −5 2 /2 1 � = − 1 diss (S9) [/formula]
where the last equality follows from Eq. (S7). Hence = 1 5 2 /2 (3 diss ) � . Upon defining = diss ⁄ , this yields = − 5 2 /2 /3 and ℎ = ln( 3 ⁄ ) + 5 2 2 ⁄ . Since = 1/( √2),
Eq. (S6) takes the form:
[formula] sink poly = 1 2 � � 3 � 3 =0 exp � ( 2 − 5 + 6) 2 2 � �− 3 � 3− × erfc � 2 ln( 3 ⁄ )⁄ + (5 − 2 ) 2√2 �. (S10) [/formula]
This is the final result.
## S.2 dissolution under non-sink conditions
Note that Eq. (12) in the main text can be rephrased as
[formula] d d = d d � d ( ) = 1 1 d d = − �1 − � (S11) where = � d ( ) 1 (S12) [/formula]
is a monotonic and thus invertible function of . Hence, dissolution from a given initial value of during a small time interval d under non-sink conditions corresponds to exactly the same change in as does dissolution under sink conditions during a another time interval d ′ given by
[formula] d ′ = �1 − � d .(S13) [/formula]
Since ( ) is invertible, the same is true for and the assertion follows. Using Eq. (9) in the main text, Eq. (S13) can be integrated to give
[formula] ′ = − 1 � ( ) 0 d = − ( ) . [/formula]
(S14)
Hence, the fraction of solid drug remaining after dissolution under non-sink conditions equals the fraction of drug remaining after dissolution under sink conditions provided the latter is evaluated at the retarded time ′ , i.e. nonsink ( ) = sink ( ′ ) = sink � − �.
(S15)
This important results is valid when (1) the dissolution rate is proportional to the difference between the drug solubility and the bulk concentration (i.e. to − ) as in the Noyes-Whitney equation) and (2) the permeation rate is proportional to the bulk concentration (i.e., to ). |
Effects of adenotonsillectomy on the growth of children with obstructive sleep apnoea-hypopnea syndrome (OSAHS): protocol for a systematic review
## Reviewer
## Jesús lópez alcalde cochrane associate centre of madrid (instituto ramón y cajal de investigación sanitaria / universidad francisco de vitoria) review returned
17-May-2019 GENERAL COMMENTS 1. Is the research question or study objective clearly defined? NO The authors state that "The aim of this systematic review is to comprehensively review the literature and synthesise relevant data to determine the effects of AT on the growth of children with OSAHS." a. Please consider keeping succinct the main objective: "The main objective of this review is to assess the effects of AT on the growth of children with OSAHS." b. The authors consider "AT related adverse events" as a secondary outcome. I suggest that you describe this as a secondary objective as well: "The secondary objective of this review is to determine AT related adverse effects in children with OSAHS."
3. Is the study design appropriate to answer the research question? NO a. The authors should justify why non-randomised designs are eligible for this review. b. I suggest that eligibility criteria emphasise specific features of study design (e.g. which parts of the study were prospectively designed) rather than 'labels' for study designs (such as prospective cohort studies). See examples in http://handbook-5-1.cochrane.org/chapter_13/13_2_2_guidance_and_resources_ava ilable_to_support_review.htm c. I suggest that authors detail if they will combine randomised and non-randomised studies in the same meta-analysis (which is usually discouraged). d. "For prospective cohort studies, healthy subjects (with age under 18 but without OSAHS) in a concurrent control group will also be considered." I recommend against including studies with healthy subjects, that is, without OSAHS, as these participants do not meet the review inclusion criteria (children with a diagnosis of OSAHS). 12. Are the study limitations discussed adequately? NO a. The authors should highlight that the potential for bias is likely to be greater for non-randomized studies compared with randomised trials.
## Version 1 -author response
Response to Reviewers bmjopen-2019-030866
Comments from Reviewer #1:
1. Review the quality of English language.
Response: Thank you for your comments. We have checked the manuscript and revised it to improve its language quality. Response: Thank you. We have added a sentence in the 'description of the intervention' section, referring to these articles that you have recommended.
## The introduction section is insufficient and needs
3. Bibliography needs to be updated. I recommend to the authors to insert in the references list the mentioned above articles.
Response: Thank you. We have replaced the previous references #2, #3, and #5 with more recent articles. We have also added the articles that you have recommended into the reference list.
Comments from Reviewer #2:
4. Is the research question or study objective clearly defined? NO
The authors state that "The aim of this systematic review is to comprehensively review the literature and synthesise relevant data to determine the effects of AT on the growth of children with OSAHS." a. Please consider keeping succinct the main objective:
"The main objective of this review is to assess the effects of AT on the growth of children with OSAHS."
Response: Thank you very much for your comments. Revision done.
b. The authors consider "AT related adverse events" as a secondary outcome. I suggest that you describe this as a secondary objective as well:
"The secondary objective of this review is to determine AT related adverse effects in children with OSAHS."
Response: Thank you very much.
1) We agree that it will be ideal to look at both benefits and harms at the same time, but after thorough consideration, we believe it is very difficult (if not impossible) to include all the benefits and harms of AT in a single systematic review.
2) In effect, there are many AT related complications and they have been summarized by recent systematic reviews specifically on this topic.1
3) Plus, our search strategy was designed to identify all relevant literature related to AT and growth, which means that it is not sensitive enough to identify all articles regarding harms of AT.
4) Therefore, we have deleted 'AT related adverse events' from our list of outcome measures.
2. Is the study design appropriate to answer the research question? NO a. The authors should justify why non-randomised designs are eligible for this review.
Response: As shown in a recent article regarding AT and craniofacial development, most of the relevant studies were observational.2 The main reason behind this is that it will be unethical to randomise children with OSAHS to a control group (without treatment). In addition, although RCTs are the gold standard to evaluate the effectiveness of interventions, prospective cohort can also provide valuable information and real-world evidence.
b. I suggest that eligibility criteria emphasise specific features of study design (e.g. which parts of the study were prospectively designed) rather than 'labels' for study designs (such as prospective cohort studies c. I suggest that authors detail if they will combine randomised and non-randomised studies in the same meta-analysis (which is usually discouraged).
Response: We have added one sentence to the data synthesis section to emphasize that data from RCTs and those from cohort studies will not be combined in any meta-analysis. As stated previously, we plan to explore the influence of study design (e.g. RCTs vs. cohort studies) in a subgroup analysis.
d. "For prospective cohort studies, healthy subjects (with age under 18 but without OSAHS) in a concurrent control group will also be considered."
I recommend against including studies with healthy subjects, that is, without OSAHS, as these participants do not meet the review inclusion criteria (children with a diagnosis of OSAHS).
Response: Thank you very much. We have removed healthy subjects from the 'types of participants' section. 1. We agree that statistical significance and clinical significance are different, and that the use of minimum clinically important difference (MCID) will be beneficial for interpreting results of our systematic review. However, we have failed to find from the literature any commonly accepted MCID for each of our outcome measures, in the context of our research question. This may be due to the low quantity of studies regarding this research question, and the fact that different studies have adopted different study design, outcome measure, source of samples (e.g. geographical location, age range), length of follow-up and methods to calculate an appropriate sample size. 4,5 We have added one sentence to the Article Summary table to reflect this limitation.
2. The lack of standard / agreed MCID is relatively common across medical areas.6 Therefore, the PRISMA guideline does not require the reporting of MCIDs. 7
3. However, we will consider the clinical significance of our findings in our assessment of evidence certainty using the standard GRADE approach, which is also a recommended and method to interpret clinical significance.7,8 e. "Growth related biomarker concentration or concentration Z score" I suggest that you define the biomarker/s that you will consider. a. The authors should justify why non-randomised designs are eligible Thank you for introducing the paragraph: "According to the Cochrane Handbook, in this review, prospective cohort studies should include two or more groups of participants receiving different intervention. At the same time, their identification of participants, assessment of baseline and allocation to intervention, assessment of outcomes, as well as the generation of hypotheses should be prospective.27"
First, I would propose to rewrite the sentence as now it seems to affirm that the Cochrane Handbook states that cohort studies should have two arms. However, chapter 13 of the Cochrane Handbook highlights that when including non-randomised studies in a review a clear description of the eligibility criteria should be provided, instead of providing just the 'study design labels'. The following sentence may reflect this: "For a prospective cohort study to be eligible in the review it should meet the following criteria: 1) it should include two or more groups of participants; 2) the identification of participants, the assessment of baseline, the allocation to intervention, and the assessment of outcomes should be done prospectively. We will not require that the study hypothesis was generated prospectively as this aspect is generally poorly reported." 4. Are the methods described sufficiently to allow the study to be repeated? : Search strategy I would propose to revise the search strategy as some aspects may be incorrect: 1)
Step #5, which correctly reflects the concept "Tonsillectomy", is not considered in step #16 2)
Step #16 is not complete as it should include #11 as well: #10 AND (#11 OR #12 OR #13) AND (#14 OR #15) Thus, I propose that #10 should read as follows: #5 AND #10 AND (#11 OR #12 OR #13) AND (#14 OR #15)
Also, if possible, I would propose to delete steps #14 and #15 from the search strategy: if they are maintained, eligible studies that did not contain these terms in the title or abstract will be lost.
## Version 2 -author response
Response to Reviewers bmjopen-2019-030866.R1
Comments from Reviewer #2:
2. Is the study design appropriate to answer the research question?
a. The authors should justify why non-randomised designs are eligible for this review.
Thank you for introducing the paragraph: "According to the Cochrane Handbook, in this review, prospective cohort studies should include two or more groups of participants receiving different intervention. At the same time, their identification of participants, assessment of baseline and allocation to intervention, assessment of outcomes, as well as the generation of hypotheses should be prospective.27" First, I would propose to rewrite the sentence as now it seems to affirm that the Cochrane Handbook states that cohort studies should have two arms. However, chapter 13 of the Cochrane Handbook highlights that when including non-randomised studies in a review a clear description of the eligibility criteria should be provided, instead of providing just the 'study design labels'. The following sentence may reflect this: "For a prospective cohort study to be eligible in the review it should meet the following criteria: 1) it should include two or more groups of participants; 2) the identification of participants, the assessment of baseline, the allocation to intervention, and the assessment of outcomes should be done prospectively. We will not require that the study hypothesis was generated prospectively as this aspect is generally poorly reported." Response: Thank you very much for your comments! We have revised the sentence and added the reference according to your suggestion. 4. Are the methods described sufficiently to allow the study to be repeated? : Search strategy I would propose to revise the search strategy as some aspects may be incorrect:Step #5, which correctly reflects the concept "Tonsillectomy", is not considered in step #16
## 2)
Step #16 is not complete as it should include #11 as well: #10 AND (#12 OR #13) AND (#14 OR #15) Thus, I propose that #10 should read as follows: #5 AND #10 AND (#11 OR #12 OR #13) AND (#14 OR #15) Also, if possible, I would propose to delete steps #14 and #15 from the search strategy: if they are maintained, eligible studies that did not contain these terms in the title or abstract will be lost.
Response: Thank you very much. We have corrected those mistakes and deleted steps #14 and #15. |
Cell-Based Transplantation versus Cell Homing Approaches for Pulp-Dentin Complex Regeneration
Regenerative dentistry has paved the way for a new era for the replacement of damaged dental tissues. Whether the causative factor is dental caries, trauma, or chemical insult, the loss of the pulp vitality constitutes one of the major health problems worldwide. Two regenerative therapies were introduced for a fully functional pulp-dentin complex regeneration, namely, cellbased (cell transplantation) and cell homing (through revascularization or homing by injection of stem cells in situ or intravenously) therapies, with each demonstrating advantages as well as drawbacks, especially in clinical application. The present review is aimed at elaborating on these two techniques in the treatment of irreversibly inflamed or necrotic pulp, which is aimed at regenerating a fully functional pulp-dentin complex.
# Introduction
Dental tissue regeneration requires the presence of specialized cells capable of the production of a tissue-specific extracellular matrix (ECM) [bib_ref] Engineering craniofacial structures: facing the challenge, Zaky [/bib_ref] [bib_ref] Comparison of the effect of PRP, PRF and induced bleeding in the..., Shivashankar [/bib_ref]. Stem/progenitor cells used in regenerative medicine are nonspecialized cells, demonstrating the ability of self-renewal and multilineage differentiation [bib_ref] Adult stem cell lines in regenerative medicine and reconstructive surgery, Conrad [/bib_ref]. The potential of stem/progenitor cells, whether endogenous or exogenous, to adapt to various environmental niche could be exploited in regenerative endodontics and pulp-dentin tissue regeneration [bib_ref] Stem/progenitor cell-mediated pulpal tissue regeneration: a systematic review and meta-analysis, El-Sayed [/bib_ref] [bib_ref] Adjunctive systemic antimicrobials for the non-surgical treatment of periodontitis, Khattri [/bib_ref] [bib_ref] Revascularization in immature and mature teeth with necrotic pulp: a clinical study, Narayan [/bib_ref]. Therapeutic application of stem/progenitor cells is mainly dependent on the utilization of the transplanted cells, on suitable scaffolds and in combination with various growth factors to generate fully functional biological tissues. Recently, the success demonstrated in animal models to repair/regenerate dental structures has paved the way for pulp-dentin organ regeneration approaches [bib_ref] Regeneration of tooth with allogenous, autoclaved treated dentin matrix with dental pulpal..., Chang [/bib_ref]. [fig_ref] Table 1: Summary of cell-based transplantation studies for pulp-dentin complex regeneration [/fig_ref] and [fig_ref] Figure 1: Cell-based transplantation method and sources of stem cells used for pulp-dentin complex... [/fig_ref]. A suggested approach to address problems related to pulp-dentin tissue regeneration relied principally on the use of various sources of stem/progenitor cells, combined with multiple scaffold systems and growth factors [bib_ref] Stem cell transplantation for pulpal regeneration: a systematic review, El-Sayed [/bib_ref]. Human mesenchymal stem/progenitor cells (MSCs) have been extracted from many areas of the human body, including the bone marrow, the skin as well as the perivascular, the adipose, and the dental tissues [bib_ref] Regenerative endodontics: barriers and strategies for clinical translation, Mao [/bib_ref] [bib_ref] Multilineage potential of adult human mesenchymal stem cells, Pittenger [/bib_ref] [bib_ref] Adult mesenchymal stem cells explored in the dental field, El-Sayed [/bib_ref]. Early trials and continuous animal studies were directed to investigate the effectiveness of cell-based transplantation on pulp healing and dentin regeneration [bib_ref] The interplay of dental pulp stem cells and endothelial cells in an..., Dissanayaka [/bib_ref] [bib_ref] Hydrogels and dentin-pulp complex regeneration: from the benchtop to clinical translation, Abbass [/bib_ref]. Autologous transplanted constructs of dental pulp stem/progenitor cells (DPSCs) in combination with platelet-rich fibrin (PRF) proved to promote the regeneration of pulp-dentin-like tissue inside dogs' root canals [bib_ref] Potential dental pulp revascularization and odonto-/osteogenic capacity of a novel transplant combined..., Chen [/bib_ref]. A further animal study employing human DPSCs and platelet-derived growth factor
## Cell-based transplantation for pulp-dentin complex regeneration
## Stem cells international
## Stem cells international
(PDGF) constructs transplanted into the emptied root canals of rats induced the creation of globular dentin-like structure with odontoblastic cells and pulp-like tissues [bib_ref] PDGFRβ+/c-kit+ pulp cells are odontoblastic progenitors capable of producing dentin-like structure in..., Cai [/bib_ref]. A trial to treat deliberately perforated pulp space of premolars of dogs using autogenous DPSCs, embedded in tricalcium phosphate (TCP) or treated dentin matrix (TDM) scaffolds, showed no dentin formation in all groups while cementum and vascular connective tissues were evident in all specimens [bib_ref] Histologic tissue response to furcation perforation repair using mineral trioxide aggregate or..., Bakhtiar [/bib_ref]. A further study examined microvascular endothelial cells (ECs) coimplanted with rat bone marrow MSCs in pulpotomized rat models. Interestingly, after 14 days, immunohistochemical examination demonstrated healing of the pulp with complete dentin bridge formation in teeth implanted with MSCs and ECs, while those implanted with MSCs lacked the completion of the formed dentin bridge . A further noninvasive regenerative pulpal approach was tested, using mobilized DPSCs freshly extracted from upper canine teeth of dogs, followed by autologous DPSCs transplantation in pulpectomized permanent teeth with apical closure. The study revealed that pulp tissue was completely regenerated 90 days following cell transplantation [bib_ref] Assessment of pulp regeneration induced by stem cell therapy by magnetic resonance..., Iohara [/bib_ref]. A novel trial on a rat model for dental pulp regeneration employed pulpotomized rat teeth, which were treated using buildups of rat bone marrow mesenchymal stem cells (BMMSCs). The tested buildups were implanted into the pulpotomized pulp chambers for 3, 7, or 14 days and then examined immunohistochemically. At 7 days, the pulp tissue was regenerated in almost the whole implantation area and regeneration continued to progress for 14 days with differentiation of odontoblast-like cells beneath the dentin at the margin of the implanted area evidenced by a detected nestin expression. Also, quantitative gene expression analysis disclosed the expression of sialophosphoprotein mRNA in the implanted area, suggesting the abundance of odontoblasts . Chitosan hydrogel scaffold containing autologous DPSCs was further transplanted in the necrotic immature permanent teeth of dogs, regenerating pulp-and dentin-like tissues with complete root maturation radiographically and histologically [bib_ref] Tissue engineering of necrotic dental pulp of immature teeth with apical periodontitis..., El Ashiry [/bib_ref]. However, not all the reported studies were successful. Implanting DPCs in TCP and TDM scaffolds, combined with transforming growth factor β, ascorbic acid 2-phosphate, and ascorbic acid 3-phosphate, did not promote the formation of a dentin bridge [bib_ref] Histologic tissue response to furcation perforation repair using mineral trioxide aggregate or..., Bakhtiar [/bib_ref]. Also, porcine
## Stem cells international
DPCs failed to heal or regenerate partial pulpotomy defects of minipigs. Moreover, hyperemia in the residual pulp and external root resorptions were evident in the radicular area of all the treated teeth [bib_ref] Implanted dental pulp cells fail to induce regeneration in partial pulpotomies, Mangione [/bib_ref]. On the contrary, another investigation demonstrates that when combining collagen scaffold with granulocyte colony-stimulating factor (G-CSF), a total recovery of the pulp tissue was achievable in the pulpectomized teeth [bib_ref] Assessment of pulp regeneration induced by stem cell therapy by magnetic resonance..., Iohara [/bib_ref].
It was appealing to seek more uncommon supplementary derivatives to enhance stem/progenitor cells' activation and differentiation, dragging attention towards nondental medications. An animal study reported that a common drug used to treat hyperlipidemia, Simvastatin (SIM), succeeded in stimulating canine DPSCs, promoting pulp and dentin regeneration following pulpotomy [bib_ref] Simvastatin Promotes Dental Pulp Stem Cell-induced Coronal Pulp Regeneration in Pulpotomized Teeth, Jia [/bib_ref]. Further animal studies suggested using glycogen synthase kinase (GSK-3) antagonists, a drug usually applied for the treatment of neurological disorders, which proved successful as a capping material of the pulpal exposure site, promoting dentin formation [bib_ref] Promotion of natural tooth repair by small molecule GSK3 antagonists, Neves [/bib_ref] [bib_ref] Translation approach for dentine regeneration using GSK-3 antagonists, Zaugg [/bib_ref]. Another animal study proved that pulp regeneration was enhanced in aged dogs' teeth by trypsin pretreatment of allogenically transplanted mobilized DPSCs [bib_ref] Treatment of pulpectomized teeth with trypsin prior to transplantation of mobilized dental..., Iohara [/bib_ref].
A case report treating accidental root perforation of a mature permanent tooth, utilizing allogenic umbilical cord mesenchymal stem cells (UCMSCs) encapsulated in a plateletpoor plasma-(PPP-) based bio scaffold, demonstrated a clinically normal pulpal tissue in terms of vitality testing, palpation, and percussion testing at six-month and one-year follow-ups [bib_ref] Allogeneic cellular therapy in a mature tooth with apical periodontitis and accidental..., Cordero [/bib_ref]. Moreover, two case reports showed a successful management of periapical lesions in permanent teeth treated with stem/progenitor cells from human exfoliated deciduous teeth (SHED), with the treated teeth responding normally to electric pulp testing and periapical tissue healing observed and maintained up to one year [bib_ref] Allogeneic stem cells derived from human exfoliated deciduous teeth (SHED) for the..., Prasad [/bib_ref].
Collectively, cell-based therapeutic applications in the dental field and specifically dentin-pulp tissue regeneration still face a number of challenges. Future strategies should be directed towards overcoming these challenges and obstacles using an ideal combination of growth factors with properly matching scaffolds [bib_ref] Histologic tissue response to furcation perforation repair using mineral trioxide aggregate or..., Bakhtiar [/bib_ref] [bib_ref] Implanted dental pulp cells fail to induce regeneration in partial pulpotomies, Mangione [/bib_ref]. Secure and controllable practice must be strictly followed to translate stem/progenitor cell research into human models, starting from protocols of stem/progenitor cells' tissue harvesting, the biocompatibility of the used scaffolds and biomaterials involved, and the safety of the technique itself and the predicted outcome [bib_ref] Strategic tools in regenerative and translational dentistry, Tatullo [/bib_ref] [bib_ref] Recent challenges with stem cell banking, Dricu [/bib_ref]. Finally, the endless mix and match trials between scaffolds of different origins, as well as electing the suitable growth factor/biological mediator, could govern the success or failure of regenerating a specialized tissue when employing the stem cell-based therapy [bib_ref] Dental pulp stem cells: advances to Applications, Tsutsui [/bib_ref].
1.2. Stem/Progenitor Cell Homing. As mentioned above for pulp-dentin complex regeneration, two strategies could be applied, namely, the cell-based transplantation therapy or the cell homing. In the latter, the regeneration is accomplished via chemotaxis of host endogenous cells to the injured tissue via biological signaling molecules. Stem/progenitor cell homing can be defined as the potential of stem/progenitor cells, whether endogenous or exogenous, to migrate into an environmental niche. MSCs can be delivered in situ or intrave-nously, or they can be recruited to sites of injury, through migration and homing [bib_ref] Mesenchymal stem cell biodistribution, migration, and homing in vivo, Zhao [/bib_ref]. Clinically, cell homing for pulpdentin complex regeneration might be simpler and more economical to perform compared to the cell-based therapy and readily performed by clinicians without special training. [fig_ref] Figure 2: MSC homing mechanisms and different approaches for enhancing MSC homing [/fig_ref]. Homing approaches can be either systemic or nonsystemic. In nonsystemic homing, MSCs are locally transplanted at the selected tissue and are then directed to the region of injury through a chemokine gradient. Oppositely, in systemic homing, MSCs are delivered or recruited endogenously into the circulation and then undergo a series of processes, leaving the bloodstream and moving towards the site of injury. These complex processes involve tethering and rolling, activation, arrest, transmigration or diapedesis, and migration [bib_ref] The lymphocyte homing receptors: gatekeepers of the multistep paradigm, Sackstein [/bib_ref]. Tethering is mediated by selectins on endothelial cells. MSCs exhibit CD44, which binds to the selectins and starts rolling along blood vessels [bib_ref] Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell..., Sackstein [/bib_ref]. This is followed by chemokinemediated activation [bib_ref] Local irradiation not only induces homing of human mesenchymal stem cells at..., François [/bib_ref]. MSCs express the chemokine receptors CXCR4 [bib_ref] A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4..., Wynn [/bib_ref] and CXCR7 [bib_ref] CXCR-7 receptor promotes SDF-1α-induced migration of bone marrow mesenchymal stem cells in..., Wang [/bib_ref] [bib_ref] Overexpression of CXCR7 promotes mesenchymal stem cells to repair phosgene-induced acute lung..., Shao [/bib_ref]. The stromal cellderived factor (SDF-1) is the ligand to these receptors, where it binds to them to enhance homing to different tissues. Then, comes the process of arrest mediated by integrins, mainly by CD49d (α4β1), which unites with VCAM-1 (CD106) present on endothelial cells [bib_ref] Microenvironmental interaction between hypoxia and endothelial cells controls the migration ability of..., Choi [/bib_ref]. In order to cut across the endothelial basement membrane, a process known as diapedesis or transmigration, MSCs produce matrix metalloproteinases (MMPs) mainly MMP-1, which plays a crucial role in tissue infiltration by MSCs [bib_ref] IL-1β-induced matrix metalloprotease-1 promotes mesenchymal stem cell migration via PAR1 and Gprotein-coupled..., Chen [/bib_ref]. Finally, MSCs migrate to injury sites. This step is regulated by chemotactic signals, produced as a reaction to tissue impairment. Numerous growth factors, such as insulinlike growth factor IGF-1 and platelet-derived growth factor (PDGF), can act as chemokines for MSCs [bib_ref] The in vitro migration capacity of human bone marrow mesenchymal stem cells:..., Ponte [/bib_ref]. Moreover, tumor necrosis factor (TNF-α) increases MSCs movement towards chemokines by increasing their expression of CCR3, CCR4, and CCR2 receptors [bib_ref] Stem/progenitor cell-mediated pulpal tissue regeneration: a systematic review and meta-analysis, El-Sayed [/bib_ref] [bib_ref] The periodontal stem/progenitor cell inflammatory-regenerative cross talk: a new perspective, Fawzy El-Sayed [/bib_ref] [bib_ref] Oral mesenchymal stem/progenitor cells: the immunomodulatory masters, Zhou [/bib_ref]. In addition, the inflammatory cytokine interleukin-(IL-) 8 was proved to enhance migration of MSCs towards regions of injury [bib_ref] Kidney cancer cells secrete IL-8 to activate Akt and promote migration of..., Liang-Kuan [/bib_ref] [bib_ref] IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory..., Bayo [/bib_ref] and further promotes them to produce regenerative growth factors, such as vascular endothelial growth factor (VEGF) [bib_ref] IL-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells..., Hou [/bib_ref].
## Stem/progenitor cell homing mechanisms
## Routes of administration and delivery methods.
One important point in MSCs transplantation and their consequent therapeutic efficiency is the route of administration to provide the ultimate regenerative benefits with the least adverse effects. The most common delivery methods for MSCs are either by intravenous (IV) or intra-arterial infusion (IA) or by direct intratissue injection [bib_ref] Mesenchymal stem cell delivery routes and fate, Kurtz [/bib_ref]. Several experimental studies proved the superiority of IA and IV delivery modes over other delivery routes [bib_ref] Efficacy and dosedependent safety of intra-arterial delivery of mesenchymal stem cells in..., Yavagal [/bib_ref] [bib_ref] Effects of intravenous administration of allogenic bone marrow-and adipose tissue-derived mesenchymal stem..., Gutiérrez-Fernández [/bib_ref]. The IV route was proved to be the most convenient route for MSCs transplantation. It is less traumatic and reproducible and enhances widespread distribution in the affected regions, enhancing various biological effects [bib_ref] Stem cell therapy in ischemic stroke: role of IV and intra-arterial therapy, Misra [/bib_ref]. However, this delivery method in nearly all cases causes entrapment of MSCs in the lungs, causing undesirable adverse effects, including embolisms. The reason for this lung entrapment relies probably on the amalgamation of physiological and mechanical factors, such as [bib_ref] Revascularization in immature and mature teeth with necrotic pulp: a clinical study, Narayan [/bib_ref] Stem Cells International the small size of blood capillaries, the vast network of capillaries, and the great adhesive characteristics of MSCs. It was also demonstrated that some cells could produce calcium deposits within the capillaries [bib_ref] Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the..., Aguilar [/bib_ref]. On the contrary, the IA route can be more efficient, as it provides a straightforward route to the injury site with an increased degree of cellular endurance and engraftment [bib_ref] Effects of administration route on migration and distribution of neural progenitor cells..., Li [/bib_ref] [bib_ref] Pulmonary passage is a major obstacle for intravenous stem cell delivery: the..., Fischer [/bib_ref]. Several studies proved the superiority of IA delivery route over the IV one. They demonstrated enhanced functional and histological results in IA delivery compared with IV injection of MSCs [bib_ref] Efficacy and dosedependent safety of intra-arterial delivery of mesenchymal stem cells in..., Yavagal [/bib_ref] [bib_ref] Intra-arterial transplantation of bone marrow mononuclear cells immediately after reperfusion decreases brain..., Kamiya [/bib_ref]. IA transplantation of MSCs increases cellular migration, cellular density, and the number of homing MSCs to the target tissue, when compared to IV injection [bib_ref] Increased survival and migration of engrafted mesenchymal bone marrow stem cells in..., Hellmann [/bib_ref] [bib_ref] Feasibility and efficacy of intraarterial administration of mesenchymal stem cells in an..., Kim [/bib_ref]. Du et al. in a comparative study demonstrated greater angiogenesis and increased functional recovery with IA transplantation compared to IV injection utilizing human BM-MSCs in a rat model of ischemia [bib_ref] Intra-arterial delivery of human bone marrow mesenchymal stem cells is a safe..., Du [/bib_ref]. Lundberg et al. confirmed these findings in a model of traumatic brain injury [bib_ref] Targeted intra-arterial transplantation of stem cells to the injured CNS is more..., Lundberg [/bib_ref]. The main reason for the superiority of IA transport over IV mode is that the IA approach can bypass the pulmonary circulation and filtering organs, such as liver and spleen [bib_ref] Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia, Pendharkar [/bib_ref] , thereby avoiding MSCs entrapment in lungs and liver [bib_ref] Pulmonary passage is a major obstacle for intravenous stem cell delivery: the..., Fischer [/bib_ref] , with a significant rise in number of cells with a more consistent cellular dissemination in target tissues [bib_ref] Intracarotid transplantation of bone marrow stromal cells increases axon-myelin remodeling after stroke, Shen [/bib_ref] [bib_ref] Effects of granulocyte-colony stimulating factor on the repair of balloon-injured arteries, Wu [/bib_ref]. This will eventually lead to increased cell homing and improved therapeutic outcomes [bib_ref] Intra-arterial delivery of human bone marrow mesenchymal stem cells is a safe..., Du [/bib_ref].
However, a probable limitation for the IA route is the possibility of vascular blockage in small arterioles and capillaries resulting in strokes. This may be attributed to the existence of large MSCs in the 20-50 μ size range [bib_ref] Cell size and velocity of injection are major determinants of the safety..., Janowski [/bib_ref] [bib_ref] The size of mesenchymal stem cells is a significant cause of vascular..., Ge [/bib_ref]. Several attempts have been performed to enhance the safety of IA transplantation via regulating infusion velocity and cell dosage [bib_ref] Cell size and velocity of injection are major determinants of the safety..., Janowski [/bib_ref] [bib_ref] The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal..., Cui [/bib_ref]. Moreover, real-time MRI could provide a useful tool in making the procedure more accurate and predictable, which is of ultimate importance for translation to clinical practice [bib_ref] Real-time MRI for precise and predictable intra-arterial stem cell delivery to the..., Walczak [/bib_ref].
Direct injection delivery mode has the advantage of accurate localization of cells, despite being invasive. However, it has been proved that aside from the delivery route, only 1∼5% of delivered cells disseminate within the target region for regeneration. The count of cells in the target region may thus be enhanced by maximizing the injection volume or enriching the cell concentration in the injectable volume [bib_ref] A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery..., Freyman [/bib_ref] [bib_ref] Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow..., Müller-Ehmsen [/bib_ref] [bib_ref] Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous Delivery, Hou [/bib_ref]. In addition, the expression of adhesion molecules can promote homing of delivered MSCs [bib_ref] Overexpression of VLA-4 in glial-restricted precursors enhances their endothelial docking and induces..., Jablonska [/bib_ref] [bib_ref] Single-cell, high-throughput analysis of cell docking to vessel wall, Andrzejewska [/bib_ref]. In this context, several approaches have been made to enhance MSC homing efficacy. [fig_ref] Figure 2: MSC homing mechanisms and different approaches for enhancing MSC homing [/fig_ref]. Cellular homing relies principally on specialized molecular interactions, not just passive diffusion. One of the main challenges facing MSCs therapeutic applications is enhancing their homing abilities [bib_ref] Non-invasive imaging reveals conditions that impact distribution and persistence of cells after..., Scarfe [/bib_ref].
## Enhancing msc
## Stem cells international
Among the challenges is the fact that the expression of homing molecules, as CXCR4, is relatively low on MSCs [bib_ref] A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4..., Wynn [/bib_ref] [bib_ref] Human adult CD34-progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7,..., Lüttichau [/bib_ref] , and the in vitro expansion of MSCs further decreases the expression of their homing molecules [bib_ref] Human bone marrow stromal cells express a distinct set of biologically functional..., Honczarenko [/bib_ref] [bib_ref] Primary murine MSC show highly efficient homing to the bone marrow but..., Rombouts [/bib_ref]. Thus, numerous approaches have been suggested to enhance MSC homing. Among these is targeted delivery, which relies on direct delivery of MSCs into the target region, employing nonsystemic rather than systemic homing [bib_ref] Effects of different mesenchymal stromal cell sources and delivery routes in experimental..., Antunes [/bib_ref]. In addition, magnetic guidance of MSCs to target tissues proved greater homing efficiency [bib_ref] Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by..., Vittorio [/bib_ref]. Furthermore, genetic modifications of MSCs via overexpression of homing factors such as VLA-4 and CXCR4 through viral transduction proved increased efficiency [bib_ref] Engineered mesenchymal stem cells for targeting solid tumors: therapeutic potential beyond regenerative..., Cheng [/bib_ref] [bib_ref] Integrin α4 overexpression on rat mesenchymal stem cells enhances transmigration and reduces..., Cui [/bib_ref]. Cell surface engineering approaches were suggested to modify the selectin ligand CD44, via transforming it into HCELL (the ligand for E-and L-selectin that MSCs utilize for homing), as MSCs normally express CD44, but not HCELL [bib_ref] Mesenchymal stem cells transmigrate between and directly through tumor necrosis factor-α-activated endothelial..., Teo [/bib_ref]. It was further demonstrated that coating MSCs with hyaluronic acid could upregulate CD44 expression [bib_ref] Hyaluronic acid coatings as a simple and efficient approach to improve MSC..., Corradetti [/bib_ref]. Moreover, hypoxic conditions enhance hypoxia-inducible factor-(HIF-) 1a, which upregulates the expression of CXCR4 [bib_ref] Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation, Annabi [/bib_ref] , CX3CR1 [bib_ref] Shortterm exposure of multipotent stromal cells to low oxygen increases their expression..., Hung [/bib_ref] , and CXCR7 [bib_ref] Hypoxic preconditioning advances CXCR4 and CXCR7 expression by activating HIF-1α in MSCs, Liu [/bib_ref] [bib_ref] LincRNA-p21 promotes mesenchymal stem cell migration capacity and survival through hypoxic preconditioning, Meng [/bib_ref].
A further strategy addressed modifying the target tissues, via overexpression of chemokines or via implantation of chemokine-coated scaffolds [bib_ref] Biodegradable gelatin hydrogels incorporating fibroblast growth factor 2 promote healing of horizontal..., Narita [/bib_ref]. This allows tissues to be a more appealing target for homing MSCs. Moreover, irradiation of target tissues increases the expression of SDF-1, upregulating in MSC engraftment [bib_ref] Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells..., Chapel [/bib_ref] [bib_ref] Human mesenchymal stem cells home specifically to radiation-injured tissues in a non-obese..., Mouiseddine [/bib_ref] and homing [bib_ref] Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem..., Ponomaryov [/bib_ref]. Pulsed ultrasound applied to the target tissue may also enhance MSC homing [bib_ref] Improving the therapeutic efficacy of mesenchymal stromal cells to restore perfusion in..., Tebebi [/bib_ref] , via altering gene expression of cytokines as bone morphogenic protein-2 (BMP-2), interleukins (IL-1α, IL-6, and IL-10), TNF-α, and growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), VEGF, and PDGF [bib_ref] The role of ultrasound in enhancing mesenchymal stromal cell-based therapies, Liu [/bib_ref] , causing disorganization of endothelial linings, enhancing vascular permeability, increasing secretion of SDF-1 on the tissue of interest, and upregulating CXCR4 expression [bib_ref] Ultrasound-targeted microbubble destruction improves the migration and homing of mesenchymal stem cells..., Li [/bib_ref].
1.6. Cell Homing for Pulp-Dentin Complex Regeneration (Revascularization) (Table 2 and [fig_ref] Figure 3: Different sources of stem/progenitor cells in the oral cavity and steps of... [/fig_ref]. Regenerative endodontics represents an alternative to root canal treatment, which is aimed at replacing the inflamed and necrotic pulp tissue with regenerated pulp-like tissue [bib_ref] Investigation of the regenerative potential of necrotic mature teeth following different revascularisation..., Fahmy [/bib_ref]. In this context, revascularization approaches of affected dental pulp were suggested as an innovative strategy to overcome the drawbacks associated with classical root canal treatment methods (e.g., fracture of the teeth and loss of vitality) [bib_ref] Current and future options for dental pulp therapy, Morotomi [/bib_ref]. A human study on mature necrotic teeth with large radiolucency concluded that the regenerative endodontic approaches have a success rate similar to nonsurgical endodontic treatment as a therapeutic alternative for mature necrotic teeth with radiolucency [bib_ref] Regenerative endodontic procedures in necrotic mature teeth with periapical radiolucencies: a preliminary..., Arslan [/bib_ref]. It could maintain the pulp vitality, leading to a reduction of apical periodontitis and enhance the periapical healing mechanism [bib_ref] Outcome of secondary root canal treatment: a systematic review of the literature, Ng [/bib_ref]. Basically, pulp revascularization is the reestablishment of angiogenesis inside the root canal but without the repopulation of odontoblasts, while the pulp regeneration means angiogenesis with presence of odontoblastic layer lining the dentinal surface, nociceptive as well as parasympathetic and sympathetic nerve fibers, interstitial fibroblasts, and stem/progenitor cells, which replenish the pulp cells in the newly regenerated pulp tissue [bib_ref] Regenerative endodontics: a paradigm shift in clinical endodontics, Pulyodan [/bib_ref]. According to American Association of Endodontists' (AAE) Clinical Considerations for a Regenerative Procedure, the primary goal should be the resolution of clinical symptoms/signs and elimination of apical periodontitis. The secondary goal should address the canal wall thickening and/or continued root maturation [bib_ref] May JADA Cover, Gluskin [/bib_ref].
Pulp revascularization could be considered a type of cell homing strategy for pulp-dentin complex regeneration. This clinical procedure depends on the delivery of a blood clot (scaffold) inside the root canal, growth factors (mainly from platelets and dentin), and stem/progenitor cells. The stem/progenitor cells of interest in revascularization are SCAP (stem cells of apical papilla) because of their anatomical positioning immediately adjacent to the termination of the root canal system, permitting easy cell delivery to the root canal [bib_ref] Evaluation of the delivery of mesenchymal stem cells into the root canal..., Lovelace [/bib_ref] [bib_ref] Tolllike receptor expression profile of human stem/progenitor cells form the apical papilla, Fehrmann [/bib_ref] and the greater superiority for dentin-like tissue formation [bib_ref] Mesenchymal stem cells derived from dental Tissuesvs. those from other sources: their..., Huang [/bib_ref] [bib_ref] Stem cells from the apical papilla (SCAP) as a tool for endogenous..., Nada [/bib_ref]. The root canal system is first disinfected with a combination of antibiotics or calcium hydroxide. In the second visit, the irrigation protocol during this clinical procedure is very critical as for the regeneration procedure to be successful; the irrigants should have bactericidal/bacteriostatic properties as well as an ability to promote survival and proliferative capacity of the patient's stem/progenitor cells. The irrigation protocols that include 17% EDTA promoted SCAP survival and attachment to the root canal dentinal wall [bib_ref] Effect of irrigants on the survival of human stem cells of the..., Trevino [/bib_ref].
Animal studies were performed to examine the tissues formed after revascularization, demonstrating ingrowth of cellular cementum-like tissues, formation of pulp-like tissue, thickening of the canal walls, closure of the root apex, and disappearance of periapical radiolucency [bib_ref] Histologic comparison between platelet-rich plasma and blood clot in regenerative endodontic treatment:..., Zhang [/bib_ref] [bib_ref] Pulp revascularization of immature dog teeth with apical periodontitis, Thibodeau [/bib_ref]. Histological sections were also performed in humans after fracture of a revascularized immature tooth (3.5 weeks after revascularization), showing that the canal was filled with loose connective tissue and a layer of flattened odontoblast-like cells lined along the predentin. Layers of epithelial-like cells, similar to the Hertwig's epithelial root sheath, further surrounded the root apex [bib_ref] Histologic observation of a human immature permanent tooth with irreversible pulpitis after..., Shimizu [/bib_ref]. Alternative endodontic therapy is now possible, using the patient's own blood samples, where PRF and PRP are introduced inside the root canal. Easier and successful efforts for pulp revascularization and pulp tissue regeneration were reported by using evoked bleeding (EB), where the blood clot acts as a protein scaffold and interacts with endogenous stem cells and growth factors already abundant in the adjacent bone marrow tissues [bib_ref] Pulp revascularization/revitalization of bilateral upper necrotic immature permanent central incisors with blood..., Rizk [/bib_ref]. The highest reported cytokines and growth factors found in PRF are IL-1β, IL-6, IL-4, TNF-α, PDGF, VEGF, IGF-1, EGF, and transforming growth factor β1 (TGFβ1) [bib_ref] Platelet-rich fibrin: basics of biological actions and protocol modifications, Pavlovic [/bib_ref] , while PRP contains FGF, PDGF, VEGF, IGF-1, EGF, and TGFβ1 [bib_ref] Platelet rich plasma: a short overview of certain bioactive components, Pavlovic [/bib_ref]. The superiority of PRP came from releasing an elevated number of proteins at early time intervals whereas PRF showed a sustained production of bioactive molecules throughout a duration of 10 days [bib_ref] Comparative release of growth factors from PRP, PRF, and advanced-PRF, Kobayashi [/bib_ref]. In the blood clot technique, the growth factors are released from the dentin matrix after conditioning of the dentin using EDTA (ethylene diamine tetra acetic acid) 17%-pH 7.2 during the revascularization technique. Thus, the dentin matrix acts as a reservoir of bioactive molecules, which provides a vital source of cell signaling molecules for initiating repair, including TGFβ1, bone [bib_ref] Regeneration of tooth with allogenous, autoclaved treated dentin matrix with dental pulpal..., Chang [/bib_ref] Stem Cells International 14 Stem Cells International morphogenetic proteins (BMPs), and VEGF [bib_ref] Growth factor liberation and DPSC response following dentine conditioning, Sadaghiani [/bib_ref]. PRF has proved to be an appropriate substitute to the blood clot technique, especially in cases where bleeding was very difficult to be obtained [bib_ref] Pulp revascularization/revitalization of bilateral upper necrotic immature permanent central incisors with blood..., Rizk [/bib_ref]. PRP and blood clotting technique used as scaffolds in immature traumatized permanent teeth with necrotic pulps also gave very good results [bib_ref] The effect of platelet-rich plasma as a scaffold in regeneration/revitalization endodontics of..., Elsheshtawy [/bib_ref]. In a clinical study on 30 patients with maxillary necrotic permanent immature central incisors, treating one group with PRP and the other with PRF scaffolds, teeth survived during the 12-month follow-up period. The teeth revealed marginal increase in radiographic root width and length, an increased periapical bone density, and a narrowing in apical diameter [bib_ref] Comparative evaluation of platelet rich plasma (PRP) versus platelet rich fibrin (PRF)..., Rizk [/bib_ref]. Other studies compared the effect of PRF, PRP, and the blood clot technique in the revascularization of necrotic teeth with open apex, demonstrating continued root development and maintenance of functionality, following different follow-up periods, yet with some teeth not responding to vital testing [bib_ref] Comparison of the effect of PRP, PRF and induced bleeding in the..., Shivashankar [/bib_ref] [bib_ref] Adjunctive systemic antimicrobials for the non-surgical treatment of periodontitis, Khattri [/bib_ref] [bib_ref] Revascularization in immature and mature teeth with necrotic pulp: a clinical study, Narayan [/bib_ref] [bib_ref] Dental pulp revascularization of necrotic permanent teeth with immature apices, El Ashiry [/bib_ref] [bib_ref] Comparative study between revitalization of necrotic immature permanent anterior teeth with and..., Ragab [/bib_ref] [bib_ref] Revascularization with and without platelet-rich plasma in nonvital, immature, anterior teeth: a..., Jadhav [/bib_ref] [bib_ref] Regenerative evaluation of immature roots using PRF and artificial scaffolds in necrotic..., Mittal [/bib_ref] [bib_ref] Efficacy of revascularization to induce apexification/apexogensis in infected, nonvital, immature teeth: a..., Shah [/bib_ref] [bib_ref] Pulp revascularization of immature teeth with apical periodontitis: a clinical study, Ding [/bib_ref] [bib_ref] Pulp and periodontal regeneration of an avulsed permanent mature incisor using platelet-rich..., Priya [/bib_ref] [bib_ref] Platelet-rich fibrinmediated revitalization of immature necrotic tooth, Narang [/bib_ref] [bib_ref] Comparative evaluation of platelet-rich fibrin and mineral trioxide aggregate as pulpotomy agents..., Keswani [/bib_ref]. A further investigation induced bleeding in root canals and used PRF in mature necrotic teeth, showing a regain in pulp sensibility [bib_ref] Assessment of regaining pulp sensibility in mature necrotic teeth using a modified..., Nageh [/bib_ref].
In a further study, Kim et al. were able to regenerate toothlike structure using cell homing approach [bib_ref] Anatomically shaped tooth and periodontal regeneration by cell homing, Kim [/bib_ref].
## Pixagon art
BMMSCs are able to differentiate towards connective tissue, bone, adipose, muscle tissues, cartilage and endothelium and also towards other lineages such as renal, lung, hepatic and neural cells.
## Stem cells international
Still, one of the drawbacks of the revascularization found among cases treated with this approach is the occasional intracanal calcification, which in some cases may progress to complete obliteration of root canals, affecting the normal function of the dental pulp tissues. This drawback could be attributed to multiple contributing factors such as the type of medicaments and the induction of intracanal bleeding [bib_ref] Revascularization-associated intracanal calcification: assessment of prevalence and contributing factors, Song [/bib_ref] [bib_ref] Responses of immature permanent teeth with infected necrotic pulp tissue and apical..., Chen [/bib_ref]. A recent review article evaluated the long-term outcomes of the apexification and the regenerative techniques in treating traumatized immature teeth with pulp necrosis and apical periodontitis, showing that the endodontic regenerative techniques appeared superior to apexification techniques in terms of root lengthening and root wall thickening [bib_ref] What is the best long-term treatment modality for immature permanent teeth with..., Wikström [/bib_ref].
## Cell-free approach for pulp-dentin complex regeneration.
Relying on "cell homing" concept, the cell-free approach is aimed at regeneration by enhancing proliferation, migration, and differentiation of intuitive stem/progenitor cells present near the root apex [bib_ref] Dental pulp regenerationviacell homing, Eramo [/bib_ref]. It was proposed that stem/progenitor cells' niches could initiate an appropriate microenvironment by releasing immunoregulatory molecules and enhancing paracrine effects to promote the differentiation of endogenous stem cells [bib_ref] Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications, Wang [/bib_ref] [bib_ref] Mesenchymal stem cells as promoters, enhancers, and playmakers of the translational regenerative..., Ballini [/bib_ref]. Additionally, natural molecules and bioactive compounds have been proved to promote dentinogenesis [bib_ref] Polydatin, natural precursor of resveratrol, promotes osteogenic differentiation of mesenchymal stem cells, Di Benedetto [/bib_ref] [bib_ref] Exosomes as biomimetic tools for stem cell differentiation: applications in dental pulp..., Huang [/bib_ref].
Conditioned medium (CM) can be described as the molecules released from living cells into the surrounding extracellular environment [bib_ref] Cell secretome based drug substances in regenerative medicine: when regulatory affairs meet..., Beer [/bib_ref]. CM was found to stimulate cellular immunomodulation, proliferation, migration, and tissue regeneration [bib_ref] Cell secretome based drug substances in regenerative medicine: when regulatory affairs meet..., Beer [/bib_ref] [bib_ref] Stem cell paracrine actions and tissue regeneration, Baraniak [/bib_ref] [bib_ref] A review of therapeutic effects of mesenchymal stem cell secretions and induction..., Madrigal [/bib_ref] as it contains abundant amounts of proteins, lipids, nucleic acid, growth factors, cytokines, chemokines, and extracellular vesicles [bib_ref] Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease, Ranganath [/bib_ref]. A recent study combined hDPSC conditioned medium with MTA for direct vital pulp therapy. It was assumed that the abundance of angiogenic growth factors such as PDGF, FGF, and VEGF [bib_ref] Quantification of angiogenic growth factors released by human dental cells after injury, Tran-Hung [/bib_ref] and immunomodulatory cytokines such as IL-6 and IL-8 [bib_ref] Taking a bite out of spinal cord injury: do dental stem cells..., Bianco [/bib_ref] secreted by DPSCs and collected in hDPSCs' conditioned medium could modulate the inflammatory and regenerative processes in the dental pulp tissue, improve the orientation of the newly formed hard tissue, and enhance formation of dentin bridges [bib_ref] Effect of human dental pulp stem cell conditioned medium in the dentin-pulp..., Sarra [/bib_ref].
Extracellular vesicles (EVs) derived from MSCs function as paracrine mediators in tissue regeneration and repair and resemble to a great extent the therapeutic efficacy of parental MSCs [bib_ref] Concise review: MSCderived exosomes for cell-free therapy, Phinney [/bib_ref]. Extracellular vesicles (EVs) are defined by the MISEV2014 and the updated MISEV2018 as "particles naturally released from the cell that are delimited by a lipid bilayer membrane and are incapable of self-replication, i.e., do not contain a functional nucleus." EVs are a collective name including many subtypes of cell-released, membranous particles, known as microvesicles, microparticles, exosomes, oncosomes, ectosomes, and apoptotic bodies. EVs are characterized by the presence of luminal and transmembrane proteins and attenuation of extracellular or cellular non-EV proteins [bib_ref] Minimal experimental requirements for definition of extracellular vesicles and their functions: a..., Lötvall [/bib_ref] [bib_ref] Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement..., Théry [/bib_ref]. The term "exosomes" usually refers to EVs that are formed by the endosomal system, opposite to ectosomes (microparticles and microvesicles) that bud from the plasma membrane. Particularly, intraluminal vesicles are unleashed into the extracellular environ-ment as exosomes when the multivesicular body coalesces with the plasma membrane [bib_ref] Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications, Witwer [/bib_ref]. Exosomes are identified by their small diameter (40-100 nm) [bib_ref] Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy, Lee [/bib_ref]. Moreover, they possess large amounts of tetraspanins (CD81, CD9, and CD63) and annexins, which are commonly used for their characterization [bib_ref] Therapeutic potential of mesenchymal stem cell-derived microvesicles, Biancone [/bib_ref].
Additionally, exosome vesicles were claimed to possess the ability to induce odontogenesis and augment dental pulp regeneration [bib_ref] Dental stem cellderived secretome/conditioned medium: the future for 22 Stem Cells International..., Moshy [/bib_ref]. Accordingly, a study based on extracted exosome-like vesicles from rat Hertwig's epithelial root sheath (HERS) was tested. Dental pulp cells (DPCs) were united with HERS cell-derived exosome-like vesicles in an in vivo tooth root slice model, triggering the regeneration of hard reparative dentin-like tissue and soft tissue rich in blood vessels and neurons [bib_ref] Exosome-like vesicles derived from Hertwig's epithelial root sheath cells promote the regeneration..., Zhang [/bib_ref]. Moreover, in an interesting study, when SCAPderived exosomes (SCAP-Exo) were put into a root slice containing BMMSCs and transplanted into immunocompromised mice, dentin and dental pulp-like tissues were formed in the root canal. Besides, when SCAP-Exo were evaluated in vitro, it was reported that dentin sialophosphoprotein expression and hard tissue deposition in BMMSCs treated with SCAP-Exo were significantly upregulated [bib_ref] Exosomes derived from stem cells from the apical papilla promote dentine-pulp complex..., Zhuang [/bib_ref]. In another study, EVs were derived from DPSCs and EVsfibrin gel constructs were manufactured as an in situ delivery system. Afterwards, DPSCs and endothelial cells were cocultured in the constructs. It was reported that EVs-fibrin gels promoted dental pulp regeneration by stimulating collagen deposition and enhancing angiogenesis through upregulating the expression of VEGF [bib_ref] Extracellular vesicles-loaded fibrin gel supports rapid neovascularization for dental pulp regeneration, Zhang [/bib_ref].
It is further well established that the usage of MSC-derived EVs possesses numerous advantages. First, it overcomes the ethical issues that limit the clinical translation of MSCs. Second, transplanting cells, which might have mutated DNA, can be avoided. Third, the dose of delivered MSCs rapidly declines posttransplant, in contrast to MSC-derived vesicles, which could attain a higher dose. Fourth, EVs are relatively small and can circulate easily, opposite to MSCs, which are too large to circulate smoothly via capillaries. However, the main disadvantage of utilizing MSC-derived vesicles is that they are static and cannot be produced in vivo. Moreover, the efficacy of EVs requires standard parameters to produce EVs of known content, develop storage techniques that preserve vesicle efficacy, and assess their therapeutic potential in well-controlled clinical trials [bib_ref] Concise review: MSCderived exosomes for cell-free therapy, Phinney [/bib_ref].
# Conclusion
Regenerative dentistry is no longer a dream, thanks to the current efforts to imply stem/progenitor cell-based techniques to enhance the regeneration of the pulp-dentin complex and to replace conventional endodontic pulp therapy. Yet, such novel therapies dictate careful testing first in vitro and in animal models, prior to human clinical translation [bib_ref] Mesenchymal stem/progenitor cells: the prospect of human clinical translation, Rady [/bib_ref]. Cell-based therapies still face many challenges, mainly economical and ethical concerns. Thus, efforts started to target cell homing for pulp-dentin complex regeneration as a simpler, safer, and reasonably priced approach compared to the cell-based transplantation therapy. However, the success and safety of MSCs administered via IV or IA routs, as well as directing such cells [bib_ref] PDGFRβ+/c-kit+ pulp cells are odontoblastic progenitors capable of producing dentin-like structure in..., Cai [/bib_ref] Stem Cells International towards the injured tissues, are not always guaranteed. Despite the great advancements in pulp-dentin complex regeneration through cell homing in the past years, they require further investigations and development. Cell homing techniques need to be examined in more realistic models, starting with animals then humans. Moreover, clinical trials are crucial to point out possible indications and contraindications. Thus, numerous aspects still need to be resolved to make it applicable and with predictable outcomes in clinical dental practice. The perspective of replacing conventional endodontic therapy, while retaining the tooth vitality in a practical and relatively safe way, provides hope for the clinical dental practice. Finally, any minor step towards the future is counted as an additional profit that must be preciously handled and searched thoroughly to be utilized later in the field of regenerative dentistry.
## Abbreviations
## Aae:
American Association of Endodontists BMMSCs:
Bone marrow mesenchymal stem cells BMPs:
Bone morphogenetic proteins BMP-2:
Bone morphogenic protein-2 CCR2:
C-C chemokine receptor type 2 CCR3:
C-C chemokine receptor type 3 CCR4:
C-C chemokine receptor type 4 CD105:
Cluster of differentiation 105 CD44:
Cluster of differentiation 44 CD49d (α4β1):
Integrin α4 CD73:
Cluster of differentiation 73 cDPSCs:
Canine dental pulp stem cells CRCT:
Conventional root canal treatment CX3CR1:
CX3 chemokine receptor 1 CXCR4:
C-X-C chemokine receptor type 4 CXCR7:
C-X-C chemokine receptor type 7 DPCs:
Dental pulp cells DPSCs:
Dental pulp stem cells EB:
Evoked bleeding ECs:
Endothelial cells EDTA:
Ethylenediaminetetraacetic acid EGF:
Epidermal growth factor FGF:
Fibroblast growth factor G-CSF:
Granulocyte colony-stimulating factor GSK-3:
Glycogen synthase kinase HCELL:
Hematopoietic cell E-/L-selectin ligand hDPSCs:
Human dental pulp stem cells HERS:
Hertwig's epithelial root sheath HIF-1a:
Hypoxia-inducible factor-1a IA:
Intra-arterial IGF-1:
Insulin-like growth factor-1 IL-1α:
Interleukin-1 alpha IL-1β:
Interleukin-1 beta IL-4:
Interleukin-4 IL-6:
Interleukin-6 IL-8:
Interleukin-8 IL-10:
Interleukin-10 IV:
Intravenous MDPSCs:
Mobilized dental pulp stem cells MMP-1:
Matrix metalloproteinase-1 MMPs:
Matrix metalloproteinases MRI: Magnetic resonance imaging MSCs:
Mesenchymal stem/progenitor cells MTA:
Mineral trioxide aggregate PDGF:
Platelet-derived growth factor pDPSCs:
Pocrine dental pulp stem cells PLLA:
Poly L-lactic acid PPP:
Platelet-poor plasma PRF:
Platelet-rich fibrin PRP:
Platelet-rich plasma RBMMSC:
Rat bone marrow mesenchymal stem cells REP:
Regenerative endodontic procedures SC:
Stem cell SCAP:
Stem cells of apical papilla SDF-1:
Stromal cell-derived factor SHED:
Stem cells from human exfoliated deciduous teeth SI:
Signal intensity SIM:
Simvastatin STRO-1:
Stromal cell surface marker-1 TCP:
Tricalcium phosphate TDM:
Treated dentin matrix TGFβ1:
Transforming growth factor beta 1 TNF-α:
Tumor necrosis factor UCMSCs:
Umbilical cord mesenchymal stem cells VCAM-1 (CD106): Vascular cell adhesion molecule 1 VEGF:
Vascular endothelial growth factor VLA-4:
Integrin VLA-4.
## Data availability
Data are available on request.
## Conflicts of interest
The authors declare that they have no conflicts of interest. Stem Cells International
[fig] Figure 1: Cell-based transplantation method and sources of stem cells used for pulp-dentin complex regeneration. [/fig]
[fig] Figure 2: MSC homing mechanisms and different approaches for enhancing MSC homing. [/fig]
[fig] Figure 3: Different sources of stem/progenitor cells in the oral cavity and steps of revascularization. [/fig]
[table] Table 1: Summary of cell-based transplantation studies for pulp-dentin complex regeneration. [/table]
[table] Table 2: Summary of cell homing studies for pulp-dentin complex regeneration. [/table]
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Identifying Probable Suicide Clusters in Wales Using National Mortality Data
Background: Up to 2% of suicides in young people may occur in clusters i.e., close together in time and space. In early 2008 unprecedented attention was given by national and international news media to a suspected suicide cluster among young people living in Bridgend, Wales. This paper investigates the strength of statistical evidence for this apparent cluster, its size, and temporal and geographical limits.Methods and findings:The analysis is based on official mortality statistics for Wales for 2000-2009 provided by the UK's Office for National Statistics (ONS). Temporo-spatial analysis was performed using Space Time Permutation Scan Statistics with SaTScan v9.1 for suicide deaths aged 15 and over, with a sub-group analysis focussing on cases aged 15-34 years. These analyses were conducted for deaths coded by ONS as: (i) suicide or of undetermined intent (probable suicides) and (ii) for a combination of suicide, undetermined, and accidental poisoning and hanging (possible suicides). The temporo-spatial analysis did not identify any clusters of suicide or undetermined intent deaths (probable suicides). However, analysis of all deaths by suicide, undetermined intent, accidental poisoning and accidental hanging (possible suicides) identified a temporo-spatial cluster (p = 0.029) involving 10 deaths amongst 15-34 year olds centred on the County Borough of Bridgend for the period 27 th December 2007 to 19 th February 2008. Less than 1% of possible suicides in younger people in Wales in the ten year period were identified as being cluster-related.Conclusions:There was a possible suicide cluster in young people in Bridgend between December 2007 and February 2008. This cluster was smaller, shorter in duration, and predominantly later than the phenomenon that was reported in national and international print media. Further investigation of factors leading to the onset and termination of this series of deaths, in particular the role of the media, is required.
# Introduction
Suicide is one of the leading causes of death in young people. In Wales, the country in the United Kingdom (UK) where this study is based, suicide accounts for almost one in five deaths among men aged 15-24 and almost one in 10 deaths among women of that age.
A suicide cluster can be defined as an excessive number of suicides occurring in close temporal and geographical proximity. A recent analysis using space-time (temporo-spatial) models over an [bib_ref] Impact of the growing use of narrative verdicts by coroners on geographic..., Carroll [/bib_ref] year period in New Zealand found that 1.3% of probable suicides occurred in clusters. In the United States (U.S.) it has been estimated that at least 2% of teenage suicides occur in temporo-spatial clusters; clustering is thought to be two to four times more common among young people (aged 15-24 years) than among other age groups [bib_ref] Time-space clustering of teenage suicide, Gould [/bib_ref] [bib_ref] Suicide clusters: an examination of age-specific effects, Gould [/bib_ref]. Temporo-spatial analyses of specific groups of people at risk of suicide have identified 'point clusters': particularly in those who have contact with mental health services [bib_ref] Clustering of suicides among people with mental illness, Mckenzie [/bib_ref] or are in psychiatric hospitals [bib_ref] A cluster of suicides at a London psychiatric unit, Haw [/bib_ref] ; prisons [bib_ref] Contribution of imitative suicide to the suicide rate in prisons, Mckenzie [/bib_ref] ; and schools [bib_ref] An outbreak of suicide and suicidal behaviour in a high school, Brent [/bib_ref].
Our understanding of what triggers a suicide cluster, what causes it to continue and eventually subside, is limited. Joiner [bib_ref] The clustering and contagion of suicide, Joiner [/bib_ref] theorises that already vulnerable individuals, who are socially connected through shared characteristics, are those most affected by the suicide of a peer. Most researchers, however, have used the analogy of contagious illness, suggesting that there is imitation of suicidal behaviour, with social learning theory [bib_ref] The cultural dynamics of copycat suicide, Mesoudi [/bib_ref] being the dominant theoretical perspective. As well as local social networks, media reporting [bib_ref] Role of media reports in completed and prevented suicide: Werther v. Popageno..., Niederkrotenthaler [/bib_ref] , and the internet [bib_ref] Media influences on suicide and attempted suicide, Pirkis [/bib_ref] have been seen as important channels of transmission for suicide contagion.
In January 2008 the UK news media began reporting on a series of deaths amongst young people in South Wales, speculating that the town of Bridgend was experiencing a suicide epidemic (South Wales Echo January 17 th 2008; The Mirror January 23 rd 2008; Daily Mail, January 23 rd 2008). The intensity of the reporting remained high for several weeks, and the numbers of cases reported in the media continued to rise.
With the development of geospatial analysis methods, and geographic information systems in particular, it is now possible to perform more refined analyses than used in previous studies to identify suicide clusters. In this study we report the results of a Space Time Permutation Scan Statistics analysis [bib_ref] A Space-Time Permutation Scan Statistic for Disease Outbreak Detection, Kulldorff [/bib_ref]
# Methods
## Data
Data for all deaths classified as suicide, undetermined intent, and accidental for residents of Wales between 1st January 2000 and 31st December 2009 were supplied by the Office for National Statistics, Newport. Information on each case included: date of death, age at time of death, sex, International Classification of Disease (ICD) code for cause of death, and 12-figure Ordinance Survey (OS) geographic grid reference of place of residence at the time of death.
Traditionally, official suicide statistics in the UK combine deaths that have received a coroner's verdict of suicide (intentional self-harm) as well as those with undetermined intent (open verdicts), as most such deaths are suicides [bib_ref] Suicide in a single health district: epidemiology and audit of the involvement..., Dennis [/bib_ref]. These deaths are coded by the Office for National Statistics (ONS) in England and Wales using the ICD tenth revision (ICD-10) codes X60-84 (intentional self-harm) and Y10-34 (death of undetermined intent). Our primary analysis was based on such deaths, excluding those coded Y33.9/U50.9 (pending verdicts) as a large proportion of these are subsequently found to be homicides [bib_ref] Trends in suicide by method in England and Wales, Brock [/bib_ref].
In recent years there has been an increase in the number of 'narrative verdicts' recorded by coroners in England and Wales. In a narrative verdict the conclusions of the coroner are recorded as a brief summary, usually several sentences describing the circumstances of the death [bib_ref] Coroners' verdicts and suicide statistics in England and Wales, Gunnell [/bib_ref] [bib_ref] Narrative verdicts and their impact on mortality statistics in England and Wales, Hill [/bib_ref]. The Office for National Statistics (ONS), code deaths given narrative verdicts on the basis of information on intent noted by the coroner. If no intent is specified in deaths from injury or poisoning then they are coded as accidents [bib_ref] Coroners' verdicts and suicide statistics in England and Wales, Gunnell [/bib_ref] [bib_ref] Narrative verdicts and their impact on mortality statistics in England and Wales, Hill [/bib_ref]. The increased use of narrative verdicts could thereby potentially increase the official rate of accidental deaths at the expense of those classified as intentional self-harm or undeter-mined intent, leading to an underestimate of probable suicide [bib_ref] Coroners' verdicts and suicide statistics in England and Wales, Gunnell [/bib_ref] [bib_ref] Impact of the growing use of narrative verdicts by coroners on geographic..., Carroll [/bib_ref]. Furthermore, there is evidence that a high proportion of deaths from poisoning and hanging that receive accidental verdicts, are found, when subjected to clinical review, to be suicides [bib_ref] Time trends in coroners' use of different verdicts for possible suicides and..., Gunnell [/bib_ref]. For this reason we carried out secondary analyses that also included deaths coded as accidental hanging and strangulation (W75 & 76) and accidental poisoning from substances other than narcotics and psychodysleptics/hallucinogens (X40 & 41, 43-49). Accidental deaths from poisoning by narcotics and psychodysleptics/hallucinogens (X42) were excluded as these are common drugs of misuse, and intent may be particularly difficult to determine for such deaths.
# Analysis
The age and sex distribution of probable and possible suicides in Wales from 2000 to 2009 were described graphically and chi square statistics were used to compare proportions. Rates of probable and possible suicide were calculated using mid-year population estimates.
Space Time Permutation Scan Statistics (STPSS) were used to test for the presence of temporo-spatial clusters across Wales for the period 1st January 2000 to 31st December 2009 [bib_ref] A Space-Time Permutation Scan Statistic for Disease Outbreak Detection, Kulldorff [/bib_ref]. Monte Carlo ranking is used to assess the strength of statistical evidence (p-value) for the occurrence of clustering. The analysis was carried out in SaTScan v9.1. In order to run a temporo-spatial analysis SaTScan needs both the time and geo-location for each event. Time of event was recorded as the day of death and the place of residence as a 12 figure Ordinance Survey grid reference, pinpointing the location of each case to the nearest metre. STPSS investigates clustering within a variable time window across varying geographical areas and uses a Poisson based likelihood to compare the expected number of cases and actual number of cases inside and outside the window. The size of the time and geographical window can be varied within pre-specified limits. The result is a set of cylinders where the base represents the area of the potential cluster and the height represents the time period of the cluster.
The temporo-spatial window with the biggest likelihood ratio of cases is reported as the most likely candidate for a temporo-spatial cluster. The variation in the size of the scanning window is specified as a percentage of the map area and as a percentage of the time duration in the data. For our analysis the variable scan window was set to a maximum of 10% of the study area (approximately 50 km radius) and 10% of the study period (equal to 1 year). A systematic review of all published studies reporting suicide clusters between 1977 and 2009has indicated a median duration of 11 months for narrative reports of clusters, further justifying a 12 months scan window. The shape of the spatial scan window can be circular or elliptical. When an elliptical shaped window is selected, the scanning window varies in shape as well as in size, and is repeated for different rotations of the ellipse. The elliptical shape changes from longer and narrower towards circular, thereby reducing the likelihood of missing clusters that are associated with linear settlements, or along particular streets within larger settlements. For the analysis of clusters the system was set to allow geographic overlap between potential clusters, so that two or more clusters could include the same geographical space, provided that the centre of the less likely cluster was not within the area of a more likely cluster. This allows for the detection of neighbouring clusters that might share some of the cases. The model also adjusts for clustering in space and time alone, thereby adjusting for areas and seasons with a higher baseline rate. Temporo-spatial cluster analysis was performed on four prespecified groups of deaths: Our focus on 15-34 year olds reflected the research evidence [bib_ref] Time-space clustering of teenage suicide, Gould [/bib_ref] [bib_ref] Suicide clusters: an examination of age-specific effects, Gould [/bib_ref] that clusters occur more frequently in young people and the particular concern that the 'cluster' in Bridgend had occurred amongst the young.
# Ethical approval
This study is a component of the study entitled 'Using routinely collected data from suicide clusters to influence social and health care service delivery: an investigation of the Bridgend cluster' that received full ethical approval from the South West Wales Research Ethics Committee (REC reference: 10/WMW02/10).
# Results
There were 3943 deaths included in the analysis: 3002 (76.1%) males and 941 (23.9%) females. Altogether 2375 (60.2%) of the deaths were coded as suicide (intentional self-harm), 616 (15.6%) undetermined and 952 (24.1%) accidental poisoning (excluding narcotics) or accidental hanging [fig_ref] Table 1: Cause of death groups according to ICD codes of possible suicides for... [/fig_ref]. [fig_ref] Figure 1: Ten year rates of probable suicides [/fig_ref] shows the geographical variations in rates of probable suicide across Welsh counties. Rates of probable suicide in Bridgend (11.4/100,000/year, 95% CI 8.7-14.1) were only marginally higher than those for the whole of Wales (10.2/ 100,000/year, 95% CI 9.3-11.0). This was also the case for possible suicide (suicide, undetermined, and accidental poisoning and hanging) with a mean Bridgend rate of 15.6/100,000/year (95% CI 13.4-17.7) compared to 13.4 /100,000/year (95% CI 12.6-14.1) for the whole of Wales. The sex and cause of death distribution of deaths were similar in Bridgend to those seen for all Wales [fig_ref] Table 1: Cause of death groups according to ICD codes of possible suicides for... [/fig_ref] , with the exception that in both all ages combined and 15-34 year olds there was statistical evidence (p = 0.002 and p = 0.019, respectively) of a higher proportion of accidental hanging/strangulation compared to accidental poisoning deaths in Bridgend than the rest of Wales.
## Rate of death by cause across time for wales
Apart from a small rise in 2003, deaths coded as suicide and of undetermined intent decreased between 2000 and 2009 in Wales as a whole [fig_ref] Figure 2: Mortality statistics for Wales by cause of death group [/fig_ref]. Deaths coded as accidental poisoning and hanging decreased slightly from 2000 to 2005, before starting to increase in 2006 and rising by 61% in 2008. For possible suicides there were two peaks in the period: the first in 2003, which was due to a rise in deaths coded as suicide and of undetermined intent; and the second in 2008, which was due to a rise in deaths coded as accidental poisoning or accidental hanging. For ages 15 to 34 years there is a similar pattern but with greater variation [fig_ref] Figure 2: Mortality statistics for Wales by cause of death group [/fig_ref]. [fig_ref] Figure 4: Temporo-spatial clusters of possible suicides [/fig_ref] , but statistical evidence for these was weak (p.0.13). Two of these (clusters 2 and 5) occurred at similar time periods to the primary cluster and included cases from the primary cluster, but either related to a larger geographical area or were of longer duration [fig_ref] Figure 5: Most likely temporo-spatial clusters of possible suicide in Bridgend and surrounding boroughs... [/fig_ref]. Combining the primary and secondary cases in this area for the period 27 th December 2007 to 19 th February 2008 extends the size of the cluster to a possible 15 cases. The ten deaths in the primary cluster comprised five coded as suicide, two undetermined and three as accidental. There were another 18 deaths in the same age group elsewhere in Wales during the period of the identified cluster. The mean age of the cluster cases was 21.4 (SD 5.6) years compared to 25.7 (SD 5.8) years among non-cluster members (two-tailed t-test = 21.884; p = 0.071). There was no statistical evidence of difference between cases in and outside the cluster in respect of gender (Male 60% inside, 83% outside; Female 40% inside, 27% outside: Fisher Exact test p = 0.21), cause of death (70% suicide inside, 61% outside; Accidental 30% inside, 39% outside: Fisher Exact test p = 0.71), or method of suicide (80% hanging inside, 61% hanging outside: Fisher Exact test p = 0.42).
## Rate of death by cause across time for the county borough of bridgend
The 10 cluster deaths comprised 0.78% of possible suicide deaths in 15-34 year olds over the 10 year study period and 0.25% of total (all age) suicides.
# Discussion
## Main findings
We found statistical evidence that a cluster of 10 deaths amongst 15-34 year olds occurred in the Bridgend area of South Wales between 27 th December 2007 and 19 th February 2008 when using a broad definition of suicide. This cluster was smaller and shorter in duration than the phenomenon reported in the print media. Additionally, most deaths in the cluster occured after the commencement of the attention from the print media; much of the initial newspaper focus related to deaths in the preceeding twelve months. There was some weak statistical evidence that the cluster may have included up to 15 cases during that time. Overall only 0.78% of possible suicides in 15-34 year old Wales (0.25% of total all-age suicides) over the ten year period were identified as being cluster-related. The number of cluster related deaths may, however, be an under-estimate as it is possible that small clusters go undetected using Space Time Permutation Scan Statistics.
The analysis has highlighted several important issues in relation to the analysis of clusters of suicide. First, in this study the cluster of suicides was only detected when the analysis was restricted to cases aged 15 to 34 years. This age group accounts for only a third of all suicides and without such a sub-group analysis the possible cluster might have been overlooked. Investigation of this group was prespecified in view of both the reported characteristics of deaths associated with the cluster and previous literature suggesting that this age group may be particularly susceptible to the factors that influence cluster development [bib_ref] Suicide clusters: an examination of age-specific effects, Gould [/bib_ref].
Second, as a result of increases in the use of narrative verdicts by coroners and the consequent possible coding of some probable suicides as accidental deaths by ONS [bib_ref] Coroners' verdicts and suicide statistics in England and Wales, Gunnell [/bib_ref] [bib_ref] Narrative verdicts and their impact on mortality statistics in England and Wales, Hill [/bib_ref] as well as the use of accidental verdicts for some likely suicides [bib_ref] Suicide in a single health district: epidemiology and audit of the involvement..., Dennis [/bib_ref] [bib_ref] The limitations of official suicide statistics, O'donnell [/bib_ref] , the cluster was only detected by applying a broader definition of suicide than usually employed in epidemiological studies of suicide. In England the number of narrative verdicts has doubled in 5 years, and in Wales this has tripled. Due to variation in coroners' use of narrative verdicts, regional and local effects may be more pronounced [bib_ref] Impact of the growing use of narrative verdicts by coroners on geographic..., Carroll [/bib_ref]. Any bias introduced by the use of narrative verdicts becomes particularly important when considering the possibility of the presence of a point cluster. Furthermore, there is increasing evidence that many deaths recorded as accidental poisoning and hanging represent probable suicide [bib_ref] Time trends in coroners' use of different verdicts for possible suicides and..., Gunnell [/bib_ref]. Additionally, risk factors associated with accidental death from nonnarcotic poisoning as well as other causes of accidental death are similar to risk factors for deaths recorded as suicide [bib_ref] Shared characteristics of suicides and other unnatural deaths following non-fatal selfharm? A..., Bergen [/bib_ref].
## Comparisons with other studies
The proportion of possible suicides in younger people identified in this study occurring as part of a cluster (0.78%) is lower than previously reported in the literature. Larkin & Beautrais, in their analysis of suicide clusters in New Zealand during 1990-2007, found that 1.3% of probable suicides for all ages occurred in clusters. Other studies of the temporo-spatial clustering of suicides have used the Knox method [bib_ref] Time-space clustering of teenage suicide, Gould [/bib_ref] [bib_ref] Suicide clusters: an examination of age-specific effects, Gould [/bib_ref] and shown that at least 2% of teenage suicides in the U.S. occur in clusters; however there are a number of limitations to this method [bib_ref] The Knox method and other tests for spacetime interaction, Kulldorff [/bib_ref]. Additionally, crossnational comparisons need to be reviewed cautiously because of a variety of factors affecting cause of death recording [bib_ref] Comparison of elderly suicide rates among migrants in England and Wales with..., Shah [/bib_ref].
## Strengths and limitations of this study
There are two main strengths of the study. The first is the use of point data giving the precise location of residence for the suicide deaths, thereby avoiding the need for calculating rates for artificially defined areas. The second is our use of state of the art methods for assessing clustering -Space Time Permutation Scan Statistics [bib_ref] A Space-Time Permutation Scan Statistic for Disease Outbreak Detection, Kulldorff [/bib_ref]. The principal limitation of the study is the reliability of official suicide statistics (see above). We have attempted to minimise problems relating to the use of narrative verdicts and ONS coding by performing an analysis based on probable suicide (suicide and undetermined deaths) and possible suicide (suicide, undetermined, and accidental hanging and poisoning deaths, excluding narcotic-related mortality). Inevitably there is the possibility that some accidental deaths are not due to suicidal behaviour, and this is also true of a proportion of deaths receiving an open verdict [bib_ref] Suicide in a single health district: epidemiology and audit of the involvement..., Dennis [/bib_ref].
Analysis of clusters such as this assumes that all relevant suicides occur within relatively close geographic proximity. Given the ease of communication and travel in the 21 st century it is possible that suicides amongst those who are part of an affected friendship group may live some distance outside the geographic areas used in our scanning methodology and so not be identified as members of the cluster. Such cases may only be identifiable through local knowledge and discussion with an affected community. Additionally, approaches such as those used here may lack power to delineate all cluster-related suicides -for example point clusters in institutions when only two or three deaths occur within a single school, particularly where catchment areas for school are dispersed.
Establishing the presence of a putative suicide cluster through sophisticated analysis and geographical mapping may be of restricted value. Firstly, the limitations previously outlined, as well as the late availability and reliability of data, are a major consideration. Secondly, community perception is much more immediately relevant in implementing an appropriate response strategy [bib_ref] Responding to community-identified suicide clusters: statistical verification of the cluster is not..., O'carroll [/bib_ref]. Statistical evidence for the Bridgend cluster is relatively weak, and at the early stages of the cluster local health authorities were not convinced that the number of suicides occurring was above that expected given normal fluctuations in rare events. In contrast, there was strong concern within the community that an excess of suicides was occurring. Additionally, the local perception of clustering and consequential effects on the community may increase the contagious effects of suicide [bib_ref] Responding to community-identified suicide clusters: statistical verification of the cluster is not..., O'carroll [/bib_ref]. The presence of a pre-existing community strategy may be particularly beneficial for communities in early identification of the risk of a cluster developing, supporting vulnerable individuals, and managing relationships with the media.
## Conclusions and implications for future research
We have found evidence for the occurrence of a temporo-spatial cluster of possible suicide centred on the County Borough of Bridgend and overlapping the time period of the early media reporting of a cluster. It represented only 0.78% of possible suicides in young people in Wales over a ten year period. Factors relating to the initiation, maintenance and cessation of this suicide cluster, as with others remain unclear. A recent systematic review examining suicide cluster risk factors and mechanisms identified some important associations, particularly direct involvement with another cluster member, and other factors known to be linked to suicide generally such as a history of self harm and substance misuse [bib_ref] Suicide clusters: A review of risk factors and mechanisms, Haw [/bib_ref]. Although contagion was the most prevalent mechanism of cluster formation reported, other mechanisms such as homophily (people preferentially assorting to similar others) may also be operating independently or in combination. One important avenue for research could be interviewing individuals who survived a serious attempt at suicide and were part of a cluster of suicidal behaviour [bib_ref] Suicide clusters: A review of risk factors and mechanisms, Haw [/bib_ref]. There is unequivocal evidence from systematic reviews that media reporting of suicide may be associated with further suicides particularly using the same method [bib_ref] Suicide and the media. Part 1: reportage in non-fictional media, Pirkis [/bib_ref] [bib_ref] Influences of the media on suicide, Hawton [/bib_ref] [bib_ref] Media coverage as a risk factor in suicide, Stack [/bib_ref] [bib_ref] Media roles in Suicide Prevention: A systematic review, Sisask [/bib_ref] ; this is particularly relevant to 'mass' clusters when there is a temporary increase in the total frequency of suicides for a population relative to the time preceding and after the cluster. The role of the print media in temporo-spatial clusters, however, has not been investigated. A number of important observations suggest that the effects of media reporting for the cluster we have detected requires further scrutiny. Firstly, the cluster was predominantly later, smaller and shorter in duration than the phenomenon that was widely reported in national and international print media. Secondly there was a high profile of reporting at the time, and thirdly there were no other clusters in Wales during the ten year period, as well as no evidence of previous clusters in the same area that would indicate any specific community vulnerability.
[fig] Figure 1: Ten year rates of probable suicides (suicides and undetermined deaths) for Welsh Counties 2000-2009. The geographical variations in rates of probable suicide across Welsh counties 2000 to 2009. doi:10.1371/journal.pone.0071713.g001 [/fig]
[fig] Figure 2: Mortality statistics for Wales by cause of death group (rate per 100,000 population). Apart from a small rise in 2003, deaths coded as suicide and of undetermined intent decreased between 2000 and 2009 in Wales as a whole (Figure 2a). Deaths coded as accidental poisoning and hanging decreased slightly from 2000 to 2005, before starting to increase in 2006 and rising by 61% in 2008. For possible suicides there were two peaks in the period: the first in 2003, which was due to a rise in deaths coded as suicide and of undetermined intent; and the second in 2008, which was due to a rise in deaths coded as accidental poisoning or accidental hanging. For ages 15 to 34 years there is a similar pattern but with greater variation (Figure 2b). doi:10.1371/journal.pone.0071713.g002 34 year olds, possible suicides peaked in 2008 (figure 3b217 deaths) with a second smallerpeak of 13 deaths in 2003. Space Time Suicide Clusters When analysing deaths aged 15 and over, or when examining younger people only (aged 15-34 years), there was no statistical evidence of temporo-spatial clusters representing probable suicide (suicide and undetermined) in Wales during the period 2000-2009-the lowest p value being 0.48. When deaths from accidental poisoning and hanging were included (i.e. possible suicide; Figure 4), statistical evidence for a cluster was identified in the sub-set of data for people aged 15-34 year (p = 0.029) (see insert, Figure 4). This cluster centred on the County Borough of Bridgend but extended into the neighbouring County of Neath Port Talbot for the period 27/12/2007 to 19/2/2008. It contained 10 of the 28 suicides in 15-34 year olds in Wales during that time period. Other clusters of possible suicides in people aged 15-34 are displayed in [/fig]
[fig] Figure 3: Mortality statistics for the County Borough of Bridgend by cause of death group (rate per 100,000 population). The rate of death by cause across time for the County Borough of Bridgend (Figure 3a) was considerably more varied than the rate of death by cause across Wales, as might be expected considering the relatively small population of Bridgend. In the all-age rates (figure 3a) there was an indication of a peak in accidental deaths by hanging and poisoning in 2008 and some evidence that 15-34 year olds contributed disproportionately to this excess (figure 3b). In 15-34 year olds, possible suicides peaked in 2008 (figure 3b) with a second smaller peak in 2003. doi:10.1371/journal.pone.0071713.g003 [/fig]
[fig] Figure 4: Temporo-spatial clusters of possible suicides (suicide, undetermined, and accidental poisoning and hanging) for people 15-34 years. The temporo-spatial analysis identified several possible clusters across Wales for age group 15 to 34 years. Three of the clusters were centred on the county borough of Bridgend, but only cluster number one was statistically significant (p = 0.029). doi:10.1371/journal.pone.0071713.g004 [/fig]
[fig] Figure 5: Most likely temporo-spatial clusters of possible suicide in Bridgend and surrounding boroughs 2000-2009. The three most likely clusters for the age group 15 to 34 years centred on the county borough of Bridgend. The clusters all extended into neighbouring boroughs. doi:10.1371/journal.pone.0071713.g005 [/fig]
[table] Table 1: Cause of death groups according to ICD codes of possible suicides for Wales 2000-2009. [/table]
[table] 1: Probable suicides amongst people aged 15 years and over: intentional self-harm (ICD-10 codes X60-84), and deaths of undetermined intent (ICD-10 Y10-34 excluding Y33.9); 2. Probable suicide amongst people aged 15 to 34 years: [/table]
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Primary small bowel adenocarcinoma with loss of nuclear expression of PMS2 after resection of mucinous cholangiocarcinoma
Mucinous cholangiocarcinoma is an extremely rare form of intrahepatic cholangiocarcinoma that has been characterized by rapid growth, widespread metastasis and poor prognosis. These tumors have been shown to be a part of the Lynch syndrome tumor spectrum, however, the role of DNA mismatch repair (MMR) deficiency in their development is poorly understood. We present the case of a 74-year-old male with cholangiocarcinoma, who underwent Roux-en-Y hepaticojejunostomy and extended left hepatectomy and was diagnosed with a primary small bowel adenocarcinoma 2 years later. Immunohistochemistry testing for mismatch repair proteins was significant for the loss of nuclear expression of PMS2. Taken together, the cause of both the mucinous cholangiocarcinoma and primary small bowel adenocarcinoma with PMS2 loss in the patient presented here is likely genetic, suggestive of a cancer syndrome.
# Introduction
Mucinous cholangiocarcinoma is an extremely rare form of intrahepatic cholangiocarcinoma that has been characterized by rapid growth, widespread metastasis and poor prognosis [bib_ref] Resected primary mucinous cholangiocarcinoma of the liver, Hagiwara [/bib_ref]. These tumors have been shown to be a part of the Lynch syndrome tumor spectrum, however, the role of DNA mismatch repair (MMR) deficiency in their development is still under research [bib_ref] Biliary carcinomas: pathology and the role of DNA mismatch repair deficiency, Silva [/bib_ref]. This report presents a case of a 74-year-old male with a history of cholangiocarcinoma, who underwent Roux-en-Y hepaticojejunostomy and extended left hepatectomy and was † A. Mujeeb Ullah and A. Jaysing are both first authors. Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2022. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] diagnosed with a primary small bowel adenocarcinoma with loss of PMS2, 2 years later.
## Case report
We present a case of a 74-year-old male who presented in 2018 with abdominal pain and underwent a series of crosssectional imaging (CT-scan) which revealed an obstructed left bile duct and an associated mass. Follow-up esophagogastroduodenoscopy (EGD) and endoscopic ultrasound (EUS) confirmed the presence of a mass at the confluence of the bile ducts with possible liver invasion. A needle aspiration done was suspicious for carcinoma. Patient underwent diagnostic laparoscopy which revealed no evidence of metastatic disease, therefore, an extended left hepatectomy with a roux-en-y hepaticojejunostomy was performed. On pathological evaluation of the specimen, invasive mucinous adenocarcinoma with an intraductal papillary mucinous component, in a background of high-grade dysplasia was found involving the left hepatic lobe (Segment 4b) and left hepatic duct. Surgical margins were negative for carcinoma. No lymphovascular, perineural or lymph node involvement was seen. The tumor was classified as pT1a, pNx R0 according to the American Joint Committee of Cancer (AJCC) 8th edition.
Two years later, the patient presented again with abdominal pain and rectal bleeding. On CT scan of abdomen and pelvis a lobular circumferential enhancing mass of the proximal fourth portion of the duodenum, with shotty adjacent lymph nodes was found, that was suspicious for primary duodenal adenocarcinoma. As part of diagnostic workup, the patient underwent an EGD. The biopsy confirmed the presence of invasive moderately differentiated adenocarcinoma and immunohistochemistry testing for mismatch repair proteins (MMR) was significant for the loss of nuclear expression of PMS2 [fig_ref] Figure 3: Invasive moderately differentiated adenocarcinoma with immunohistochemistry testing for mismatch repair proteins [/fig_ref]. The patient underwent resection of the small bowel between the ligament of Treitz and the previous jejunojejunostomy site and end-to-side duodenojejunostomy was performed between the end of the fourth portion of the duodenum and the side of the distal limb of the Roux-en-Y portion of bowel that had previously been created for the hepaticojejunostomy, to reestablish intestinal continuity.
Post resection, surgical pathology specimen revealed a 5.8 cm invasive moderately differentiated adenocarcinoma with mucinous features invading the mesenteric fat of the small bowel without lymphovascular or perineural invasion. Surgical margins were negative for carcinoma. Remarkably, 1 of 25 lymph nodes was associated with acellular mucin. Multiple deeper levels and immunohistochemical stain for CAM5.2 failed to reveal isolated tumor cells. Additionally, in the absence of any neoadjuvant therapy, the acellular mucin-associated lymph node was considered uninvolved by adenocarcinoma, as per AJCC conventionsA histomorphological comparison of small bowel adenocarcinoma and prior cholangiocarcinoma was made at the intradepartmental consensus conference and the two tumors were deemed as separate primaries. The small bowel adenocarcinoma was staged as pT3N0. Genetic analysis of the tumor by sequencing confirmed the MLH1 gene [K618del] mutation, in addition, a common hereditary gene panel also revealed multiple mutations with lesser frequency. At the time of writing this report, the patient shows no radiological evidence of recurrence.
# Discussion
Mucinous cholangiocarcinoma is a rare type of intrahepatic cholangiocarcinoma, characterized by rapid growth, extensive metastasis and poor prognosis [bib_ref] Resected primary mucinous cholangiocarcinoma of the liver, Hagiwara [/bib_ref] [bib_ref] Rapidly growing mucinous cholangiocarcinoma, Motoo [/bib_ref] [bib_ref] Pathological and immunohistochemical findings in a case of mucinous cholangiocarcinoma, Sasaki [/bib_ref]. Proposed risk factors include a history of biliary disease, chronic hepatitis C and intrahepatic C. sinensis flukes [bib_ref] Mucin-producing cholangiocarcinoma: an autopsy study in Hong Kong, Chou [/bib_ref] [bib_ref] Mucinous cholangiocarcinoma: clinicopathological features of the rarest type of cholangiocarcinoma, Sumiyoshi [/bib_ref]. In addition, mismatch repair deficiency (MMR-d), which is associated with hereditary cancer syndromes such as Lynch syndrome, has been shown to be associated with mucinous cholangiocarcinoma [bib_ref] Biliary carcinomas: pathology and the role of DNA mismatch repair deficiency, Silva [/bib_ref] [bib_ref] Microsatellite instability occurs rarely in patients with cholangiocarcinoma: a retrospective study from..., Winkelmann [/bib_ref] [bib_ref] Mismatch repair protein deficiency/microsatellite instability is rare in Cholangiocarcinomas and associated with..., Ju [/bib_ref]. However, the patients with MMR-d and mucinous cholangiocarcinoma described by Ju et al. did not have any other typical Lynch syndrome-associated cancers documented. Therefore, it is possible that the first sign of a hereditary cancer syndrome in some patients is mucinous cholangiocarcinoma.
Primary small bowel adenocarcinoma is a similarly a rare gastrointestinal track malignancy characterized by nonspecific symptoms, late diagnosis and poor prognosis [bib_ref] Small bowel adenocarcinoma: epidemiology, risk factors, diagnosis and treatment, Aparicio [/bib_ref]. Risk factors include alcohol consumption, as well as Lynch syndrome, the basis of which is a germline MMR mutation [bib_ref] Is there an association between alcohol intake or smoking and small bowel..., Kaerlev [/bib_ref] [bib_ref] Prognostic factors in primary adenocarcinoma of the small intestine: 13-year single institution..., Chaiyasate [/bib_ref].
Both somatic and germline mutations in MMR genes lead to the loss of MMR function. Although there are seven known MMR proteins, MLH-1, MSH-2, MSH-6 and PMS-2 are of the most oncologic relevance [bib_ref] DNA mismatch repair proteins: scientific update and practical guide, Bateman [/bib_ref]. The result of a deficiency in any one of these proteins can be variation in the lengths of the microsatellites, termed microsatellite instability (MSI) [bib_ref] Mismatch repair pathway, genome stability and cancer, Pecina-Slaus [/bib_ref]. Though cases are limited, its suggested that MSI-H cholangiocarcinomas are more likely to be of atypical histomorphology (solid, mucinous or signet-ring) [bib_ref] Mismatch repair protein deficiency/microsatellite instability is rare in Cholangiocarcinomas and associated with..., Ju [/bib_ref]. Similarly, small bowel adenocarcinoma has a high rate of MSI [bib_ref] Genomic profiling of small-bowel adenocarcinoma, Schrock [/bib_ref].
Taken together, the cause of both the mucinous cholangiocarcinoma and primary small bowel adenocarcinoma with PMS2 loss in the patient presented here is likely genetic, suggestive of a cancer syndrome. A negative history of a hereditary cancer syndrome warrants further evaluation and judicious follow-up and surveillance to prevent recurrence.
[fig] Figure 1: (A) Computed tomography (axial): ovoid low-density mass contiguous with dilated left hepatic bile duct. (B) Histology: invasive mucinous adenocarcinoma with an intraductal papillary mucinous component, in a background of high-grade dysplasia was identified involving the left hepatic lobe (segment 4b) and left hepatic duct. [/fig]
[fig] Figure 2: (A and B) Esophagogastroduodenoscopy (EGD) and computed tomography: lobular circumferential enhancing mass of the proximal fourth portion of the duodenum. [/fig]
[fig] Figure 3: Invasive moderately differentiated adenocarcinoma with immunohistochemistry testing for mismatch repair proteins (MMR) significant for the loss of nuclear expression of PMS2 with retained strong expression of MSH2, MSH6 and MLH1. [/fig]
[fig] Figure 4: (A) Invasive moderately differentiated adenocarcinoma with mucinous features invading the mesenteric fat of the small bowel. (B) One of 25 lymph nodes associated with acellular mucin. [/fig]
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Shared properties and singularities of exoribonuclease-resistant RNAs in viruses
a b s t r a c tWhat viral RNA genomes lack in size, they make up for in intricacy. Elaborate RNA structures embedded in viral genomes can hijack essential cellular mechanisms aiding virus propagation. Exoribonucleaseresistant RNAs (xrRNAs) are an emerging class of viral elements, which resist degradation by host cellular exoribonucleases to produce viral RNAs with diverse roles during infection. Detailed three-dimensional structural studies of xrRNAs from flaviviruses and a subset of plant viruses led to a mechanistic model in which xrRNAs block enzymatic digestion using a ring-like structure that encircles the 5 0 end of the resistant structure. In this mini-review, we describe the state of our understanding of the phylogenetic distribution of xrRNAs, their structures, and their conformational dynamics. Because xrRNAs have now been found in several major superfamilies of RNA viruses, they may represent a more widely used strategy than currently appreciated. Could xrRNAs represent a 'molecular clock' that would help us understand virus evolution and pathogenicity? The more we study xrRNAs in viruses, the closer we get to finding xrRNAs within cellular RNAs.
## Introduction: xrrnas as generic mechanical blocks to exoribonucleases
The compact genomes of RNA viruses (max. 30 kb) are littered with structured elements [bib_ref] Architecture and secondary structure of an entire HIV-1 RNA genome, Watts [/bib_ref] [bib_ref] Structure mapping of dengue and Zika viruses reveals functional long-range interactions, Huber [/bib_ref]. Structured RNA elements are key contributors to all stages of viral infection and usually to pathogenicity. They are often used by viruses to manipulate transcription, translation, antiviral responses, and other cellular mechanisms [bib_ref] Viral RNA structure-based strategies to manipulate translation, Jaafar [/bib_ref]. Such elements comprise for example hairpin structures, which protect the 3 0 end and may affect translation [bib_ref] RNA regulatory processes in RNA virus biology, Cross [/bib_ref] , and pseudoknotted folds, which may start translation or alter the translation reading frame [bib_ref] Viral RNA structure-based strategies to manipulate translation, Jaafar [/bib_ref] [bib_ref] Frameshifting RNA pseudoknots: structure and mechanism, Giedroc [/bib_ref].
An emerging class of structured viral RNAs are the exoribonuclease-resistant RNAs (xrRNAs), which were first discovered in the 3 0 untranslated region (UTR) of flaviviruses and plant viruses [bib_ref] RNA structures required for production of subgenomic flavivirus RNA, Funk [/bib_ref] [bib_ref] A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay..., Iwakawa [/bib_ref] [bib_ref] A highly structured, nuclease-resistant, noncoding RNA produced by flaviviruses is required for..., Pijlman [/bib_ref]. xrRNAs resist degradation from the 5 0 direction by the host cellular exoribonuclease Xrn1 (or its homologs, like Xrn4 in plants [bib_ref] XRN 5'?3' exoribonucleases: structure, mechanisms and functions, Nagarajan [/bib_ref] and more generally by any 5 0 -to-3 0 exoribonuclease (e.g., bacterial RNase J1 and yeast decapping and exoribonuclease protein 1) [bib_ref] Functional non-coding RNAs derived from the flavivirus 3' untranslated region, Clarke [/bib_ref] [bib_ref] Subgenomic flaviviral RNAs: What do we know after the first decade of..., Slonchak [/bib_ref] [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref]. Viral xrRNAs can therefore be viewed as a generic roadblock to directional degradation.
Altogether, xrRNAs have now been reported in at least three of the five major virus branches [fig_ref] Figure 1: xrRNAs across the viral phylogeny [/fig_ref] [bib_ref] Origins and evolution of the global RNA virome, Wolf [/bib_ref]. Outside of Flaviviridae, but still within branch 3, xrRNAs have been identified in Tombusviridae and Luteoviridae [bib_ref] A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay..., Iwakawa [/bib_ref] [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref] [bib_ref] Exoribonuclease-Resistant RNAs exist within both coding and noncoding subgenomic RNAs, Steckelberg [/bib_ref] as well as in the Alphavirus supergroup [bib_ref] Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading..., Peltier [/bib_ref] [bib_ref] Beet necrotic yellow vein virus noncoding RNA production depends on a 5'?3'..., Flobinus [/bib_ref]. Putative xrRNAs have also been proposed within branch 2 (e.g., Potyviridae [bib_ref] Structural features of an Xrn1-resistant plant virus RNA, Dilweg [/bib_ref] and branch 5 (e.g., Bunyaviridae and Arenaviridae [bib_ref] Identification of phlebovirus and arenavirus RNA sequences that stall and repress the..., Charley [/bib_ref]. Overall, these findings highlight that xrRNAs are more widespread than originally thought.
Generally, xrRNAs are responsible for the production of certain subgenomic RNAs (sgRNAs), through partial degradation of the genomic RNA [fig_ref] Figure 1: xrRNAs across the viral phylogeny [/fig_ref] ; these are distinguished from sgRNAs resulting from internal transcription initiation [bib_ref] Synthesis of subgenomic RNAs by positive-strand RNA viruses, Miller [/bib_ref]. For example, in flaviviruses -where the sgRNAs are referred to as sfRNAs for 'subgenomic flaviviral RNAs'-sfRNAs act as noncoding RNAs that interact with the cellular proteome to alter antiviral responses [bib_ref] Fragile X mental retardation protein is a Zika virus restriction factor that..., Soto-Acosta [/bib_ref] [bib_ref] Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness, Manokaran [/bib_ref] , but the details of this remain poorly understood [bib_ref] Subgenomic flaviviral RNAs: What do we know after the first decade of..., Slonchak [/bib_ref]. For some plant-infecting viruses, xrRNAs are located within subgenomic RNAs (sgRNAs) that encode proteins and these sgRNAs may therefore be translated [bib_ref] Exoribonuclease-Resistant RNAs exist within both coding and noncoding subgenomic RNAs, Steckelberg [/bib_ref] [bib_ref] Opium poppy mosaic virus has an Xrnresistant, translated subgenomic RNA and a..., Ilyas [/bib_ref]. In Tombusviridae, an xrRNA is similarly responsible for the formation of a non-coding RNA with translation regulation properties [bib_ref] A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay..., Iwakawa [/bib_ref] [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref] [bib_ref] Molecular biology and epidemiology of dianthoviruses, Okuno [/bib_ref]. In Benyviridae, Betaflexiviridae and Virgaviridae, an xrRNA is sufficient to block Xrn4, leading in Benyviridae to the formation of non-coding RNA3 (ncRNA3) and RNA5 (ncRNA5), which are essential for viral long-distance movement within the infected plant [bib_ref] Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading..., Peltier [/bib_ref] [bib_ref] Beet necrotic yellow vein virus noncoding RNA production depends on a 5'?3'..., Flobinus [/bib_ref] [bib_ref] Structural features of an Xrn1-resistant plant virus RNA, Dilweg [/bib_ref] [bib_ref] Beet soilborne mosaic virus RNA-3 is replicated and encapsidated in the presence..., Ratti [/bib_ref]. Hence, xrRNAs may be important for producing or protecting a variety of viral RNAs with diverse functions; they are emerging as an important mode of viral RNA maturation and protection.
2. Encircling the 5 0 end: Different strategies for a shared feature
The three-dimensional crystal structures of xrRNAs from several viruses within the flavivirus supergroup (Murray Valley Encephalitis Virus, MVEV; Zika Virus, ZIKV; Tamana Bat Virus, TABV) offer detailed insights into the molecular basis of exoribonuclease resistance [bib_ref] The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production, Chapman [/bib_ref] [bib_ref] Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a..., Akiyama [/bib_ref]. They reveal that xrRNAs adopt a ringlike structure through which the 5 0 end threads [fig_ref] Figure 2: Ring-like structures in RNA [/fig_ref]. In all cases observed thus far, the ring comprises 15-16 nucleotides. Complex tertiary interactions including pseudoknots, noncanonical base-pairs, bound metal ions, and intricate stacking and hydrogen bonding schemes stabilize the ring feature. Some of these interactions form patterns that are specific to different xrRNA classes [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref] ; see next section). The resulting compact fold braces against the surface of the enzyme approaching from the 5 0 side, preventing further progression of the enzyme through the xrRNA [bib_ref] The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production, Chapman [/bib_ref].
The importance of a ring structure for blocking the enzyme is further supported by structures from xrRNAs outside the flaviviruses. Specifically, plant-infecting viruses from Tombusviridae and Luteoviridae [fig_ref] Figure 1: xrRNAs across the viral phylogeny [/fig_ref] , have xrRNAs with sequences and secondary structures that differ from flaviviral xrRNAs [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref] [bib_ref] The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production, Chapman [/bib_ref] [bib_ref] Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a..., Akiyama [/bib_ref]. Although the crystal structures of xrRNAs from Sweet Clover Necrotic Mosaic Virus (SCNMV; Tombusviridae) and Potato Leaf Roll Virus (PLRV; Luteoviridae) did not capture the RNA in its active ring-supported conformation, a ring-like structure could be modeled that also contained 15 nucleotides encircling the 5 0 end [fig_ref] Figure 2: Ring-like structures in RNA [/fig_ref]. As in flaviviruses, this ring depends on the formation of a pseudoknot whose importance was verified by site-directed mutagenesis and via an infection system [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref]. The ring-like architecture in both flavivirus and plant virus xrRNAs requires at least one pseudoknot. In the flavivirus xrRNAs, the ring topology appears to be formed by interactions between a three-way junction and the 5 0 end, forming the PK1 pseudoknot [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref]. A second pseudoknot forms to ''latch" the structure closed [bib_ref] New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: conservation,..., Kieft [/bib_ref] [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref]. In plant virus xrRNAs, formation of PK1 (Figs. [bib_ref] Viral RNA structure-based strategies to manipulate translation, Jaafar [/bib_ref] [bib_ref] RNA regulatory processes in RNA virus biology, Cross [/bib_ref] and the ring follows degradation by Xrn1 through the P1 stem, leading to the exoribonuclease resistant fold, as shown by Förster resonance energy transfer (FRET; [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref]. A similar phenomenon of structural remodeling could occur within other xrRNA families, particularly when alternative pairing schemes are proposed, which may result in the Xrn1 halt site being located within a predicted stem (compare for example secondary structure predictions in [bib_ref] Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs, Macfadden [/bib_ref] [bib_ref] Functional RNA structures in the 3'UTR of tick-borne, insect-specific and no-known-vector flaviviruses, Ochsenreiter [/bib_ref] [bib_ref] Xrn1-resistant RNA structures are well-conserved within the genus flavivirus, Dilweg [/bib_ref]. In short, just like 'co-transcription folding' can occur as an RNA chain is progressively extended [bib_ref] Direct observation of cotranscriptional folding in an adenine riboswitch, Frieda [/bib_ref] , progressive degradation of an RNA in the 5' to 3' direction can create 'co-degradational' stabilization of a specific structure. While these proposed folding pathways remain to be fully tested, they may present two different ways to solve the topological challenge of 'threading' a single strand of RNA through a ring.
## How unique are xrrna ring-like structures?
Ring-like structures in xrRNAs beg the question as to how common these folds are. In fact, ring-like topologies have been previously observed in RNA structures. For example, double-stranded regions may form a ring-shaped super-structure, as in the packaging motor prohead RNA [bib_ref] Structure and assembly of the essential RNA ring component of a viral..., Ding [/bib_ref]. Ring structures are also appealing for synthetic biology [bib_ref] Ring Catalog: A resource for designing self-assembling RNA nanostructures, Parlea [/bib_ref] , which focuses on the design RNA nanoparticle for disease diagnosis and drug delivery [bib_ref] The emerging field of RNA nanotechnology, Guo [/bib_ref]. Such RNA rings combine several molecules, which is different from the situation in xrRNAs, where a single continuous section of an RNA strand encircles a single-stranded element. The resulting structure is ''knot-like" and thus far seems unique to xrRNAs [fig_ref] Figure 2: Ring-like structures in RNA [/fig_ref]. The Alu region from the bacterial single recognition particle (SRP) shows a stunningly similar ring architecture to that of xrRNAs, but no RNA threads through the ring [bib_ref] Structure of the complete bacterial SRP Alu domain, Kempf [/bib_ref] [fig_ref] Figure 2: Ring-like structures in RNA [/fig_ref]. In the case of the SARS-CoV-2 frameshift stimulation element, a ring was proposed to represent a critical structural feature, but that ring is assembled from several parts of the RNA [fig_ref] Figure 2: Ring-like structures in RNA [/fig_ref]. Additional evidence may be required to support a ring-like status for features that essentially constitute tightly packed RNA. Whether the topology seen in xrRNAs represents a distinct and defining characteristic of xrRNAs remains to be determined as more xrRNA structures are solved, or perhaps similar features are identified in other RNAs.
## Flavivirus xrrna structures in context
xrRNAs form discrete folded structures, which are often found in series in the 3 0 UTRs of many flaviviruses [bib_ref] Exoribonuclease-Resistant RNAs exist within both coding and noncoding subgenomic RNAs, Steckelberg [/bib_ref] [bib_ref] Structural features of an Xrn1-resistant plant virus RNA, Dilweg [/bib_ref] [bib_ref] Dengue virus RNA structure specialization facilitates host adaptation, Villordo [/bib_ref]. While these 'tandem' xrRNAs might initially seem independent and redundant, evidence suggests that the structural integrity of one can affect the function of the other [bib_ref] The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production, Chapman [/bib_ref] [bib_ref] Zika virus subgenomic flavivirus RNA generation requires cooperativity between duplicated RNA structures..., Pallarés [/bib_ref] [bib_ref] RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus..., Chapman [/bib_ref]. This may be an important virological feature, as studies of the Dengue virus (DENV) and Zika show tandem xrRNA1 and xrRNA2 allow for a higher fitness as the virus cycles between hosts (such as mosquito and human) [bib_ref] Dengue virus RNA structure specialization facilitates host adaptation, Villordo [/bib_ref] , where sfRNAs may play different roles. When two xrRNA folds are present in tandem, the pressure to maintain one of them may be relaxed to accommodate mutations that would be beneficial for that particular environment. How do two tandem xrRNAs communicate to couple their function? The most straightforward hypothesis is through a direct physical interaction. Consistent with this, small-angle X-ray scattering (SAXS) revealed that tandem xrRNAs from ZIKV, DENV2 and WNV have a defined envelope and thus may have a preferred relative orientation, but that they do not appear to be in intimate contact [bib_ref] Long non-coding subgenomic flavivirus RNAs have extended 3D structures and are flexible..., Zhang [/bib_ref]. However, because SAXS envelopes reflect the populationally weighted average of the structural ensemble, more data are needed at higher resolution to fully assess the presence and nature of likely physical contacts between individual xrRNAs in tandem. Overall, these current data suggest that flavivirus 3 0 UTRs and sfRNAs are best described as a structural ensemble rather than by a single conformation [bib_ref] Long non-coding subgenomic flavivirus RNAs have extended 3D structures and are flexible..., Zhang [/bib_ref]. This characteristic may be important for the virus, allowing alternative folds to be adopted in different environments or at different stages of the viral life cycle to coordinate different processes important for infection.
## A budding structure-based taxonomy of xrrnas
Within the flavivirus supergroup where they were originally found, xrRNAs have now been identified and characterized in all genera of Flaviviridae [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref] [bib_ref] Subgenomic flaviviral RNAs: What do we know after the first decade of..., Slonchak [/bib_ref] [bib_ref] Functional RNA structures in the 3'UTR of tick-borne, insect-specific and no-known-vector flaviviruses, Ochsenreiter [/bib_ref] [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref]. Initial characterization of these xrRNAs based on proposed secondary structures, conserved sequences, and the halt point of the enzyme suggested two major genera-specific classes [bib_ref] Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs, Macfadden [/bib_ref] , both organized around a threeway junction (3WJ [bib_ref] Topology of three-way junctions in folded RNAs, Lescoute [/bib_ref] and at least one pseudoknot [bib_ref] Structure and function of pseudoknots involved in gene expression control, Peselis [/bib_ref] [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref]. The high-resolution structures of class 1 (from MVE and ZIKV) revealed additional tertiary features, including a conserved basetriple interaction (BT; [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref] supplemented with additional non-canonical interactions necessary to support such intricate folds. A three-dimensional structure of a class 2 xrRNA (e.g., within Tick-Borne Encephalitis Virus, Langat Virus and Powassan Virus) has yet to be solved, but alignments of the conserved xrRNA fea- [bib_ref] The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production, Chapman [/bib_ref] [bib_ref] Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a..., Akiyama [/bib_ref] (32 sequences and models from [bib_ref] New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: conservation,..., Kieft [/bib_ref] [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref]. Core features (P1, P3, PK1, BT and the discriminating position (in orange)) are horizontally aligned between panels A-C. (B) 3-D based secondary structure of subclass 1b xrRNAs(87 sequences from [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref]. (C) Predicted secondary structure of class 2 xrRNAs (28 sequences from [bib_ref] Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs, Macfadden [/bib_ref] [bib_ref] Xrn1-resistant RNA structures are well-conserved within the genus flavivirus, Dilweg [/bib_ref] , with automatic improvement of covariation patterns using R-scape [bib_ref] A statistical test for conserved RNA structure shows lack of evidence for..., Rivas [/bib_ref] [bib_ref] RNA structure prediction using positive and negative evolutionary information, Rivas [/bib_ref]. (D) 3-D based secondary structure of xrRNAs from Luteoviridae and Tombusviridae (55 sequences from [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref]. (E) Predicted secondary structure of xrRNAs from Benyviridae, Betaflexiviridae and Virgaviridae (original 10-12 sequence alignment [bib_ref] Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading..., Peltier [/bib_ref] [bib_ref] Structural features of an Xrn1-resistant plant virus RNA, Dilweg [/bib_ref] expanded to 47 sequences from searching the viral sequence databaseusing a published methodology [bib_ref] Exoribonuclease-Resistant RNAs exist within both coding and noncoding subgenomic RNAs, Steckelberg [/bib_ref] [bib_ref] Group I introns are widespread in archaea, Nawrocki [/bib_ref] [bib_ref] Infernal 1.1,. 100-fold faster RNA homology searches, Nawrocki [/bib_ref] tures in both classes have been harnessed by a search algorithm for automatic identification of xrRNAs [bib_ref] A database of flavivirus RNA structures with a search algorithm for pseudoknots..., Zammit [/bib_ref]. Overall, a taxonomy of xrRNAs emerges from the knowledge of their features at the secondary and tertiary structure levels [bib_ref] Functional RNA structures in the 3'UTR of tick-borne, insect-specific and no-known-vector flaviviruses, Ochsenreiter [/bib_ref] [bib_ref] Xrn1-resistant RNA structures are well-conserved within the genus flavivirus, Dilweg [/bib_ref] [bib_ref] Information encoded by the flavivirus genomes beyond the nucleotide sequence, Ramos-Lorente [/bib_ref] [bib_ref] All genera of Flaviviridae host a conserved Xrn1-resistant RNA motif, Dilweg [/bib_ref]. By definition, the current classification for xrRNAs will be amended as more xrRNAs are discovered. Specifically, class 1 xrRNAs are distinguished from class 2 xrRNAs on the basis of (1) their 3WJ configuration, (2) a longer distance between the regions forming the PK2 pseudoknot in 2, and (3) a longer P4 in 2 [bib_ref] Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs, Macfadden [/bib_ref] [bib_ref] Xrn1-resistant RNA structures are well-conserved within the genus flavivirus, Dilweg [/bib_ref] [bib_ref] Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses, Schnettler [/bib_ref]. xrRNAs originally described as belonging to class 1 are now further divided into subclasses 1a (32 sequences, from e.g., MVEV, ZIKV, and DENV [bib_ref] New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: conservation,..., Kieft [/bib_ref] [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref] and 1b (87 sequences, from e.g., Culex Flavivirus, Cell Fusing Agent Virus and TABV [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref] : (1) in 1b, the P1 and P3 stems are shorter than in 1a, (2) characteristic non-Watson-Crick pairs in P1 are found in 1b but not 1a, (3) a P4 stem may not always be present in 1b, and (4) the BT interaction tends to be U-A-U for 1a but C-G-C + for 1b [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref] [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref]. Whether the BT interaction is present in 2 remains an open question. (Sub)classes 1a, 1b and 2 are distinct on the basis of bioinformatic and structural analysis; in the case of subclasses 1a and 1b this extends to distinct tertiary interactions beyond Watson-Crick base-pairing.
In addition, the region of the 3WJ between stems P2 and P3 (which comprises either a single nucleotide or no nucleotide) acts as a convenient discriminator between classes and subclasses (orange on [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref] ,D and 4A-C). A~90% conserved C in 1a forms tertiary contacts at the core of the fold [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref] ; replacing it with a G or deleting it abrogates exoribonuclease resistance [bib_ref] The structural basis of pathogenic subgenomic flavivirus RNA (sfRNA) production, Chapman [/bib_ref] [bib_ref] Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a..., Akiyama [/bib_ref]. Conversely, no nucleotide is present at the equivalent position in 1b [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref] and introducing one leads to loss of resistance. Hence, subclasses 1a and 1b achieve a similar fold and architecture with different sets of tertiary interactions. In class 2, a highly conserved A is found at what is proposed to be the analogous location [fig_ref] Figure 4: Covariation of xrRNAs [/fig_ref] , which can be hypothesized to similarly support the architecture of the 3WJ, although this has not yet been visualized in a structure.
Overall, using the solved crystal structures (for 1a and 1b representatives), comparative sequence alignments, site-directed mutagenesis, and chemical mapping data (for all classes), we can hypothesize that all xrRNAs from the Flavivirus supergroup fold around a generally similar core [fig_ref] Figure 4: Covariation of xrRNAs [/fig_ref]. Locally, key interactions differ, leading to the classification into classes and subclasses, but globally, core ring structures are similar. 1b is more compact than 1a, while 2 presents a longer PK1, with additional pairing upstream of PK1 as well as 10-15 nucleotides within P1 [fig_ref] Figure 4: Covariation of xrRNAs [/fig_ref] [bib_ref] Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs, Macfadden [/bib_ref] [bib_ref] Xrn1-resistant RNA structures are well-conserved within the genus flavivirus, Dilweg [/bib_ref]. The hypothesis that xrRNAs fold around a common core remains to be validated through structural analysis. In particular, the three-dimensional structure of a class 2 representative would illustrate the role of the discriminating A between P2 and P3 and would indicate whether the conserved but covarying BT interaction in other classes is present in 2.
The xrRNAs from Luteoviridae and Tombusviridae adopt distinct secondary structures from the flavivirus xrRNAs, with no central 3WJ [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref]. The crystal structures of Tombusviridae xrRNAs in an inactive conformation revealed a stem-loop structure with appendages so the apical loop 'falls back' to make contacts with an internal loop region [bib_ref] A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally..., Steckelberg [/bib_ref] [fig_ref] Figure 4: Covariation of xrRNAs [/fig_ref]. These xrRNAs nonetheless form a functionally important and hence conserved pseudoknot [fig_ref] Figure 4: Covariation of xrRNAs [/fig_ref]. In contrast, the Benyviridae xrRNAs contain neither a 3WJ, nor a pseudoknot [fig_ref] Figure 3: Towards a structure-based taxonomy of xrRNAs [/fig_ref]. The currently accepted secondary structure model for the Benyviridae xrRNA has two stem loops separated by a 10 nucleotide-long linker [bib_ref] Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading..., Peltier [/bib_ref] [bib_ref] Structural features of an Xrn1-resistant plant virus RNA, Dilweg [/bib_ref] [fig_ref] Figure 4: Covariation of xrRNAs [/fig_ref]. The simplicity of that arrangement does not account for the complex fold needed to block an exoribonuclease. In fact, the strong conservation of the first~20 nucleotides neither supports nor disproves this secondary structure. More xrRNAs have been identified with even more distinct predicted structures [bib_ref] Identification of phlebovirus and arenavirus RNA sequences that stall and repress the..., Charley [/bib_ref] [bib_ref] Investigation of novel RNA elements in the 3'UTR of tobacco necrosis virus-D, Newburn [/bib_ref]. Whether these more 'exotic' xrRNAs form more distinct classes remains uncertain until their tertiary folds have been visualized.
## Footprints of evolution?
The presence of diverse xrRNAs in divergent RNA virus clades raises the question of their origins and evolutionary relationships. It seems likely that within the Flavivirus supergroup, xrRNA classes 1 and 2 evolved from a common fold, then the basic core structure supporting the ring-like structure diversified as new lineages arose. This idea is highlighted by the structures of class 1 xrRNAs, which showed variations within a similar overall fold. Furthermore, these variations led to distinct subclasses based on diverged tertiary interactions [bib_ref] Functional RNA structures in the 3'UTR of tick-borne, insect-specific and no-known-vector flaviviruses, Ochsenreiter [/bib_ref] [bib_ref] A New subclass of exoribonuclease-resistant RNA found in multiple genera of flaviviridae, Szucs [/bib_ref]. However, except for class 1 flavivirus xrRNAs, we do not know what the active fold of xrRNAs looks like, which limits fully understanding xrRNA evolution. Solving the structures of xrRNAs from distinct viral families and lineages remains a key to determining how these RNAs evolved.
Likewise, determining how widely xrRNAs are distributed across viruses would offer a valuable reference for better seizing the evolutionary relationships between viruses. If the proposition that viruses have a common ancestor is correct [bib_ref] Origins and evolution of the global RNA virome, Wolf [/bib_ref] , xrRNAs may represent 'molecular clocks' that would help us understand virus evolution and pathogenicity. xrRNAs may also have emerged independently in different lineages, due to the high mutation rates and effective population size of viral particles. Again, a full accounting of xrRNAs and their structures can inform this discussion. Whether, for example, xrRNAs from the Flavivirus supergroup and from Tombusviridae / Luteoviridae are related would be worth investigating. Similarly, searching the recently reported class of Jingmenviruses for xrRNAs ought to be informative, as they are more closely related to Flaviviruses than Hepaci-, Pegi-and Pestiviruses [bib_ref] A Multicomponent animal virus isolated from mosquitoes, Ladner [/bib_ref] [bib_ref] A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral..., Qin [/bib_ref]. Unfortunately, currently available database entries for these viruses lack the 3 0 UTR sequences. Continuing to expand the catalogue of xrRNAs remains a necessity to increase our chances of finding how widespread these folds are.
Tracking evolutionary relationships from comparing xrRNAs could directly inform studies of structured RNA elements in human pathogens outside flaviviruses. Extensively studying xrRNAs from plant viruses for example from Benyviridae, Virgaviridae and Poleroviruses makes sense, as these viruses are closely related to Hepeviridae, Togaviridae and Coronaviridae (based on RNAdependent RNA polymerase sequences, [fig_ref] Figure 1: xrRNAs across the viral phylogeny [/fig_ref] , which comprise human pathogens like hepatitis E viruses, chikungunya viruses, and coronaviruses. The chikungunya virus and coronaviruses possess structured 3WJ RNA elements with currently unknown functions [bib_ref] Updated phylogeny of chikungunya virus suggests lineage-specific RNA architecture, Schneider Adb [/bib_ref] [bib_ref] RNA recombination at Chikungunya virus 3'UTR as an evolutionary mechanism that provides..., Filomatori [/bib_ref] [bib_ref] Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy, Wacker [/bib_ref] , and the hepatitis E virus arose from recombinations of Benyviridae viruses [bib_ref] Ancient recombination events and the origins of hepatitis E virus, Kelly [/bib_ref]. Such putative elements could be systematically tested for exoribonuclease resistance, for example by transposing to viral sequences a genome-wide assay for Xrn1 resistance that was recently reported for a human genomic library [bib_ref] Hovlinc is a recently evolved class of ribozyme found in human lncRNA, Chen [/bib_ref].
## Xrrnas within eukaryotic genomes?
Up to now, xrRNAs have only been characterized in viruses. However, expecting xrRNAs in eukaryotic cells makes sense, because their ability to block exoribonucleases could be a desired feature for some cellular RNAs. In addition, eukaryotic genomes are known to embed viral sequences [bib_ref] Initial sequencing and analysis of the human genome, Lander [/bib_ref] , which would then be passed across species through horizontal gene transfer [bib_ref] Horizontal gene transfer in eukaryotic evolution, Keeling [/bib_ref]. Such sequences of viral origin could comprise xrRNAs. In fact, flaviviral sequences exist in host mosquito genomes Learning more about viral xrRNA folds, their variety, and their evolutionary relationships better equips us to search for similar folds in eukaryotic genomes. Searching for xrRNAs both computationally and experimentally may lead to the discovery of folds with a quite different mechanism to block Xrn1 than their viral counterparts. Such structures could have arisen within eukaryotes without transfer from viruses. Alternatively, ring-like folds may be found within regulatory RNAs like long non-coding RNAs, similarly to folds which promote catalytic activities and which are also found in a variety of genetic contexts [bib_ref] Hovlinc is a recently evolved class of ribozyme found in human lncRNA, Chen [/bib_ref] [bib_ref] An allosteric self-splicing ribozyme triggered by a bacterial second messenger, Lee [/bib_ref].
## Summary and outlook
xrRNAs are examples of viral elements that use complex threedimensional structures to usurp cellular mechanisms. Emerging detailed three-dimensional structural information gives deep insights into the molecular mechanism of these elements, and also allows for new interpretations of biochemical, virological and phylogenetic information. We are now in a position to better explore how widespread and diverse xrRNAs are, and how they are evolving. Doing so has the potential to illuminate the biology of diverse but evolutionarily related viruses, expand our overall knowledge of RNA structure in the viral world, and motivate futures studies into the roles of xrRNAs in virus-induced disease. Because xrRNAs are found in all major superfamilies of RNA viruses, they may represent an ancient feature that existed in the common ancestor to these superfamilies [bib_ref] Origins and evolution of viruses of eukaryotes: The ultimate modularity, Koonin [/bib_ref] [bib_ref] Origin of viruses: primordial replicators recruiting capsids from hosts, Krupovic [/bib_ref]. Active xrRNAs could also be found in cells, where they may have other functions in addition to blocking exoribonucleases.
CRediT authorship contribution statement Quentin Vicens: Conceptualization, Funding acquisition, Writing -original draft, Writing -review & editing. Jeffrey S. Kieft: Conceptualization, Funding acquisition, Writing -review & editing.
## 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 1: xrRNAs across the viral phylogeny. (A) Relationship between viruses based on RNA-dependent RNA polymerase sequences (adapted from [13]). The five main branches stemming from a putative common ancestor are shown by colored boxes. Only relevant viral families are shown, the rest are symbolized as dashed lines. Color coding for the viral families: bold & black, contains an xrRNA; black, representative families within each branch in which xrRNAs have not (yet?) been found; grey, human pathogen closely related to viruses containing xrRNAs. (B) Schematic exoribonuclease-mediated degradation pathway. The xrRNA element blocks the exoribonuclease. [/fig]
[fig] Figure 2: Ring-like structures in RNA. (A) The~15 nucleotides forming a ring around the 5 0 end are highlighted in bold and purple on the three-dimensional structure of xrRNAs from MVE, ZIKV and TABV (PDB ID: 4PQV, 5TPY, 7K16). See Fig. 3B for corresponding secondary structure cartoons. (B) 3D model computed for the SCNMV xrRNA. (C) Ringlike topology in the bacterial SRP Alu RNA (PDB ID 4WFL). (D) Helical packing leading to a ring-like arrangement within the SARS-CoV-2 frameshift stimulation element (PDB ID 6XRZ). The 5 0 end and strand directionality around the ring structure are indicated in all panels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) [/fig]
[fig] Figure 3: Towards a structure-based taxonomy of xrRNAs. (A) Phylogeny within the Flavivirus supergroup, based on the NS5 sequence (adapted from [69]). Genera belong to one of three (sub)classes: 1a, 1b, or 2. The position between P2 and P3 (orange circle) is a key discriminator between xrRNA (sub)classes of xrRNAs. NKV, no known vector flavivirus; TBFV, tick-borne flavivirus; MBFV, mosquito-borne flavivirus; ISFV, insect-specific flavivirus. (B) Cartoon representation of the secondary structure of xrRNAs from the Flavivirus supergroup. P, paired region; PK, pseudoknot; BT, base triple. (C) BT interactions visualized in the crystal structures of the xrRNAs from ZIKV (PDB ID 5TPY) and TABV (PDB ID 7K16). (D) Close up on the discriminating position (orange) in the ZIKV and TABV structures. (E) Cartoon representation of the secondary structure of an xrRNA from Luteoviridae. (F) Cartoon representation of the secondary structure of an xrRNA from Benyviridae. Double arrow in panels B, E, F: Xrn1 halt site. Regions forming the ring as highlighted in Fig. 2 are shown in purple in panels B, E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) [/fig]
[fig] Figure 4: Covariation of xrRNAs. (A) Secondary structure of subclass 1a xrRNAs emphasizing the three-dimensional architecture [/fig]
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Widowhood and Mortality: A Meta-Analysis
Background: While the "widowhood effect" is well known, there is substantial heterogeneity in the magnitude of effects reported in different studies. We conducted a meta-analysis of widowhood and mortality, focusing on longitudinal studies with follow-up from the time of bereavement.Methods and Findings: A random-effects meta-analysis was conducted to calculate the overall relative risk (RR) for subsequent mortality among 2,263,888 subjects from 15 prospective cohort studies. We found a statistically significant positive association between widowhood and mortality, but the widowhood effect was stronger in the period earlier than six months since bereavement (overall RR = 1.41, 95% CI: 1.26, 1.57) compared to the effect after six months (overall RR = 1.14, 95% CI: 1.10, 1.18). Meta-regression showed that the widowhood effect was not different for those aged younger than 65 years compared to those older than 65 (P = 0.25). There was, however, a difference in the magnitude of the widowhood effect by gender; for women the RR was not statistically significantly different from the null (overall RR = 1.04, 95% CI: 1.00, 1.08), while it was for men (overall RR = 1.23, 95% CI: 1.18, 1.28).Conclusions:The results suggest that further studies should focus more on the mechanisms that generate this association especially among men.
# Introduction
The death of a spouse is a common experience in old age, and predicts immediate elevation of mortality risk for the surviving spouse [bib_ref] Marital biography and health at mid-life, Hughes [/bib_ref]. The ''widowhood effect,'' describing the increased probability of death among those experiencing recent spousal bereavement, is well known. The widowhood effect has been found in bereaved men and women of all ages around the world, particularly in the western industrialized world, using both crosssectional and longitudinal data, with and without controlling for covariates, and using diverse statistical methodologies [bib_ref] Mortality differentials by marital status: an international comparison, Hu [/bib_ref] [bib_ref] Causes of death in a widowed population, Helsing [/bib_ref] [bib_ref] Til Death Do Us Part: Marital Disruption and Mortality, Lillard [/bib_ref] [bib_ref] Some epidemiologic aspects of the high mortality rate in the young widowed..., Krause [/bib_ref] [bib_ref] Broken Heart: A Statistical Study of Increased Mortality among Widowers, Parkes [/bib_ref]. Longitudinal studies put the long-term excess risk of death associated with widowhood compared to marriage at about 15%, net of controls, while estimates of short-term effects during the first few months' immediately post-bereavement range from 50% to 90% [bib_ref] Mortality following conjugal bereavement and the effects of a shared environment, Schaefer [/bib_ref] [bib_ref] Widowhood and Race, Elwert [/bib_ref] [bib_ref] Mortality after the death of a spouse: rates and causes of death..., Martikainen [/bib_ref]. However, there is substantial heterogeneity in the magnitude of effects reported in different studies [bib_ref] Mortality following conjugal bereavement and the effects of a shared environment, Schaefer [/bib_ref] [bib_ref] Mortality after the death of a spouse: rates and causes of death..., Martikainen [/bib_ref]. A recent meta-analysis of the literature on marital status (including widowhood) and mortality, reported that widowed persons had an 11% higher risk of mortality when compared to married persons after adjustment for age and additional covariates. This metaanalysis compared mortality among elderly widowed and nonwidowed and thus left two important questions unanswered. First, does the relationship hold in younger populations? Second, what is the magnitude of the relationship when analyses focus on prospective studies with follow-up from the time of bereavement? Because of the evidence that the risk associated with widowhood changes as time since bereavement elapses, comparing mortality experience of widowers to non-widowers can give very biased estimates of the effect of widowhood. In any existing population, the majority of widowers have already survived the highest risk period, which occurs immediately post-bereavement. It is therefore important to focus on studies that begin follow-up at the time of bereavement, rather than comparing the mortality experience of widowers to non-widowers. We therefore conducted a meta-analysis of 2,263,888 subjects based on similar study selection criteria as the review by Stroebe and colleagues [bib_ref] Health outcomes of bereavement, Stroebe [/bib_ref] , but focusing on longitudinal studies with follow-up from the time of bereavement and including publications since early 2006. We aimed to assess the overall relationship between bereavement and mortality based only on longitudinal studies and also explore the factors explaining heterogeneity among studies published so far. Understanding the sources of such between-study heterogeneity in the existing results is important for interpreting meta-analysis results.
# Methods
## Study selection
We followed published guidelines for meta-analyses of observational studies [bib_ref] Meta-analysis of observational studies in epidemiology: a proposal for reporting, Stroup [/bib_ref]. The literature review was conducted in published scientific work to look at the relation between bereavement and mortality. The most valid and reliable information is provided in longitudinal investigations comparing newly bereaved with non-bereaved counterparts, controlling for several confounders such as socioeconomic and lifestyle/behavioral factors the bereaved spouse would have shared with their deceased partner, which could affect the bereaved spouse's health as well.
Studies evaluating widowhood effect and mortality were initially searched using online search engines such as PubMed, Medline, PsycINFO and ISI Web of Science (Thomson Reuters) with the terms ''bereavement/grief/grieving/mourn/mourning,'' ''widow/ widows/widowed/ widowhood,'' ''spouse/spousal'' and ''mortality/death/survival/longevity'' for reports published since 1960 (last searched October 2009; see [fig_ref] Table S1 PubMed: Search Strategy [/fig_ref]. During the time period prior to 1990, we found that there were few longitudinal studies. From the studies within this search period, a study was included if it 1) investigated the relationship between mortality and widowhood status; 2) used a population-based sample, i.e., the majority of participants were non-institutionalized and nonhospitalized healthy individuals; 3) reported quantitative data adjusted for or stratified by at least age and gender; 4) was a prospective, longitudinal study beginning mortality follow-up for the widowed group at the time of bereavement; 5) met quality criteria including follow-up time of at least 3 years, response rate of at least 75%, use of standardized measurements, reporting sufficient data to calculate common effect estimates, and use of a control group of non-bereaved individuals; and 6) was published in English. Finally, to avoid duplication of data from the same subjects, information were selected from only one report/analysis of the same dataset. [fig_ref] Figure 1: Flow Chart of Study Selection Process [/fig_ref] summarizes the process of selecting studies for the meta-analysis; see for the list of extracted studies.
## Data extraction
Data from each included article was independently evaluated and extracted by two reviewers (RM and NK), including information on study design, data sources, country of data origin, sample size, the number of cases, age, gender, estimations, response rate, follow-up duration, outcome, adjustment variables, and statistical modeling strategies. In addition, all data needed for the quality assessment and statistical analysis, including relative risk (RR) estimates (in terms of odds ratio, OR; hazard ratio, HR; risk ratio or incidence rate ratio, IRR, and standardized mortality rate, SMR), relative 95% confidence intervals (95% CI) or standard error (SE) for each exposed group were extracted from published reports. Data were also extracted for the sub-studies generated from each main study, and the analysis was stratified by gender and time since the spousal death.
When enrollment spanned over several years, we used the midpoint of the range for analyses that stratified on the study year. In order to analyze whether the effects of bereavement differed by age, we dichotomized age at 65 because this was available for the majority of the studies, i.e., $65 = yes = 1; ,65 = 0. However, two studies had age dichotomization at 69 [bib_ref] A twin study of mortality after spousal bereavement, Lichtenstein [/bib_ref] or at 74 [bib_ref] Widowhood and mortality risk in a community sample of the elderly: a..., De Leon [/bib_ref] , and we used those ages instead.
# Statistical analysis
A meta-analysis is an analysis of analyses; it is a statistical method of combining the results of independent studies that address a set of related research hypotheses, exploring heterogeneity, and synthesizing summaries if appropriate, generally aiming at producing a single effect estimate for the purpose of integrating findings [bib_ref] Primary, secondary, and meta-analysis of research, Glass [/bib_ref]. The main objectives of a meta-analysis are to 1) to derive a meaningful overall estimate of effect by increasing power and thus reducing the possibility of concluding that there is no statistical association between widowhood and mortality when in fact there is such an association (type 2 error), and to 2) explore potential sources of heterogeneity of results in existing studies and to discover potential patterns of risk among our study results [bib_ref] Systematic reviews in health care: metaanalysis in context, Egger [/bib_ref]. Resulting overall averages when controlling for study characteristics can be considered meta-effect sizes, which are more powerful estimates of the true effect size than those derived in a single study under a given single set of assumptions and conditions. Although a relationship between widowhood and mortality has long been recognized, only one prior study [bib_ref] Marital status and mortality in the elderly: a systematic review and meta-analysis, Manzoli [/bib_ref] has provided a summary estimate of the strength of the association.
As a primary analysis, we estimated the overall relative risk for subsequent mortality among all cohort studies (cohort RR), adjusting for age and gender only. All of the studies we used for our investigation were different in their choices of stratification strategy, whether by age groups (and which age groups), gender, socioeconomic status, or time since bereavement. Not all of them had their ''primary'' analysis using the overall population controlled for age and gender only. To be more specific, there were 2 studies that showed RR for overall population (male and female combined) for all age groups (Hart, Kaprio); 2 studies with overall population for all age groups stratified by time since bereavement ; 3 studies with gender-stratified population for all age groups ; 3 studies with gender-stratified population by time since bereavement (Martikainen, Kaprio, Nagata); 3 studies with genderstratified population by age groups (Martikainen, Mineau, Smith); 1 study with gender-stratified population by age groups and socioeconomic status (Martikainen); 1 study with gender-stratified population of all age groups by other charactistics (Christakis); 3 studies with gender-stratified population of all age groups by timesince -bereavement (Christakis, Nagata, Schaefer); 4 studies with gender-stratified population by age groups and time since bereavement (Kaprio, Lichtenstein, Manor, De Leon); and 2 studies with gender-stratified population by age group, time since bereavement and other characteristics . From the RRs adjusted by age and gender only, whether from overall analysis or stratified analysis, we chose from each study a unique set of RRs with the greatest number of deaths, without overlapping the population in order to avoid double-counting. Our primary analysis was conducted using the set of RRs chosen this way. We used the available information from the studies to calculate and convert various effect measures (OR, RR, SMR) to a common effect measure. In other words, we combined the RRs that are already adjusted for or stratified by age and gender, by using the adjusted or age-and/or gender-specific RRs and then combining them using meta-analytic technique. We then estimated the RR for the main effect study from each analysis used. In secondary analyses, we estimated the RRs for subsequent mortality among all cohort studies in the same way, which included additional covariate adjustment. The exact covariates used varied across studies, but included: financial strain, race/ ethnicity, US-born, health behaviors (smoking, drinking), FEV1, cholesterol, body mass index (BMI), co-morbidities (diabetes, angina, ischemia, previous myocardial infarction (MI), other cardiovascular diseases, respiratory diseases and other chronic diseases), social class, education, occupation, deprivation categories, relative ages of each spouse, and the number of children. For subgroup analyses, we selected specific models reporting RRs for men or women only, age ,65 or $65 years only, and time since bereavement ,6 months or $6 months only, and separately conducted meta-analyses. We evaluated between-study heteroge-neity using I 2 statistics which provides a measure of the percentage of total variability due to between-study heterogeneity, and Cochran Q test which is an extension to chi-square tests for related samples that provides a method for testing for differences between three or more matched sets of frequencies or proportions) [bib_ref] Quantifying heterogeneity in a meta-analysis, Higgins [/bib_ref] [bib_ref] The combination of estimates from different experiments, Cochran [/bib_ref]. We conducted a sensitivity analysis of whether our primary results were dependent on any single study by repeating analyses excluding each study one at a time. We also conducted a meta-regression, by regressing study-specific effect estimates chosen for our primary analysis on study characteristics as explanatory variables. It should be noted that we used a different set of RRs to conduct the meta-regression on time-sincebereavement variable. These RRs were again a unique set of effect estimates; however, for the purpose of focused analysis on time since bereavement, we chose those with the time-sincebereavement information from each study that had the information available without the regard to the number of deaths. We did this because the set of estimates used in our primary analysis did not include a sufficient number of stratified estimates by ,6 months/$6 months. Finally, we used funnel plots to assess reporting bias, based on the effect estimates used in our primary analysis (stratified single-study results). Begg's and Egger's tests were conducted to measure the degree of funnel plot asymmetry [bib_ref] Bias in meta-analysis detected by a simple, graphical test, Egger [/bib_ref] [bib_ref] Investigating and dealing with publication and other biases in meta-analysis, Sterne [/bib_ref]. One of the weaknesses of meta-analysis is the heavy reliance on published studies, which may create exaggerated outcomes, as it is very hard to publish studies that show no significant results. Such a reporting bias results in the distribution of effect sizes that are biased, skewed or cut off, creating a serious base rate fallacy, in which the significance of the published studies is over-estimated.
The meta-analyses were performed in STATA v.11.1 (STATA Corporation, College Station, TX, 2003), including the creation of plots and estimate SE from 95% CI. [fig_ref] Table S3: Summary of Selected Studies on Mortality of Bereavement [/fig_ref] presents an overview of findings of 15 longitudinal studies published since 1960 on the mortality associated with bereavement. The 15 studies got consolidated into 12 studies, because we consolidated 1) three studies by same authors (Martikainen, et al) using the same dataset and 2) two studies by same authors . The main meta-analysis compared the risk of all-cause mortality of currently married (or persons with a partner) vs. widowed individuals. There was only one study examining the main effect in men and women combined. All other studies gave the results stratified by gender, to show the modifying impact of gender on the outcome. The data were also stratified by age group (dichotomized at 65 years of age), country, and time since bereavement. The studies included in the meta-analysis have the following characteristics. Datasets for the studies come from the USA (7) [bib_ref] Mortality following conjugal bereavement and the effects of a shared environment, Schaefer [/bib_ref] [bib_ref] Widowhood and Race, Elwert [/bib_ref] [bib_ref] Widowhood and mortality risk in a community sample of the elderly: a..., De Leon [/bib_ref] [bib_ref] Mortality after the Hospitalization of a Spouse, Christakis [/bib_ref] [bib_ref] Risk of mortality following widowhood: Age and sex differences by mode of..., Smith [/bib_ref] [bib_ref] Historical trends of survival among widows and widowers, Mineau [/bib_ref] [bib_ref] Risk of mortality related to widowhood in older Mexican Americans, Stimpson [/bib_ref] , Finland (4) [bib_ref] Mortality after the death of a spouse: rates and causes of death..., Martikainen [/bib_ref] [bib_ref] Do education and income buffer the effects of death of spouse on..., Martikainen [/bib_ref] [bib_ref] Mortality after bereavement: a prospective study of 95,647 widowed persons, Kaprio [/bib_ref] [bib_ref] Mortality after death of spouse in relation to duration of bereavement in..., Martikainen [/bib_ref] , UK/Scotland (1) [bib_ref] Effect of conjugal bereavement on mortality of the bereaved spouse in participants..., Hart [/bib_ref] , Sweden (1) [bib_ref] A twin study of mortality after spousal bereavement, Lichtenstein [/bib_ref] , Japan (1) [bib_ref] The Impact of Changes in Marital Status on the Mortality of Elderly..., Nagata [/bib_ref] , and Israel (1) [bib_ref] Mortality after spousal loss: are there sociodemographic differences?, Manor [/bib_ref]. Of the 15 studies we used (which eventually became consolidated into 12 distinct studies), thirteen were reported as nationally representative. The number of years of follow-up ranged from 3 to 95 years. The causes of death investigated were mostly all-cause mortality, with 3 studies looking at cardiovascular and cerebrovascular diseases. Most of the studies showed significant excess mortality associated with widowhood, especially in the first month to 6 months following the spousal death. Excess mortality during this period spanned around 40% to 50% on average, for both men and women. Not all studies provided the age-and gender-adjusted data; 8 studies out of 15 only provided the age-and gender-stratified results. Of the 15 studies, 13 papers have some levels of individual socio-economic status (SES) control, using occupation, education, gross income, poverty status and/or housing tenure and condition. Even though SES controls were used for these studies, the level of control was rather coarse, often using dichotomous measures (e.g., below or above the federal poverty line, crude educational categories, etc.). Co-morbidities were controlled in 8 studies, including one study that controlled only the psychiatric disorder prior to the partner's death and only two studies using the Charlson score, the more comprehensive co-morbidities score.
# Results
The random effect meta-analysis of all eligible age-and genderadjusted estimates provided an overall RR of bereavement of 1.12 (95% CI: 1.10, 1.15; P,0.0001). This result is consistent with the previous meta-analysis result. The studies included in the metaanalysis were highly heterogeneous (I 2 = 88; 95% CI: 85, 90; Heterogeneity P,0.0001). Gender-specific analysis shows that males have overall RR of 1.22 (95% CI: 1.18, 1.26), and females 1.03 (95% CI: 1.00, 1.07). Age-specific analysis shows that those under age 65 years had an overall RR of 1.18 (95% CI: 1.17, 1.25), while those 65 years and above had an overall RR of 1.10 (95% CI: 1.07, 1.13). The overall RR did not change when we selected only those studies that adjusted for additional covariates, e.g., income, education, race/ethnicity, US-born, health behaviors, co-morbidities, social class, occupation, number of children, etc. (overall RR 1.14; 95% CI: 1.11, 1.18; P,0.0001; . This was confirmed by meta-regressions that stratified the primary meta-analysis by various covariates. The test of no difference between eight age-and gender-adjusted models and the four models additionally adjusted for other covariates was not statistically significant (P = 0.74) (See . As the source of between-study heterogeneity, we found that the widowhood effect was stronger in the period earlier than six months since bereavement (overall RR = 1.41, 95% CI: 1.26, 1.57) compared to the effect in later time (overall RR = 1.14, 95% CI: 1.10, 1.18; P,0.0001 for the difference) (See [fig_ref] Figure 2: Forest Plot for Time Since Bereavement of ,6 months [/fig_ref]. In addition, the widowhood effect was not different for those aged younger than 65 years compared to those older than 65 (P = 0.25). There was, however, a difference in the magnitude of the widowhood effect by gender; the widowhood effect for women was not statistically significantly different from the null (overall RR = 1.04, 95% CI: 1.00, 1.08, see [fig_ref] Figure 4: Forest Plot for Men [/fig_ref] , while it was statistically significant for men (overall RR = 1.23, 95% CI: 1.18, 1.28; P,0.0001 for the difference). The baseline years of our studies varied from 1860 to 1993. To understand whether there is a cohort effect, we examined the difference in RR depending on the baseline year. There was a small but significant incremental increase in the widowhood effect with calendar year (overall RR = 1.05, 95% CI: 1.01, 1.09). Finally, between-country difference for US vs. non-US was not significant, nor was for US vs. Europe vs. Japan (see .
A series of sensitivity analyses was conducted by omitting one study at a time from the selection pool. The results showed that there was not a particularly influential study among all selected studies (see [fig_ref] Figure 6: Funnel Plot of Reporting Bias [/fig_ref]. The overall RRs ranged from 1.12 to 1.22. The funnel plot constructed to gauge reporting bias was fairly balanced and the formal statistical test also did not detect any significant reporting bias (Egger's test P = 0.35, Begg's test P = 0.64). Although producing a funnel plot based on subgroups and not on studies may obscure reporting bias by just including a lot of small non-significant subgroups, in many cases the authors of the selected studies only calculate RRs in subgroups. In this case, the ''subgroup analysis'' estimates are the appropriate data points for the funnel plot. Further, we had no way of using overall effect size in the funnel plot if the paper does not provide such information.
# Discussion
In our meta-analysis of cohort studies with 2,263,888 subjects, we found that people experiencing widowhood have an excess risk for premature mortality independent of their age and gender. A unique strength of our analysis and selection criteria was that datasets are mostly nationally representative with sufficient sample sizes. The results, all of which come from longitudinal studies identified in our literature review, seem consistent in showing the widowhood effect, i.e., bereavement is associated with an increased risk of mortality from many causes including suicide. Only limited controls for socioeconomic confounders were available in most studies, and generally even less information was available to adjust for co-morbidities and lifestyle factors. Our results indicate that the effect of widowhood was not significant in studies that controlled for more covariates than age and gender. Although this was based on a low number of included studies that control for other covariates, the result indicates that caution is still needed when discussing whether there is an association of widowhood with mortality or not.
The overall cohort relative risk should also be interpreted with caution, given the substantial heterogeneity detected between studies. Several local factors may be taken into account for this heterogeneity, including the spatial unit across which the widowhood effect was evaluated. Although not evaluated in this study, other contextual characteristics such as national and local social security policies and neighborhood/residential area factors could also explain the heterogeneity between studies. Given the existence of substantial heterogeneity, our primary purpose of this meta-analysis was to formally and quantitatively test the hypotheses on the source of the heterogeneity rather than to get the overall estimates of the effect size. Our analysis by metaregressions shows quite reasonable results.
Of the significant moderators of the widowhood effect in our results, the most notable one was the time since bereavement. The effect immediately following the spousal loss seems to be more detrimental (,6 months) compared to the longer-term effect, which is consistent in all of the individual studies. The gender difference is also consistently significant in our analysis as well as the individual studies. The difference in baseline year, though fairly small, suggests modest cohort effect.
There are several potential pathways via which marriage, as opposed to widowhood, may protect health [bib_ref] The impact of the family on health: The decade in review, Ross [/bib_ref]. First of all, marriage may increase dispensable income through the principles of economies of scale and specialization of spouses to market and family work, which in turn leads to better health insofar as access to material resources [bib_ref] Reconceptualizing marital status as a continuum of social attachment, Ross [/bib_ref]. Second, marriage could provide additional social and emotional support, which may act as a buffer against harmful effects of various psychosocial stress. The guardian role theory suggests that spouses monitor each other, encouraging healthy behaviors of the partner, such as compliance with medical regimens, as well as discouraging unhealthy and risky ones, such as alcohol and other substance use and smoking [bib_ref] The impact of the family on health: The decade in review, Ross [/bib_ref] [bib_ref] Family status and health behaviors: social control as a dimension of social..., Umberson [/bib_ref]. Married people, particularly males, have been shown to die less often because of accidents, suicide, homicide, cirrhosis of the liver and diabetes compared to the unmarried [bib_ref] Marriage, sex, and mortality, Rogers [/bib_ref]. A final hypothesis is that grief associated with the loss of a loved one increases mortality risk directly via psychoneuroimmune pathways. This is especially plausible given that widowhood effect is markedly stronger in the months immediately following the spouse's death than in the years following. Behavioral pathways, and pathways mediated by material resources are likely to influence health in a long-term, cumulative fashion. However, the long-term excess risk associated with widowhood appears quite modest, conditional on surviving the immediate bereavement period.
The following limitations need to be considered while interpreting our findings. First and foremost, all meta-analyses of observational studies are inherently vulnerable to the biases in the original studies [bib_ref] Meta-analysis -Bias in location and selection of studies, Egger [/bib_ref]. For example, although we evaluated multiple models using alternative sets of covariates, the estimates from the original studies might have been biased by residual confounding. Most of the studies done so far do not seem to have robust socioeconomic controls (i.e., income, wealth, education) at the individual level, all of which are likely to influence mortality and could confound the relationship between widowhood and mortality. Further, there has been very little control of comorbidities in the existing studies, which exacerbates the bias from inadequate socioeconomic controls. There is a need to learn more about co-determinants of the differential outcomes of bereavement, to understand how the circumstances of bereavement may interact with pre-bereavement experiences, personal socioeconomic and health factors, and ways of coping with grief to cause difficulties. Second, a significant number of studies was excluded because they did not report the necessary information to permit us to include them in the meta-analysis. The omission of such studies might have influenced our conclusions. Third, some of the papers we used only provided age-and gender-stratified analyses but no further control by any other variable, which may have potentially contributed to the high degree of heterogeneity. However, we do not think it seriously compromises the credibility of our findings; rather, our analytical strategy was typical of published metaanalyses. A random-effects meta-analysis addresses the heterogeneity even due to the differences in population (i.e., age groups and gender), under certain assumptions. In general, combining stratified effect estimates is an appropriate way to handle confounding by the stratification variable, assuming no treatment effect heterogeneity, with the only disadvantage that age is a continuous covariate and the stratified models usually categorize age into only a few groups. However, many of the papers we used did not provide a single age-adjusted RR. Fourth, none of the studies done included information on marriage quality or the length of the marriage prior to the study period. Both are potential confounders or modifiers of the observed relationship between individual widowhood and mortality. Widowhood is a common experience that represents not only a source of personal grief but also appears to predict elevated mortality risk in men and women in diverse populations. The overall association between widowhood and mortality is now well established. The next generation of studies should move beyond this finding and focus on identifying opportunities to improve the health consequences of widowhood. Towards this goal, we identify four important gaps in research on widowhood and mortality. First, further exploration needs to be made on potential mediation and/or confounding. Most prior studies have inadequately controlled for socioeconomic confounders, especially childhood and lifecourse socioeconomic conditions in addition to income, wealth and education, thus leading to uncertainty about whether the widowhood effect is causal. Second, related, the fundamental issue around more meticulous measure of confounders is likely the original study design, rather than the selection of (or lack thereof) covariates at the time of analysis. Cohort studies that have only one-time measurement of exposures, for example measuring exposures at baseline only, cannot handle the confounding and/or mediation issues. More sophisticated longitudinal studies need to be conducted that follow married individuals over time with measurements at many regular intervals, before and after the widowhood event.
Third, very few studies have examined physiologic changes underlying the widowhood effect; such analyses could both help improve the causal basis for interpreting widowhood-mortality association, and identify points of intervention. Fourth, despite heterogeneity in widowhood effect sizes, as revealed in prior research, surprisingly little attention has been paid to identifying modifying factors at the individual, familial or contextual level. Identifying modifiers is crucial to understand how to reduce the adverse health consequences of bereavement and as a marker for especially at-risk groups. It is known that the widowhood effect is highest immediately after spousal loss and declines thereafter. Studies that average the effects across multiple time periods obscure the nature of the relationship between widowhood and mortality. Because most widowed individuals at any given moment in time lost their spouse one or more years earlier, studies contrasting currently widowed to currently married will generally produce downwardly biased estimates of the widowhood effect compared to models based on ''incident'' widowhood. The selection of models is often driven by the available data. In many data sets, the data structure is not appropriate to follow individuals longitudinally from marriage to widowhood. In conclusion, in a large meta-analysis of prospective studies we observed a robust association between widowhood and mortality. An examination of how far social exposures such as widowhood may vary across individuals and across different contextual factors (including time and place) is critical for improving our understanding of the specific pathways, through which social exposures influence health.
## Supporting information
[fig] Figure 1: Flow Chart of Study Selection Process. doi:10.1371/journal.pone.0023465.g001 [/fig]
[fig] Figure 2: Forest Plot for Time Since Bereavement of ,6 months. doi:10.1371/journal.pone.0023465.g002 [/fig]
[fig] Figure 3: Forest Plot for Time Since Bereavement of $6 months. doi:10.1371/journal.pone.0023465.g003 [/fig]
[fig] Figure 4: Forest Plot for Men. doi:10.1371/journal.pone.0023465.g004 [/fig]
[fig] Figure 5: Forest Plot for Women. doi:10.1371/journal.pone.0023465.g005 [/fig]
[fig] Figure 6: Funnel Plot of Reporting Bias. doi:10.1371/journal.pone.0023465.g006 [/fig]
[table] Table 1 Table 2: Result of the Primary Meta-Analysis and Stratified Meta-Analyses. Result of the Meta-Regression: Test of Interaction Effect between the Widowhood and Various Study Characteristics. [/table]
[table] Table S1 PubMed: Search Strategy. (DOCX)Table S2 List of extracted studies. (DOCX) [/table]
[table] Table S3: Summary of Selected Studies on Mortality of Bereavement. (DOCX) [/table]
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The sound of trustworthiness: Acoustic-based modulation of perceived voice personality
When we hear a new voice we automatically form a "first impression" of the voice owner's personality; a single word is sufficient to yield ratings highly consistent across listeners. Past studies have shown correlations between personality ratings and acoustical parameters of voice, suggesting a potential acoustical basis for voice personality impressions, but its nature and extent remain unclear. Here we used data-driven voice computational modelling to investigate the link between acoustics and perceived trustworthiness in the single word "hello". Two prototypical voice stimuli were generated based on the acoustical features of voices rated low or high in perceived trustworthiness, respectively, as well as a continuum of stimuli inter-and extrapolated between these two prototypes. Five hundred listeners provided trustworthiness ratings on the stimuli via an online interface. We observed an extremely tight relationship between trustworthiness ratings and position along the trustworthiness continuum (r = 0.99). Not only were trustworthiness ratings higher for the high-than the low-prototypes, but the difference could be modulated quasi-linearly by reducing or exaggerating the acoustical difference between the prototypes, resulting in a strong caricaturing effect. The f0 trajectory, or intonation, appeared a parameter of particular relevance: hellos rated high in trustworthiness were characterized by a high starting f0 then a marked decrease at mid-utterance to finish on a strong rise. These results demonstrate a strong acoustical basis for voice personality impressions, opening the door to multiple potential applications.OPEN ACCESSCitation: Belin P, Boehme B, McAleer P (2017) The sound of trustworthiness: Acoustic-based modulation of perceived voice personality. PLoS ONE 12(10): e0185651. https://doi.org/10.
# Introduction
When we see a new face or hear a new voice, we automatically form a first impression of the person's personality, inferring traits such as trustworthiness, competence, attractiveness, etc. [bib_ref] The functional basis of face evaluation, Oosterhof [/bib_ref] [bib_ref] Judging personality from voice: a cross-cultural approach to an old issue in..., Scherer [/bib_ref] [bib_ref] Voice of Personality-Stereotyped Judgments and Their Relation to Voice Quality and Sex..., Aronovitch [/bib_ref] [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] [bib_ref] The role of femininity and averageness of voice pitch in aesthetic judgments..., Feinberg [/bib_ref]. These personality impressions may not be accurate but they are robust, showing high inter-rater agreement [bib_ref] The functional basis of face evaluation, Oosterhof [/bib_ref] [bib_ref] Voice of Personality-Stereotyped Judgments and Their Relation to Voice Quality and Sex..., Aronovitch [/bib_ref] [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] [bib_ref] Personality Inference from Voice Quality-Loud Voice of Extroversion, Scherer [/bib_ref] ; they also form very rapidly as sub-second exposure to face or voice is sufficient to yield consistent judgments [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] [bib_ref] First impressions: making up your mind after a 100-ms exposure to a..., Willis [/bib_ref].
For both faces and voices, first impressions are well summarized by a 2-dimensional « social space » with axes mapping onto perceived trustworthiness and dominance, respectively [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] [bib_ref] Personality Inference from Voice Quality-Loud Voice of Extroversion, Scherer [/bib_ref]. These two dimensions are thought to reflect evolutionary ancient mechanisms, developed under selective pressures of living in large social groups, to rapidly assess the dispositions (trustworthiness) and ability to act on these dispositions (dominance) of an unfamiliar conspecific individual [bib_ref] Accurate social perception at zero acquaintance: the affordances of a Gibsonian approach...., Zebrowitz [/bib_ref]. For faces, data-driven computational modelling has demonstrated a very tight relationship between perceived personality impressions and specific facial features [bib_ref] The functional basis of face evaluation, Oosterhof [/bib_ref] [bib_ref] Social attributions from faces: determinants, consequences, accuracy, and functional significance, Todorov [/bib_ref]. The generation of model faces at different positions of the "social face space" shows that the main dimensions of trustworthiness and dominance map onto very different sets of facial features, such as jaw shape vs. eyebrow shape.
For voices, significant correlations between personality ratings and acoustical parameters such as average fundamental frequency (f0) have been observed suggesting an acoustical basis for voice personality judgments by which principled changes in voice acoustics could potentially map onto desired changes in perceived personality. But until now the correlational approach has fallen short of precisely characterizing this acoustical basis. How exactly should one say "hello" to be perceived as e.g., trustworthy by new listeners?
Here we used voice computational modelling to investigate more closely the acoustical basis for perceived voice personality. We focused on perceived trustworthiness because due to its evolutionary link to cooperation and survival, trust is a central aspect of human communication, the foundation for functioning relationships, interpersonal interaction, effective collaboration [bib_ref] Facial and Vocal Cues in Perception of Trustworthiness, Tsankova [/bib_ref] as well as the cooperation and mutual adaptation needed to surmount problems [bib_ref] Affect-and cognition-based trust as foundations for interpersonal cooperation in organizations, Mcallister [/bib_ref]. Ratings along that trait were found in a previous study to correlate most strongly with the first principal component explaining more than half the variance in overall voice personality ratings [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] , indicating the foremost role of perceived trustworthiness in overall voice personality impressions.
We first asked whether voices modeled as the acoustical average of several low-and hightrustworthy voices would also be rated with low and high perceived trustworthiness-potential evidence of a mapping between acoustical and social voice spaces. We further explored the possible parametric nature of the acoustics-personality link by collecting trustworthiness ratings for voice stimuli generated in voice space along the direction defined by the low-and high-trustworthiness prototypes, including caricatures of one relative to the other.
Thirty-two male voices saying the word "hello" were ranked by their average trustworthiness ratings collected as part of a previous study [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] [fig_ref] Fig 1: Generation of the voice trustworthiness continuum [/fig_ref]. A low-and a high-trustworthiness prototypical model voice stimuli were generated via high-quality analysis/resynthesis [bib_ref] Auditory morphing based on an elastic perceptual distance metric in an interference-free..., Kawahara [/bib_ref] based on the average acoustical characteristics of the eight voices (25%) rated lowest and highest on perceived trustworthiness, respectively [fig_ref] Fig 1: Generation of the voice trustworthiness continuum [/fig_ref]. Methods). This resulted in two natural-sounding "hellos" (cf. audio files in Supporting Information) with perceptually different voice qualities and which we hypothesized would yield different trustworthiness ratings.
Next we generated via morphing a 9-stimulus "Trustworthiness continuum" in acoustically equal steps that contained the low-and high-trustworthiness prototypes at intermediate positions (stimuli S3 and S7) and that inter-and extrapolated their acoustical difference resulting at the extreme in low-and high-trustworthiness caricatures (stimuli S1 and S9; [fig_ref] Fig 1: Generation of the voice trustworthiness continuum [/fig_ref]. To provide a set of control stimuli generated similarly but unrelated to trustworthiness we generated a second 9-stimulus "Control continuum" based on a random selection of a set of 32 female voices (S2 files in Supporting Information).
Five hundred listeners (146 males, 354 females; aged 19-65 years old, median age = 24) each provided 18 trustworthiness ratings using a visual analogue scale via an online interface, 9 for the morphed voice stimuli of the Trustworthiness continuum and 9 for those of the control continuum (stimuli presented in random orders in counterbalanced blocks; cf. Methods).
Ratings were z-scored within participant prior to analysis to account for inter-individual variation in rating scale use and because we were primarily interested in rating variations within the morphed continua rather than in their absolute values.
# Results and discussion
We observed a compelling relationship between average trustworthiness rating z-scores and position along the Trustworthiness, but not the Control, continuum [fig_ref] Fig 2: Acoustic-based modulation of perceived voice trustworthiness [/fig_ref]. Ratings for the low-and high-trustworthiness prototypes (stimuli S3 and S7) differed highly significantly with lower average ratings for S3 than S7 (S3: z = -0.39; S7: z = 0.40; paired t-test: t(499) = 14.2, p = 1.38e-38) while no such difference was apparent for control stimuli (S3: z = 0.03; S7:z = -0.09; t(499) = 2.08, p = 0.038 n.s. after Bonferroni correction).
We also observed a strong caricaturing effect, as exaggerating acoustical differences between S3 and S7 yielded higher average trustworthiness ratings for S9 than S7 (S7: z = 0.40; S9; z = 0.80; t(499) = 7.40, p = 5.93e-13), and lower average trustworthiness ratings for S1 than for S3 (S1: z = -0.67; S3: z = -0.38; t(499) = 6.06; p = 2.62e-9). In fact, the average ratings followed an extremely tight relation with acoustical differences over the whole range of the continuum generated resulting in a correlation of r = 0.99 (p = 5.03e-8) between position along These results demonstrate a strong acoustical basis for perceived voice personality-an extremely tight mapping between acoustical and social voice spaces. Not only does averaging voices from different individuals with low or high ratings trustworthiness ratings results in the predicted rating differences, but these personality impressions can be parametrically modulated, with unsuspected accuracy, by acoustical changes and considerably increased (or decreased) by caricaturing.
Furthermore the results allow listening to the prototypes and caricatures, permitting direct access to the listeners' underlying representations of voice trustworthiness in a way that correlations between traits and acoustical measures could not allow. The comparison of the prototypes and caricatures' acoustical measures not only gives insight on which acoustical parameters are relevant, it directly shows how to modulate these parameters in order to achieve the desired increase in perceived trustworthiness.
Thus, past studies have hinted at a relation between f0 variation in voice and perceived personality traits such as attractiveness [bib_ref] The role of femininity and averageness of voice pitch in aesthetic judgments..., Feinberg [/bib_ref] [bib_ref] Manipulations of fundamental and formant frequencies influence the attractiveness of human male..., Feinberg [/bib_ref] [bib_ref] Integrating fundamental and formant frequencies in women's preferences for men's voices, Feinberg [/bib_ref] or trustworthiness [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref] but they could not determine the exact f0 contour that would drive this percept. What intonation should one use when saying "hello" to be perceived as trustworthy? [fig_ref] Fig 3: Intonation and perceived trustworthiness [/fig_ref] important elements of answer to that question by illustrating f0 contour for the 9 stimuli of the Trustworthiness continuum. F0 at mid-duration is roughly similar for all stimuli but a clear difference is apparent for starting and ending f0: while the low-trustworthiness stimuli started with a relatively low f0 remaining flat or slightly increasing over the course of the syllables, the high-trustworthiness stimuli started with a much higher f0 at first syllable then decreased markedly to reach the average f0 at mid utterance and finish on a strongly rising intonation (cf. High trustworthiness caricature, [fig_ref] Fig 3: Intonation and perceived trustworthiness [/fig_ref] audio file in Supporting Information). While f0 contour is but one of the many only acoustical parameters varying between stimuli (S1 this particular intonation likely plays a central role on yielding the observed change in perceived personality impressions. Further work should weigh the respective importance of the different parameters by selectively including them or not in the prototypical models, for instance by generating an acoustic continuum that only manipulates f0 between the different stimuli, all other parameters being kept constant.
The results could potentially generalize to other personality traits, providing unprecedented insight into the voice acoustics that drive the perception of different traits and opening numerous possibilities for research. They also open the door to practical applications where desired personality impressions need to be achieved. Indeed, there are many situations in life where voice is the only cue available to form a new person's first impression and where that first impression strongly influences future interactions. For example, telephone interviewers face the task of establishing trust a creating a positive impression with only vocal cues. Since the great majority of refusals occur within the opening phrases, a positive attitude towards both the subject and interviewer could be induced early on via acoustical modulation, resulting in increased response rates. Furthermore, speech synthesis and computer-generated voices foe e.g. social robots could be addressed as a promising area for applications in the customer service sector.
Some limitations of the study should be mentioned: They were only obtained on the word "hello", which is justified as this word is often associated with first encounters, yet it is unclear how they would generalize other words or longer utterances. Moreover, the 8-voices averages are probably but a crude approximation of the listeners' internal representations of trustworthy and untrustworthy voices; results could likely be further enhanced by using high-level reverse correlation for a more precise estimation of the listeners' internal templates [bib_ref] Using auditory classification images for the identification of fine acoustic cues used..., Varnet [/bib_ref] [bib_ref] A Psychophysical Imaging Method Evidencing Auditory Cue Extraction during Speech Perception: A..., Varnet [/bib_ref]. Our choice of female voices for the control condition and male voice for the experimental condition was entirely arbitrary; it will be important in future studies to examine the potential role of factors such as gender, but also age, ethnicity, accent, verbal content, etc. on the acoustical basis for voice personality impressions.
# Methods
Participants 500 participants (146 males, 354 females; aged 19-65 years old, median age = 24) took part in the voice rating experiment. Participants were recruited via email and social networking sites and directed to the web address of the experiment. Experimental procedures were approved by the University of Glasgow ethics committee and the study was conducted in accordance with the ethical standards laid down in the Declaration of Helsinki. Participants gave informed consent prior to the experiment, via reading a series of statements regarding freedom to withdraw, anonymity of responses and secured storage of data. As the experiment was carried out online, they confirmed that they had read and agreed to these statements by registering on the website. Participants were not permitted to take part without providing consent.
## Stimuli
Voice stimuli were modelled and generated using STRAIGHT [bib_ref] Auditory morphing based on an elastic perceptual distance metric in an interference-free..., Kawahara [/bib_ref] in Matlab (Mathworks, Inc., Natick, USA). STRAIGHT performs an instantaneous pitch-adaptive spectral smoothing in each stimulus for separation of contributions to the voice signal arising from the glottal source vs. supra-laryngeal filtering. A natural voice stimulus is decomposed by STRAIGHT into five parameters: f0, frequency, time, spectro-temporal density and aperiodicity that can be manipulated and combined across stimuli independently of one another, then synthesized into a novel voice stimulus. Time-frequency landmarks to be put in correspondence across voices during morphing were manually identified in each stimulus and corresponded to the frequencies of the first 3 formants at temporal landmarks corresponding to onset and offset of phonation or formant transitions (S1 The low-and high-trustworthiness prototypes were generated by averaging each voice parameter across the eight voices having obtained lowest or highest average trustworthiness ratings, respectively, in a previous study [bib_ref] How do you say 'hello'? Personality impressions from brief novel voices, Mcaleer [/bib_ref]. These two prototypes were then used to generate a 9-stimulus acoustic continuum in eight steps of equal acoustical distance (25% of the difference between the two prototypes) by inter-and extrapolation between the low-trustworthiness prototype in position S3 and the high-trustworthiness prototype in position S7. Thus the respective weights of S3 and S7 for the 9 stimuli of the continuum were: S1: 150%/-50%; S2: 125%:-25%;S3: 100%/0%; S4: 75%/25%; S5: 50%/50%; S6:25%/75%; S7: 0%/100%; S8: -25% 125%; S9: -50%/150%. A second continuum (Control Continuum) was generated using the exact similar approach using the female voice recordings and a random selection of the 16 underlying voices (Audio files in Supporting Information). Acoustical measures of the stimuli were obtained using VoiceSauce [bib_ref] VoiceSauce: A program for voice analysis, Shue [/bib_ref] and are shown in S1 [fig_ref] Table: Acoustical measures [/fig_ref] Spectrograms of the 9 stimuli of the Trustworthiness continuum are shown in [fig_ref] Fig 1: Generation of the voice trustworthiness continuum [/fig_ref] and their f0 contours in
## Procedures
The experiment was carried out online on Glasgow University' Institute of Neuroscience and Psychology experiments website (http://experiments.psy.gla.ac.uk/). Participants performed two blocks of ratings (order counterbalanced across subjects): they were either presented with male voice block first, followed by control voices (female) or vice versa, and asked to rate each voice on a continuum of trustworthiness represented by a visual analogue scale ranging from "very untrustworthy" to "very trustworthy". At the start of each block, participants were asked, "For each voice, please rate how trustworthy it sounds, that is, how much you would be ready to trust that person." No contextual information for the experiment was given, akin to a "no acquaintance" scenario. All voices were heard once per block with presentation order randomised in both blocks to avoid possible order effects. Participants were given the opportunity to take an untimed break between the two blocks.
Rating data are available as supporting information S1 Data.
# Analysis
Raw trustworthiness ratings (range 0-500) were converted to individual z-scores based on the mean and standard deviation of the 18 ratings provided by each participant. For both the Trustworthiness and Control continua, the distributions of individual z-scores were compared between the S3 and S7 stimuli via paired t-tests. A possible caricaturing effect was also examined via paired t-tests comparing S1 to S3 and S9 to S7 in each continuum. Bonferronni correction was applied to account for the 3 comparisons per continuum.
Supporting information S1 Audio File. Trustworthiness_continuum.wav: The 9 stimuli played in succession from S1 to S9 (windows PCM format, 16-bit, mono, 16 kHz sampling rate).
# Author contributions
Conceptualization: Pascal Belin.
## Data curation: bibi boehme.
Formal analysis: Pascal Belin, Bibi Boehme.
[fig] Fig 1: Generation of the voice trustworthiness continuum. a. Thirty-two male voices saying "hello" are represented as points in a 2D "social voice space" with axes mapping onto their perceived Trustworthiness and Dominance, respectively (cf.[4]). Coloured dots indicate voices within the bottom (blue dots) and top (red dots) 25% for Trustworthiness ratings. b. A Low-and a High-Trustworthiness prototype were generated by averaging via morphing the bottom and top 25% rated voices, respectively. c. A 9-stimulus "Trustworthiness continuum "was generated via morphing in8 acoustically equal steps using the two prototypes in intermediate positions and extrapolating to low-and high-Trustworthiness caricatures at extreme positions. https://doi.org/10.1371/journal.pone.0185651.g001 Sound of trustworthiness PLOS ONE | https://doi.org/10.1371/journal.pone.0185651 October 12, 2017 the acoustical continuum and average ratings. Again no such effect was observed for control stimuli on either end of the continuum (S1: z = 0; S3: z = 0.03; t(499) = -0.49, p = 0.62 n.s.; S7: z = -0.09; S9: z = -0.19; t(499) = 1.63, p = 0.10 n.s.). Importantly these effects were highly similar for male and female listeners (S3 Fig) suggesting the involvement of gender-independent mechanisms. [/fig]
[fig] Fig 2: Acoustic-based modulation of perceived voice trustworthiness. Filled circles indicate groupaverage Trustworthiness rating z-scores for each of the 9 stimuli of the Trustworthiness continuum (spectrograms at bottom) from low-Trustworthiness caricature (S1) to low-Trustworthiness prototype (S3; blue rectangle), to the average of the two prototypes (S5), to the high-Trustworthiness prototype (S7; red rectangle) to the high-Trustworthiness caricature (S9). Note the quasi-linear modulation of Trustworthiness ratings along the Trustworthiness continuum. Open circles show average T-rating z-scores for the control continuum. Error bars and shaded areas indicate mean ± s.e.m.https://doi.org/10.1371/journal.pone.0185651.g002 Sound of trustworthiness PLOS ONE | https://doi.org/10.1371/journal.pone.0185651 October 12, 2017 [/fig]
[fig] Fig 3: Intonation and perceived trustworthiness. The f0 contours of the 9 stimuli of the Trustworthiness continuum (from light blue for stimulus S1 to red for stimulus S9) are shown illustrating a marked change in intonation as perceived trustworthiness increases. While low trustworthiness stimuli have a flat or slightly rising f0 contour, high-trustworthiness stimuli are characterized by a marked f0 drop at the end of the first syllable to finish the second syllable on a markedly rising f0.https://doi.org/10.1371/journal.pone.0185651.g003 Sound of trustworthiness PLOS ONE | https://doi.org/10.1371/journal.pone.0185651 October 12, 2017 [/fig]
[table] Table: Acoustical measures. (PDF) [/table]
|
Viral Bad News Sent by EVAIL
Citation: Clauss, M.; Chelvanambi, S.; Cook, C.; ElMergawy, R.; Dhillon, N. Viral Bad News Sent by EVAIL. Viruses 2021, 13, 1168. https://
# Introduction
For many years, acute and chronic viral infections have been associated with the development of cardiovascular disease (CVD). For example, infectious encounters with cytomegalovirus (CMV) correlate with a 22% increased risk for future development of cardiovascular disease. However, the contribution and the biology of viruses towards CVD are incompletely understood. What is extraordinarily well studied is infection with Human Immunodeficiency Virus-1 (HIV) and how it may affect cardiovascular health. Notably, after the introduction of antiretroviral therapy (ART), cardiovascular disease (CVD) became a leading cause of death in the HIV-positive population. Because the HIV biology concerning cardiovascular and pulmonary diseases is so well understood, it may serve as a blueprint for studying other viruses with known impacts on CVD. This review focuses specifically on the role of extracellular vesicles (EVs) in HIV infection and their possible CVD contribution. Finally, using the example of the pandemic SARS-CoV-2 virus, it tries to apply what we have learned about HIV-induced EV formation to understand SARS-CoV-2 virus-induced CVD (see.
## Hiv mystery: heightened cvd risk in hiv-infected individuals on art
## Persistent cvd risk in hiv-infected individuals despite art
The HIV-associated risk of developing CVD persists despite the initiation of ART. For example, atherosclerotic conditions continue to progress more in the HIV population after ART is initiated based on arterial carotid intima-media thickness. Further, a more recent large meta-analysis combining five studies and 89,713 subjects concluded that "stroke represents a relatively common complication in young HIV patients". Another example is primary pulmonary arterial hypertension (PAH); the prevalence of PAH was determined as 0.5% during the pre-ART eraand stayed high (0.46%) in the era of ART. These are alarming numbers, given that the prevalence of PAH in the general population is 0.0015%. An overview about studies assessing the risk of HIV patients on ART for developing CVD is depicted in.
## Factors contributing to cvd risk in hiv-infected individuals on art
What are the possible explanations for this persistently increased risk for CVD in the HIV-positive population? One possibility is toxicity and dyslipidemia from antiretroviral drugs, as reviewed previously. Typically, nucleoside-and non-nucleoside-based reverse transcriptase inhibitors are combined, with or without the addition of integrase or HIV-protease-targeting inhibitors. The latter, in particular, have displayed pronounced toxicity and are now mostly out of use. Typically, newer ART regimens have less toxicity and are more efficient than early HIV replication inhibitors.
Another possible explanation why inflammation persists during ART could be that HIV infection irreversibly damages the mesenteric lymphatic system. This way, microbial translocation may continue after ART initiation, possibly leading to chronic inflammation. In this context, the link between systemic inflammation and vascular health was the topic of the "Strategic Timing of Anti-Retroviral Therapy" (START) trial, in which HIV patients on successful ART demonstrated a correlation between inflammatory plasma markers and impaired arterial elasticity. Interestingly, elevated markers of endothelial dysfunction have been found in patients with ART-controlled viral loads.
Increased CVD risk in HIV-infected patients may also be caused by virus-induced dyslipidemia, which is highly prevalent among patients living with chronic HIV infection. Interestingly, HIV-Nef was suggested as the main viral factor associated with HIV-linked dyslipidemia. Because Nef protein production appears to be relatively resistant to ART, this viral protein could explain persistent dyslipidemia in ART patients (see upcoming chapters on HIV-Nef persistence).
Last but not least, HIV in latent or actively infected cells could elicit the production and release of EVs. Studies have suggested EVs mediated cardiovascular dysfunction in transferring its protein and non-coding RNA cargo to vascular endothelial and smooth muscle cells. Several reports have demonstrated that the HIV-encoded Nef protein organizes EV formation and uses them as vehicles for dissemination. These Nefcontaining EVs are toxic to surrounding cells, including bystander CD4 positive T cells and vascular endothelial cells that absorb them and become apoptotic. These possible effects of HIV-associated EVs will be discussed in more detail in the following chapters.
## Increased ev in hiv-infected individuals despite art
## Evs are enriched in the plasma of hiv patients on art
The term extracellular vesicles (EV) defines different types of vesicles secreted outside of the cell, including exosomes, microvesicles, and apoptotic bodies. Because EVs are taken up by blood and tissue cells, they could spread proinflammatory cargo and HIV proteins throughout the body. Exosomes are reported as a subpopulation of EVs associated with HIV activities, including inflammation and dyslipidemia. Of note, Lee et al.showed that in HIV patients-whether viremic or on combined high dose antiretroviral therapy (ART)-EV numbers were approximately 20-fold higher over healthy controls. Similarly, the cargo load of these EVs from HIV increased 18-25-fold over otherwise healthy controls. As for cargo load, the authors determined the expression of miRNA and protein. Support for the significance of these findings came from further studies using unbiased proteomic approaches and miRNA analysis to demonstrate that isolated plasma EVs contained markers "associated with immune activation and oxidative stress in HIV patients on ART".
## Nef protein persists in evs from body fluids
Given the presence and upregulation of EVs in the HIV-positive population, "early response" HIV genes including Nef, tat, and rev were suspected. These "early" HIV genes decrease after initiation of ART treatment less prominently than other HIV gene products. Intracellular mRNA-encoding Nef levels were maintained during ART, while Tat and Rev showed less prominent mRNA expression.
Nef protein inside CD45+ EVs in the plasma of HIV-infected individuals was first reported in 2011 by Raymond et al.. Subsequent studies have detected Nef protein in EVs from plasma and peripheral blood mononuclear cells (PBMCs) of both treatment-naive HIV patients and HIV-infected individuals on ART with undetectable viral loads. Interestingly, HIV-Nef was detected in over half the patients on ART and in elite controllers in a study using 134 HIV-infected patients with undetectable HIV RNA. Our previous work could confirm these findings in a much smaller study of 16 patients on ART, in which also about half were HIV-Nef-positive. Interestingly, we also found high numbers of Nef protein-positive PBMCs using flow cytometry and a panel of three monoclonal antibodies targeting three different and highly conserved epitopes because of the high mutation rate of HIV.
## Nef protein in evs transfers rapidly to blood and tissue cells
The presence of Nef in both EVs and PBMC begs the question of whether mononuclear cells are the target or source of Nef-containing EVs. Apparently, Nef can be transported by EVs and transferred to PBMCs and endothelial cells because it is present to a large extent in B cells and CD4− T cells, and Nef-positive PBMCs transferred Nef rapidly to vascular endothelial cells in vitro. Further support for the hypothesis that mononuclear cells are the target of Nef-containing EVs comes from our previous work demonstrating the presence of Nef protein in EVs and cells derived from bronchoalveolar lavages (BAL). While 50% of the patients (10 out of 18) displayed Nef-EV positivity based on ELISA, Nef positive BAL cells were randomly distributed between alveolar macrophages, CD4+ T cells, and CD8+ T cells (in nine out of 15 HIV patients on ART).
To date, the source (or sources) of these surprisingly robust levels of Nef protein in successfully ART-treated patients remains unclear. Lee et al.suggested a myeloid or myeloid-like compartment as the origin of Nef-induced EV formation, based on protein array analysis of patient-derived plasma EVs, which was different from those in EVs derived from HIV-transfected Jurkat cells. A closer identification of this compartment could help in understanding HIV latency in the era of ART.
## Hiv-nef induces extracellular vesicle-assisted inflammatory load (evail)
## Specific surface proteins in evs define proinflammatory cargo associated with hiv proteins
Given the 20-fold increase in EV numbers and protein load found in plasma from HIV patients on ART, researchers asked whether HIV proteins could be linked to specific EV surface markers. To address this, Lee et al.tested a panel of 262 monoclonal antibodies coupled to magnetic beads for their ability to adsorb plasma-derived EV with HIV cargo. They could identify one antibody (clone 2H4) that captured all EVs containing HIV proteins (Nef and Vpu). Interestingly, this antibody was identified to recognize activated αvβ3, an angiogenic integrin also known as vitronectin receptor, and associated with macrophages' phagocytic activity. Further, this study found that activated αvβ3 positive EVs contained the proinflammatory matrix-metalloproteinases ADAM-17 (TACE) and ADAM-10, absent in the unbound fraction. Subsequently, Nef-and TACE-containing EVs were isolated from HIV patients, which, in turn, correlated with immune pathogenesis in chronic HIV-infected patients.
Interestingly, the presence of Extracellular Vesicle Assisted Inflammatory Load (EVAIL) includes a specific pattern consisting of cytokines, chemokines, and growth factors, such as PDGF BB and FGF-9. In conclusion, a particular EV surface protein emerged as a potential biomarker for HIV-associated EVs carrying inflammatory cargo. However, there is a gap of knowledge regarding the Nef-associated composition of non-coding RNAs, including miRNAs, which were reported to be modulated in HIV-induced EVs.
## Nef initiates endosomal routing leading to recruitment of adam-17/tace and secretion of tnf
Ostalecki et al.showed that uptake of these Nef-containing EVs induced endosomal TNF cleavage by TACE. They identified as the first step Nef-dependent internalization of TACE in Rab4+ early endosomes. This enabled compartmentalization with transmembrane TNFα, which was followed by TACE activation, TNFα cleavage, and secretion as TNFα/TACE repackaged EVs. TNFα maintains at intravesicular location, which is different from the canonical TNFα release as a soluble protein from cells. These findings are significant as TACE is the primary mechanism driving the release of mature TNFα and the primary driver of other proinflammatory pathways involved in chronic inflammation and its associated diseases. They also show that Nef is associated with vesicle sorting. It is likely that Nef orchestrates the loading of proteases and cytokines in EVs. Alternatively, the host system could interpret Nef as a danger signal to load Nef-containing EVs with proteases and cytokines in preparation to fight infectious agents.
## Pathophysiology of the hiv-nef-associated evail
The elucidation of pathogenic mechanisms in HIV-associated comorbidities in the era of ART is still incompletely understood, as is the function of HIV-released EVs. The release of pathogenic HIV proteins like Nef into the systemic circulation through ADAM17/TACEcontaining EVs could be advantageous for HIV by activating latent viral activity. Likewise, ADAM-17/TACE-induced v generation can prime resting CD4+ T lymphocytes for HIV expression and replication. As it would make evolutionary sense for the virus to promote its infectivity broadly, could it be that the same TACE-induced TNFα generation causes endothelial activation and dysfunction more like an unwanted side effect? Indeed, Nef-containing EVs can affect recipient cells both on functional and gene expression levels. Khan et al.isolated Nef-EVs from HIV patients with HIV-associated dementia (HAD) and demonstrated that they could elicit increased Aβ secretion from neuronal cells. Reportedly, Nef-containing EVs are indeed taken up by neuronal cells leading to oxidative stress.
EVs must not necessarily have to reach distal organs to increase proinflammatory pathologies. Alternatively, microglia cells could produce and release such EVs. Raymond et al.demonstrated that EVs could cross the blood-brain barrier. In addition, they found that microglial cells transfected with Nef released Nef-EV to reduce the endothelial blood-brain barrier. This loss of barrier function is likely due to Nef-EV reducing tight junction protein expression, ZO-1. Importantly, this study demonstrated that by using a specific Nef-peptide inhibitor, Nef-EV-induced disturbance of their blood-brain barrier model can be reversed. These findings indicate that HIV reservoir cells in the brain can employ HIV-Nef release to contribute to HIV-associated neurocognitive diseases (HAND). Furthermore, they could also promote cardiovascular disease as EVs produced from brain cells cross the BBB also in reverse.
As another example of the proinflammatory propensity of EVs, the addition of Nef-EVs to peripheral blood monocytes (PBMCs) led to TNFα-converting enzyme (TACE/ ADAM17) packaging into vesicles and subsequent secretion. Uptake of Nef-EVs in T cells from human PBMCs causes free radical formation and apoptosis when these cells are brought in close contact with human coronary arterial endothelial cells. This finding suggests that leukocytes can further enhance HIV-Nef-induced pathologies.
Wang et al.were the first to report that Nef is transported from T cells to endothelial cells to cause reactive oxygen species (ROS) production, upregulation of chemokines, and pro-apoptotic signaling. Interestingly, T cells infected with HIV elicited the same panel of endothelial activation markers, which was either absent or much reduced when HIV with a deleted Nef gene was used.
Further, Nef-containing EVs from BAL fluids and Nef-transfected cells were shown to induce programmed cell death in endothelial cells, dependent on induction of EMAP II. Interestingly, transgenic expression of Nef in the endothelium induced endothelial EMAP II surface expression, which could mediate the emphysema phenotype in these transgenic mice.
Furthermore, our own previous work has shown that HIV-Nef expressing T cells and HIV-Nef-induced EVs upregulated endothelial adhesion proteins and apoptosis. This upregulation occurred together with cytosolic dyes and Nef protein transfer from T to endothelial cells, dependent on Rac1-activation. These Rac1-dependent activities were characterized by the production of NADPH/NOX2-mediated reactive oxygen species (ROS). Statin treatment equally inhibited Rac1 inhibition in preventing or reversing HIV-Nef-induced ROS formation, mitochondrial polarization, and increased pro-apoptotic signaling in human coronary arterial endothelial cells. This therapeutic effect was explained by the ability of statins to block Rac1 prenylation. Indeed, geranylgeranyl transferase inhibitors significantly reduced HIV-Nef-induced ROS formation. These findings suggest a protective role of statins beyond lipidemia. Certainly, there is clinical evidence of the positive effects of statins in combination with ART in HIV/AIDS patients, thus the recommendation that statin treatment should be considered in this population after due attention to possible drug-drug interactions.
In addition to the ability of Nef-EVs to induce endothelial cell activation and increased stickiness of proinflammatory leukocytes to vascular endothelial cells even under conditions of flow, they can increase cholesterol and triglyceride levels. Cholesterol levels are known to be regulated by reverse cholesterol transport. Reportedly, Nef mediates the down-regulation of adenosine triphosphate-binding cassette transporter A1 (ABCA1) transporters by post-translational degradation. In apoE (−/−) mice, injected Nef reduced levels of ABCA1 in the liver, supporting the link between Nef and increased cholesterol plasma levels. Not surprisingly, these effects occurred together with increased atherosclerotic lesion size and blood vessel remodeling.
Furthermore, the results of Nef on dyslipidemia could be replicated in the SIV-infected macaque model. SIV infection down-regulated the ABCA1 cholesterol transporter in the liver, and serum from SIV-infected but not from healthy macaques inhibited cholesterol efflux from human cultivated macrophages. Interestingly, Nef-induced ABCA1 downregulation goes beyond regulation of lipid levels: Mukhamedova et al.could demonstrate that Nef-induced ABCA1 downregulation results in increased abundance of lipid rafts in monocytes in a Cdc42 dependent fashion. The authors also found that these changes in lipid rafts stimulate inflammation through "re-localization of TLR4 and TREM-1 to rafts".
In summary, Nef-containing EVs can contribute to CVD both by increasing proteolytic and proinflammatory cargo and by intrinsic functions of the Nef protein (see also. It would be interesting to study other viruses for a similar mechanism to employ EVs to control immune-related functions.
## Does the evail concept also hold true for other viruses? what about pandemic sars cov-2?
It would be interesting to test whether mechanisms similar to HIV-Nef occur in other virus-related diseases. The COVID-19 respiratory illness is associated with pulmonary vascular thickening, endothelial dysfunction, thrombo-inflammation, and microthrombi. Emerging findings suggest that COVID-19 patients, like HIV-infected patients, may be at higher risk of developing pulmonary hypertension in the future. When A549 lung epithelial cells were transduced with lentivirus encoding SARS-CoV-2 proteins, exosomes are released carrying these viral proteins. The addition of these exosomes to recipient pluripotent stem cell-derived cardiomyocytes resulted in the entry of viral RNA into cardiomyocytes and increased expression of inflammatory genes. It remains unclear whether these passed viral genes are biologically active and capable of inducing cell injury. It may be too speculative, but cellular recipients of EVs carrying viral proteins could be prone to becoming targets of autoimmune reactions.
Exosomes also have been reported to transfer ACE2, the receptor exploited by SARS-CoV-2 for cell entry, to recipient cells. Interestingly, a higher burden of EV-associated ACE2 is noted in SARS-CoV-2-infected patients without hypoxia than those requiring oxygen treatment. This finding suggests that EVs with ACE2 cargo might act as decoys, thereby reducing SARS-CoV-2 infection in epithelial cells. Alternatively, EVs transporting ACE2 cargo may also render ACE2-negative cells, usually not targeted by SARS-CoV-2, to become infected with this virus. Although there is no apparent means of interfering with ACE2 transport by EVs, this is an essential finding that, depending on the membrane orientation of this receptor, could lead to antibody-directed detection and potential removal of COVID-19-related EVs.
It has been shown (see previous sections on HIV) that HIV-associated EVs carry HIV proteins and proinflammatory cargo that can be transferred to endothelial and other tissue recipient cells. Therefore, it would be interesting to explore whether this EV-mediated cargo transfer can also be observed with SARS-CoV-2-associated EVs. Our recent work has shown that EVs from patients infected with SARS-CoV-2 display higher levels of specific cytokines in correlation with disease severity. For example, large EVs from the plasma of patients with COVID-19 are enriched with multiple members of the TNF superfamily and their receptors and IL-6 family proteins. These cytokines and their receptors are also characteristic of acquired respiratory distress syndrome (ARDS). Specifically, IL-6 has been associated with cytokine release in ARDS, septic shock, and COVID-19. Notably, the transfer of cytokine receptors could explain why EVs may add to inflammation despite the presence of already high concentrations of cytokines in COVID-19. This would imply that these cytokines receptors would be functionally integrated into the lipid bilayers of EVs. Of note, this is in contrast to soluble cytokine receptors in plasma, which typically result from proteolytic cleavage of the extracellular domains. Although uptake of these receptors has not yet been addressed in the study of COVID-19-associated EVs, it is a tempting speculation that COVID-19-related EVs could heighten the cytokine storm by increasing proinflammatory receptor densities in blood and other vascular cells.
The finding of increased IL-6 in COVID-19-associated EVs is significant because increased IL-6 plasma levels are part of predictor panels for cardiac events, especially in the setting of HIV infection. In addition, higher levels of pro-inflammatory RAGE and EN-RAGE in EVs correlated with COVID-19 clinical severity could further provide a positive feedback loop for generating a cytokine storm. Additionally, several proteins and proteases associated with cardiovascular disease and vascular remodeling, including prostatin, cathepsin L1, matrix metalloproteinase 9, and carboxypeptidases 1/2 (CPA1/CPB1), are also upregulated in large EVs from patients with severe infection.
EVs, transiently released from injured cardiomyocytes, contain markers of early ischemic injury. This release may comprise a mechanism by which some patients with COVID-19 experience elevated levels of circulating cardiac troponins (cTn). Lala et al. reported a significantly higher mortality risk among COVID-19 patients with even a tiny troponin leak. EV-linked tissue factor (TF), a vital driver of the extrinsic coagulation cascade, is also elevated in COVID-19 patients with moderateand severe disease. Elevated TF in EV levels have been already linked to cancer-associated thrombosisand now our work correlated EV-linked TF levels and activity with increasing COVID-19 disease severity and length of hospitalization. This suggests that the prothrombotic and antithrombotic molecular profile of EVs from SARS-CoV-2-infected individuals may be one of the factors responsible for the increased risk of microthrombosis COVID-19 infection (see. The long-term cardiovascular consequences of SARS-CoV-2 infection may result from an EV-mediated prothrombotic environment and endothelial activation and dysfunction. Notably, EVs may contribute to COVID-19 disease severity by adding increased proteolytic stress and procoagulant activation pathways to the already known increases in inflammatory cytokines. Additional research is needed to explore whether there is a role for EVs in propagating tissue destruction, inflammation, and thrombosis weeks to months out from COVID-19 diagnosis. Finally, it needs to be established whether EV cargo could become biomarkers for disease severity in COVID-19 or if distinct surface markers for COVID-19-associated EVs could be targeted for their therapeutic removal.
## Conclusions and potential for therapy
This review focuses on a single protein, Nef, as an example of how viruses and their proteins can trigger the specific release of proinflammatory EVs. Although further studies targeting these EVs are required to provide more direct links to CVD, the potential of EVAIL to activate the endothelium is intriguing. It may be feasible to target the generation of EVAIL if we better understand the generation and uptake of EVs. Interestingly, EV-associated anti-inflammatory load has been described from mesenchymal stem cells (MSCs). The anti-inflammatory activities of MSC have emerged as an attractive anti-inflammatory strategy over the past decade. In this article, MSCs were suggested to improve cardiovascular function and reduce the exuberant production of influenza-induced cytokines. MSCs are already in clinical trials for treating COVID-19, and using EVs from these cells could be added to other already used MSC-based therapeutics. In addition, the incorporation of other anti-inflammatory and immune regulatory proteins like CD24 or cytokine-specific neutralizing therapeutic antibodies could lead to a novel platform of molecular therapies mediated by EVs.
Funding: This review was in part supported by a grant from the NIHBLI: 5R01HL154859-01 (to MC and ND).
Institutional Review Board Statement: Not applicable.
## Informed consent statement: not applicable.
Data Availability Statement: Not applicable. |
A Case of Cardiac Tamponade in a Patient with Metastatic Renal Cell Carcinoma on Pazopanib Treatment
Asymptomatic minimal pericardial effusion may be frequently found in patients with hypothyroidism. Cardiac tamponade secondary to hypothyroidism is rarely referenced in medical literature. Hypothyroidism as an adverse effect of pazopanib (tyrosine kinase inhibitor) treatment leading to cardiac tamponade is an even rarer occurrence. Here, we report an unusual case of a 71-year-old male, with a case of renal cell carcinoma on pazopanib treatment presenting with shortness of breath who was found to have hypothyroidism with a large pericardial effusion leading to cardiac tamponade. The patient did not have any prior reports of thyroid-stimulating hormone (TSH) or thyroid hormone levels at presentation. No such case of cardiac tamponade due to hypothyroidism as an adverse effect of pazopanib tablet treatment has been reported to our knowledge.
# Introduction
Pericardial effusion and cardiac tamponade are caused due to a variety of etiological factors which include acute pericarditis, tumor, uremia hypothyroidism, trauma, cardiac surgery, or other inflammatory/noninflammatory conditions.
Recent studies have shown that pericardial effusions are extremely rare in hypothyroidism with an incidence of 3 to 6 percent.
Patients who are on pazopanib have been reported to develop hypothyroidism at an incidence of less than 10%.
A small pericardial effusion can cause significant cardiac tamponade when it accumulates rapidly, and hence, it is important to suspect cardiac tamponade in patients with sudden hemodynamic compromise.
Here, we report an uncommon case of hypothyroidism with cardiac tamponade.
## Case report
A 71-year-old male patient who was a known case of metastatic renal cell carcinoma on treatment with the tablet pazopanib 800 mg OD since 5 years presented to us for the first time in ER on 19.06.2018 with complaints of shortness of breath and easy fatigability.
On examination, the patient was breathless and had a sallow complexion with dry skin and peripheral edema with prominent visible neck veins.
The patient's blood pressure was 100/70 mmHg with tachycardia and oxygen saturation of 92% at room air. The patient had demonstrable pulsus paradoxus with an inspiratory drop in a blood pressure of more than 16 mmHg.
Bedsides, a 2D echocardiogram showed a large pericardial effusion with right atrial right ventricular diastolic collapse suggestive of cardiac tamponade as shown in.
A primary working diagnosis of suspected disease progression with pericardial/cardiac metastases was made.
The patient was immediately shifted to the Intensive Care Unit and underwent pericardiocentesis. About 350 ml of yellowish golden colour fluid was aspirated which was sent for cytological and biochemical investigation.
Immediately post procedure, the patient showed significant improvement in cardiopulmonary status. His tachypnea and tachycardia subsided, and the patient maintained adequate oxygen saturation on room air. Post pericardiocentesis, the 2D echocardiogram revealed minimal pericardial fluid with no evidence of cardiac tamponade.
Pericardial fluid analysis showed degenerate and mesothelial cells and no evidence of any malignancy or acid fast bacilli.
However, to rule out disease progression, the patient had an 18-fluorodeoxyglucose (FDG) PET-CT scan which showed regression of the necrotic mass of the left kidney, stable proximal left renal tumor thrombosis, stable lesion adjacent to tumor thrombosis in the anterior calyx, stable metastatic right lung nodule, and interval development of bilateral pleural effusions and mild to moderate pericardial effusion with pericardial catheter drainage tube in situ.
Considering his history and findings, we did a thyroid function test which was suggestive of severe hypothyroidism with high TSH levels (TSH > 100 mIU/L) and low T3 T4 levels.
The patient did not have any prior reports of TSH or thyroid hormone levels. Antithyroid peroxidase antibody testing was done which was negative.
The patient was treated with the tablet levothyroxine 50 micrograms daily which was later gradually increased to 125 mcg daily. The patient's condition improved significantly and was discharged in a stable condition 1 week post removal of the pericardial drainage tube.
Follow-up echocardiogram after a period of 2 months showed near total resolution of pericardial effusion. Within a few weeks, all his signs and symptoms completely resolved and the patient is currently continuing treatment on pazopanib tablet as part of his renal cell carcinoma treatment.
The patient is on regular follow-up, and his latest 2D Echo on 10.10.2018 showed minimal pericardial effusion with good systolic left ventricular function. The patient is currently hale and hearty on thyroid replacement and pazopanib tablet treatment with a normalized TSH value of 0.97 mIU/L.
# Discussion
Cardiac tamponade as a complication of hypothyroidism is very rare with few cases described in world literature.
Hypothyroidism may commonly cause asymptomatic pericardial effusions, but rarely leads to symptomatic tamponade. The mechanism of this myxomatous pericardial effusion is postulated to be due to the increased permeability of capillaries causing leakage of fluid rich in protein into the interstitial space and impaired lymphatic drainage leading to salt and water retention.
Although the definitive mechanism of tyrosine kinase inhibitor-(TKI-) induced hypothyroidism is debatable, several theories have been proposed. Case Reports in Oncological Medicine Vascular endothelial growth factor (VEGF) is a signal protein allowing formation of new blood vessels as well as formation of collateral circulation. TKIs like pazopanib reduce blood supply to the tumor leading to tumor regression; however, capillary regression is also noted in normal organs like the thyroid considering that the thyroid is an organ with the highest blood flow per unit weight.
Other mechanisms proposed include alterations in thyroxine/triiodothyronine metabolism, induction of type 3 deiodinase activity, blockage of iodine uptake, and autoimmunity where antithyroid peroxidase antibodies are detectable.
However, the latter seems highly unlikely in our patient as antithyroid peroxidase antibodies were not detected.
Another interesting extrapolation which our patient may have raised is whether adverse effects of treatment (severe hypothyroidism in our patient) translate into increased clinical benefit.
Pazopanib treatment in advanced renal cell carcinoma has been known to provide an average of 9 months of progression-free survival and 23 months of overall survival. Our patient has shown a progression-free survival of 5 years till date which is remarkable considering the much lower average.
The above has been demonstrated in previous studies showing hypothyroidism and other adverse effects of TKIs improved tumor response rates and survival time of kidney cancer patients treated with targeted therapy.
During pazopanib therapy, monitoring the thyroid function must be necessary.
Our case also highlights further the importance of stringent follow-up and monitoring to pick up adverse effects at the earliest time and prevent potentially lifethreatening events.
# Conclusion
Atypical symptoms arising in cancer patients on treatment should provoke other more easily treatable differential diagnoses like treatment-related adverse events rather than just disease progression.
Treatment-related adverse events may translate into a better response and survival rate for the patient.
## Consent
The article was initiated and written after informed consent from the patient was obtained. |
Incretin based drugs and risk of cholangiocarcinoma among patients with type 2 diabetes: population based cohort study
## Supplementary methods 1. multiple imputation
We used multiple imputation for variables with incomplete data (i.e. missing values for haemoglobin A1c, BMI and smoking) in all analyses. This method is more efficient than a complete case analysis. [bib_ref] Imputing missing covariate values for the Cox model, White [/bib_ref] To impute missing data, an ordinal regression model was used with explanatory variables and cumulative hazard, [bib_ref] Imputing missing covariate values for the Cox model, White [/bib_ref] use of DPP-4 inhibitors and GLP-1 receptor agonists at cohort entry and all confounders listed in the manuscript. Using Rubin's rules, we combined the results of five imputations to estimate the value of missing variables. 4
## Supplementary methods 2. marginal structural modelling
To address the possibility of residual time-dependent confounding associated with time-varying exposures, we repeated the analysis using a marginal structural Cox proportional hazards model. Using two pooled logistic regression models (numerator and denominator of the stabilized inverse-probability-of-treatment weights [IPTWs]), we estimated the conditional probability of being exposed to DPP-4 inhibitors and GLP-1 receptor agonists given previous treatment history in the 30-days prior. The numerator model included baseline covariates (listed in the manuscript) and follow-up time, and the denominator model included covariates (listed in the manuscript) measured at each 30-day interval and follow-up time. Follow-up was modelled using a restricted cubic spline with five knots to avoid biases from the linearity assumption. [bib_ref] Constructing inverse probability weights for marginal structural models, Cole [/bib_ref] We used similar methods to estimate inverse probability of censoring weights (IPCWs). Thus, using predicted probabilities from treatment and censoring models, we calculated stabilized IPTW and IPCW for each patient. The product of these weights was used to reweigh the cohort, in which we estimated the hazard ratios of cholangiocarcinoma associated with the use of DPP-4 inhibitors and GLP-1 receptor agonists, with 95% confidence intervals calculated using robust variance estimators. 6
## Supplementary methods 3. negative control exposure
To address the potential impact of confounding by diabetes severity, we used insulin as a negative control exposure 8 , a drug typically reserved to patients with advanced disease that has not been associated with the incidence of cholangiocarcinoma. For this analysis, we excluded patients who had previously used insulin at any time before cohort entry and modelled new use of insulin as a time-varying variable lagged by one year. Specifically, each person-day of follow-up was categorised into one of four mutually-exclusive categories: 1) use of insulin (alone or in combination with other antidiabetic drugs, excluding incretin-based drugs); 2) use of insulin in but with current or previous use of incretin-based drugs; 3) use of second or third line drugs (drugs (defined as initiation of treatment with either thiazolidinediones, prandial glucose regulators, acarbose, sodium-glucose cotransporter-2 inhibitors, insulin, or combination of oral antidiabetic drugs; or switch to or add-on of an antidiabetic drug, including insulin, after failure with metformin or sulfonylurea in monotherapy); and 4) use of first line drugs (defined as use of metformin or sulfonylurea in monotherapy). For this analysis, we compared use of insulin without incretin-based drugs to use of other second or third line antidiabetic drugs.
## Supplementary methods 4. propensity score matched analysis
To investigate the potential impact of confounding by diabetes severity, we conducted an ancillary analysis using a propensity score-matched design. This analysis was an extension of the sequence of non-randomized trials approach proposed by Hernan et al. [bib_ref] Observational studies analyzed like randomized experiments: an application to postmenopausal hormone therapy..., Hernan [/bib_ref] Using the study cohort defined in the manuscript, we identified all new users of incretin-based drugs and other second or third line drugs at each calendar month of the study accrual period (January 1, 2007 to March 31, 2017). Each calendar month generated a separate cohort. We applied the same exclusion criteria detailed in the manuscript at the time of entry into each of these 123 sequential cohorts. Patients in the comparator group who eventually added-on or switched to an incretin-based drug were allowed to contribute to the exposed group after the time of the switch. Within each cohort, we estimate the predicted probability (propensity score) of receiving an incretin-based drug versus another second or third line drug using conditional logistic regression, stratified on calendar month and conditional on the variables listed in the manuscript. Age and duration of diabetes were modelled as continuous variables using restricted cubic splines to avoid linearity assumptions. We then matched each incretin-based drug user chronologically to one patient (without replacement) initiating a secondto third-line antidiabetic drug in the same calendar month and propensity score using a calliper of 0.01 using a greedy matching algorithm, with the nearest neighbour chosen as the match. The matched sets were followed until an incident diagnosis of cholangiocarcinoma, death from any cause, end of registration with the practice, or end of the study period (March 31, 2018), whichever occurred first. We used a Cox proportional hazards model to estimate the hazard ratio and 95% confidence interval of cholangiocarcinoma, comparing use of incretin-based drugs with use of other second or third line drugs.
## Supplementary methods 5. pharmacovigilance analysis using the world health organization vigibase
Using the World Health Organization global individual case safety report database, Vigibase, we conducted a case/non-case study. [bib_ref] The history of disproportionality measures (reporting odds ratio, proportional reporting rates) in..., Moore [/bib_ref] This database includes information on over 16 million individual case safety reports forwarded to the World Health Organization Uppsala Monitoring Center by national pharmacovigilance systems from over 150 countries around the world since 1967.Individual case safety reports register information about patients, prescription drugs (classified as suspected or concomitant), suspected adverse drug reactions, and the country reporting. The likelihood that the drug has caused the reported event varies from case to case. We included all individual case safety reports from January 1, 2008 to April 1, 2018 in adults over the age of 18 and excluded all duplicate reports.
We defined cases of cholangiocarcinoma as an individual case report containing any of the following terms: bile duct adenocarcinoma, bile duct adenosquamous carcinoma, bile duct cancer/recurrent/stage 0 to IV, bile duct squamous cell carcinoma, biliary cancer metastasis, cholangiocarcinoma or biliary malignant tumours. Non-cases were all other reports in Vigibase during the same period, with known age and sex. We defined exposure among cases and non-cases as the use of dipeptidyl peptidase-4 inhibitors or glucagon-like peptide-1 receptor agonists, compared to the use of sulfonylureas or thiazolidinediones. To investigate the impact of confounding by diabetes severity, we repeated the analysis using long-acting insulin analogues as a negative control exposure.
Descriptive statistics were used to summarize the baseline characteristics of the cholangiocarcinoma cases. We performed a disproportionality analysis to estimate reporting odds ratios (RORs) of cholangiocarcinoma with 95% confidence intervals (CIs) compared to all other adverse drug reactions in Vigibase. [bib_ref] The reporting odds ratio and its advantages over the proportional reporting ratio, Rothman [/bib_ref] The ROR is the exposure odds among reported cases of cholangiocarcinoma compared with the exposure odds among reported non-cases. The ROR was calculated among users of the incretin-based drugs compared to users of sulfonylureas and/or thiazolidinediones. RORs were adjusted for age, sex, notifier type (physician, consumer or other), country of report (Americas, Asia or other) and year of report. This frequentist approach (ROR and 95% CIs) based the concept of disproportionality has been largely used in pharmacovigilance databases to detect signals of disproportionate reporting, and the performance, accuracy and reliability are usually similar to other frequentist approaches (Proportional Reporting Ratio and Relative Reporting Ratio) or Bayesian approaches. [bib_ref] A comparison of measures of disproportionality for signal detection in spontaneous reporting..., Van Puijenbroek [/bib_ref] 13 year lag period applied after each new antidiabetic prescription; patients were considered exposed to each new antidiabetic drug starting one year after their initial prescription. The solid lines represent the exposure periods. The dotted line (in green) represents the period after discontinuation of an incretin-based drug, whereby patients remain exposed until the end of the follow-up period. Finally, each event date forms a risk set, where exposure to the different antidiabetic drugs is assessed at these time points.
## Supplementary table 4. crude and adjusted hazard ratios for the association between the use of dpp-4 inhibitors and glp-1 receptor agonists and the risk of cholangiocarcinoma (lag 2 years)
## Supplementary table 5. crude and adjusted hazard ratios for the association between the use of dpp-4 inhibitors and glp-1 receptor agonists and the risk of cholangiocarcinoma (lag 3 years)
## Supplementary table 6. crude and adjusted hazard ratios for the association between the use of dpp-4 inhibitors and glp-1 receptor agonists and the risk of cholangiocarcinoma (no lag)
## Supplementary table 7. crude and adjusted hazard ratios for the association between the use of dpp-4 inhibitors and glp-1 receptor agonists and the risk of cholangiocarcinoma (competing risk)
## Supplementary table 8. crude and adjusted hazard ratios for the association between the use of dpp-4 inhibitors and glp-1 receptor agonists and the risk of cholangiocarcinoma (four prescriptions within a 12-month period)
## Supplementary table 9. crude and adjusted hazard ratios for the association between the use of dpp-4 inhibitors and glp-1 receptor agonists and the risk of cholangiocarcinoma (marginal structural model)
In the scenario above, Patient A is exposed to a second or third line antidiabetic drug (other than an incretin-based drug) and contributes an unexposed event (risk set 1) after one year of follow up. At this point, this patient's exposure is compared with the exposure of Patient B (DPP-4 inhibitors), Patient C (use of other second or third line antidiabetic drugs) and Patient D (DPP-4 inhibitors). Patient C contributes both unexposed and exposed person time to the analysis. When this patient experiences the event (risk set 3), they contribute an exposed event to the DPP-4 inhibitor analysis. At this time, the exposure of Patient C is compared with the exposure of Patient D (DPP-4 inhibitor). While Patient D had discontinued use of DPP-4 inhibitors at year 2 of follow-up, they are considered exposed until the end of follow-up. We used a similar exposure definition for the GLP-1 receptor agonist analysis.
## Supplementary figure 2: illustration of the propensity score-matched analysis
In each of the 123 months of the patient accrual period (January 1, 2007 to March 31, 2017), new users of DPP-4 inhibitors (exposed group) were matched 1:1 to new users of other second-to third-line antidiabetic drugs (comparator group) on propensity scores. For example, in January 2007 patient 1 could be matched to patient 2, 4 or 7; the patient with the closest propensity score with a maximum calliper of 0.01 could become a match. Patients in the comparator group eventually adding on or switching to a DPP-4 inhibitor could contribute to the DPP-4 inhibitor group, but only after the time of switch. For example, patient 4 stopped contributing to the comparator group and started contributing to the DPP-4 inhibitor group after June 2007 (and was matched on propensity score to patient 6 from the comparator group in the same month).
## Supplementary figure 5: array approach to quantify the effect of unmeasured confounding
We used the Array approach to estimate the effect of an unknown or unmeasured confounder on our observed estimate. [bib_ref] Sensitivity analysis and external adjustment for unmeasured confounders in epidemiologic database studies..., Schneeweiss [/bib_ref] We fixed the prevalence of a hypothetical confounder in the reference group (use of at least two antidiabetic drug classes) to 0.2 and varied the strength of the confounder-disease association (1.0 to 5.5) and the prevalence of the confounder in the group exposed to DPP-4 inhibitors (0.0 to 0.5). The relationship between these three factors is plotted in the three-dimensional graph above.
If the hypothetical confounder is equally distributed among the group exposed to DPP-4 inhibitors and the reference group, there is no bias. If the confounder is imbalanced between the two groups, the estimate will change from the observed HR. Thus, if the prevalence of the confounder in the DPP-4 inhibitor group is less than the prevalence of the confounder in the reference group, the "true" HR would be higher than the observed HR. However, if the prevalence of the confounder in the DPP-4 inhibitor group is higher than the prevalence of the confounder in the reference group, the "true" HR would be lower than the observed HR. This would require the confounder to be strongly associated with the outcome, with RR estimates ranging from 3.0 to 5.5. It is unlikely that such a confounder exists beyond what was adjusted for in the analyses.
[fig] 2 Supplementary 22 Supplementary, Figure 3 23 Supplementary, Figure 4: Table 13. Impact of Imperfect Sensitivity and Specificity on the Association between the Use of DPP-4 Inhibitors and the Risk of Cholangiocarcinoma ................................ 20 Supplementary Figure 1. Exposure Definition............................................................................. 21 Supplementary Figure 2: Illustration of the Propensity Score-Matched Analysis ....................... Flow Chart of Patients Included in the Propensity Score-Matched Analysis......................................................................................................................................... Cumulative Incidence of Incident Cholangiocarcinoma Among Users of Dipeptidyl Peptidase-4 Inhibitors and Other Second-to Third-Line Antidiabetic Drugs (Propensity Score-Matched Analysis) ......................................................................................... 24 Supplementary Figure 5: Array Approach to Quantify the Effect of Unmeasured Confounding 25 References for Online-Only Supplements .................................................................................... 26 [/fig]
[fig] Supplementary, Figure 1: summarizes the exposure definition. The dashed lines represent the one- [/fig]
[table] , 14 8: SupplementaryTable 1. British National Formulary Codes for Antidiabetic Drugs Short-acting Insulins/Diabetic Ketoacidosis 06010103/59010400 Diabetes Hypodermic Equipment/-06010200/06010203 Antidiabetic Drugs/Other Antidiabetic Drugs 06010201/06010203 Sulphonylureas/Other Antidiabetic Drugs 06010202/06010203 Biguanides/Other Antidiabetic Drugs BNF=British national formulary Supplementary Table 2. Read Codes and Associated Terms for Cholangiocarcinoma Supplementary Table 3. Crude and Adjusted Hazard Ratios for the Association between the Use of DPP-4 Inhibitors According to Cumulative Duration of Use, Time since Initiation and Drug Type GLP-1=glucagon-like peptide-1; HR=hazard ratio; CI=confidence interval. a Use of other anti-diabetic drugs was considered in the models, but not presented in the table. b Per 100,000 Person-Years. c Adjusted for age, sex, year of cohort entry, obesity, smoking status, alcohol-related disorders, Charlson comorbidity index score, inflammatory bowel disease, gallbladder disease, haemoglobin A1c, and duration of diabetes. d Including initiating on combination therapy or switching to or adding-on a new antidiabetic drug class. e Renal excretion: sitagliptin, saxagliptin and alogliptin. Biliary excretion: linagliptin and vildagliptin. S* Numbers less than 5 are not displayed, as per the confidentiality policies of the Clinical Practice Research Datalink. [/table]
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Mapping human vulnerability to climate change in the Brazilian Amazon: The construction of a municipal vulnerability index
Vulnerability, understood as the propensity to be adversely affected, has attained importance in the context of climate change by helping to understand what makes populations and territories predisposed to its impacts. Conditions of vulnerability may vary depending on the characteristics of each territory studied-social, environmental, infrastructural, public policies, among others. Thus, the present study aimed to evaluate what makes the municipalities of the state of Amazonas, Brazil, vulnerable to climate change in the context of the largest tropical forest in the world, and which regions of the State are the most susceptible. A Municipal Vulnerability Index was developed, which was used to associate current socioenvironmental characteristics of municipalities with climate change scenarios in order to identify those that may be most affected by climate change. The results showed that poor adaptive capacity and poverty had the most influence on current vulnerability of the municipalities of Amazonas with the most vulnerable areas being the southern, northern, and eastern regions of the state. When current vulnerability was related to future climate change projections, the most vulnerable areas were the northern, northeastern, extreme southern, and southwestern regions. From a socio-environmental and climatic point of view, these regions should be a priority for public policy efforts to reduce their vulnerability and prepare them to cope with the adverse aspects of climate change.
# Introduction
The Intergovernmental Panel on Climate Change (IPCC) defines vulnerability as "the propensity or predisposition to be adversely affected" [bib_ref] Contribution of Working Groups I. II and III to the Fifth Assessment..., Ipcc [/bib_ref] , which encompasses the basic components of exposure, sensitivity and adaptive capacity. These three components are capable of influencing vulnerability and may increase or decrease it according to characteristics inherent to the human or natural system of interest. In recent years, vulnerability analysis has followed a multifactorial approach that incorporates distinct social, political, economic and environmental a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 conditions, including future climate change scenarios [bib_ref] Mapping vulnerability to multiple stressors: climate change and globalization in India, O'brien [/bib_ref]. In this sense, vulnerability assessments have been effective tools for enabling, among other uses, vulnerability mapping [bib_ref] Mapping vulnerability to multiple stressors: climate change and globalization in India, O'brien [/bib_ref] [bib_ref] Putting vulnerability to climate change on the map: A review of approaches,..., Preston [/bib_ref]. Among the advantages that understanding the geography of vulnerability to climate change provides are enhanced disaster risk management; reduced exposure of human and ecological assets; and identification of particularly vulnerable populations [bib_ref] Putting vulnerability to climate change on the map: A review of approaches,..., Preston [/bib_ref].
Indicators have been commonly used as tools for assessing the vulnerability of populations and territories. They not only allow comparisons to be made among the evaluated systems, but also facilitate the visualization of information about what needs to be adapted in a simple and objective way [bib_ref] Mapping vulnerability to multiple stressors: climate change and globalization in India, O'brien [/bib_ref] [bib_ref] Climate change vulnerability assessments: An evolution of conceptual thinking, Füssel [/bib_ref] [bib_ref] Assessing the vulnerability of social-environmental systems, Eakin [/bib_ref] [bib_ref] Unpacking governance: building adaptive capacity to climate change of river basins in..., Engle [/bib_ref]. This method has been applied in different ways by various authors, either on a regional or national scale [bib_ref] Revealing the vulnerability of people and places: a case study of Georgetown..., Cutter [/bib_ref] [bib_ref] Indicators of vulnerability and adaptive capacity: Towards a clarification of the science-policy..., Hinkel [/bib_ref]. Using a quantitative approach, Brooks et al. [bib_ref] The determinants of vulnerability and adaptive capacity at the national level and..., Brooks [/bib_ref] and Moss et al.established a series of key indicators for assessing the vulnerability and adaptive capacity of countries throughout the world. On a regional scale, Sullivan & Meigh [bib_ref] Targeting attention on local vulnerabilities using an integrated index approach: The example..., Sullivan [/bib_ref] demonstrated the applicability of the Climate Vulnerability Index for identifying populations that are most susceptible to climate impacts; Yusuf & Franciscocombined climate risk assessments, human and ecological sensitivity, and adaptive capacity to produce maps of vulnerability to climate change for Southeast Asia; and Thornton et almapped vulnerability and human poverty in Africa by identifying key elements to included in their vulnerability indicator.
In Brazil, the indicator method has also been applied to assessing vulnerability to climate change. Studies have related the impacts of climate change to increased vulnerability to hydrometeorological events at the national level [bib_ref] An index of Brazil's vulnerability to expected increases in natural flash flooding..., Debortoli [/bib_ref] , the demographic and migration processes in various regions of the country [bib_ref] Climate change and population migration in Brazil's Northeast: scenarios for 2025-2050, Barbieri [/bib_ref] [bib_ref] Population transitions and temperature change in Minas Gerais, Brazil: a multidimensional approach, Barbieri [/bib_ref] , and to the socioeconomic conditions of Brazilian municipalities that create socio-climatic hotspots [bib_ref] Socio-climatic hotspots in Brazil, Torres [/bib_ref]. A methodology involving synthetic indicators was developed and refined by the Oswaldo Cruz Foundation since the 2000s, which began with the construction of an indicator of vulnerability of the Brazilian population to climate impacts on health. Development of this methodology included the study of social, environmental and health vulnerabilities of the Northeast Region of Brazil [bib_ref] Social, environmental and health vulnerability to climate change in the Brazilian Northeastern..., Confalonieri [/bib_ref] , of the municipalities of the state of Rio de Janeiroand, more recently, of the state of Minas Gerais [bib_ref] Social, Environmental, and Health Vulnerability to Climate Change: The Case of the..., Quintão [/bib_ref].
The Amazon Region has been facing challenges inherent to the development projects taking place in the countries that comprise it, such as combining the need to promote social and economic progress with environmental conservation, which includes climatic issues. On the one hand, ecosystems of the Amazon are important strongholds of biodiversity and natural resources and play an active role in biosphere-atmosphere exchange on both regional and global scales, and thus serve an essential function in climatic balance [bib_ref] The Amazon basin in transition, Davidson [/bib_ref] [bib_ref] Amazonian deforestation and regional climate change, Nobre [/bib_ref]. On the other hand, the socioeconomic conditions of human populations in the Amazon are at a great disparity with national averages of their respective countries, which is even more severe when analyzed from the perspective of traditional populations, whose estimated contingent is 1.6 million people. Overcoming this condition finds obstacles in the current anthropic pressures placed upon the Amazon due to the expansion of agriculture and "economic development"-both have profoundly reduced biodiversity, cultural assets and ecosystem services. The effects of the combination of these elements with global climate change are apparent in the region. In the case of the Brazilian Amazon, the last 10 years have experienced the most severe events of the century related to droughts (2005 and 2010) and floods (2009 and 2012) due to changes in rainfall in the Amazon Basin [bib_ref] The Amazon basin in transition, Davidson [/bib_ref] [bib_ref] The drought of 2010 in the context of historical droughts in the..., Marengo [/bib_ref] [bib_ref] Extreme climatic events in the Amazon basin: climatological and hydrological context of..., Marengo [/bib_ref] [bib_ref] Intensification of the Amazon hydrological cycle over the last two decades, Gloor [/bib_ref] [bib_ref] Potential effects of climate change on inundation patterns in the Amazon Basin, Langerwisch [/bib_ref] [bib_ref] Extreme seasonal droughts and floods in Amazonia: causes, trends and impacts, Marengo [/bib_ref].
The state of Amazonas, Brazil, exhibits the same social and environmental patterns observed in other regions of the Amazon. The state is, for the most part, covered by forest and faces important issues related to: 1) land use policies associated with farming and the construction of roads and hydroelectric dams; 2) health, due to high incidence rates of infectious diseases and the need to adapt infrastructure to peculiar socio-spatial conditions-mobility is is related to the intrinsic characteristics of the impacted system. The present study worked with the indirect effects of climate resulting from the possible damage that can occur, more specifically on specific population groups, due to either their social or financial condition. Adaptive capacity is related to the ability of populations to adjust to possible damage and respond to the consequence of climate impacts [bib_ref] Contribution of Working Groups I. II and III to the Fifth Assessment..., Ipcc [/bib_ref] , and which focuses on the conditions of community organization, such as the role of institutions, governance and management.
It was assumed that the human populations at each location are subjected to multiple and specific tensions that combine with each other to determine climate impacts on a regional scale, with feedback between human and natural systems. Considering that the division between these systems is only arbitrary, since anthropic actions and social structure are not dissociated from the natural environment, the present study considered the "municipality" as the unit of analysis since it represents an integrated system in which impacts will occur. In this sense, the present work has developed a conceptual framework for the municipalities of the state of Amazonas, which considers systems that possess some peculiar characteristics that shape their vulnerability profile and that may be exacerbated by future climate change .
In the circle represents a system, such as a municipality, in which various physical, natural, economic, social, political and structural conditions shape the vulnerability profile of a municipality. This profile is, somehow, determined by exposure and sensitivity, since these components contribute to determining the magnitude of the potential impacts of future climate change. The magnitude of impacts may depend on the capacity of a population and of its municipality to cope with the adverse effects of climate. The adaptive capacity is strictly linked to the sensitivity of the system, since this component includes certain characteristics intrinsic to a population that determine its ability to recover from impacts. Thus, the vulnerability profile constructed may be altered by the influence of climate change, here represented by gradual alterations in temperature and precipitation over time (from the present to the period of 2041-2070).
Concepts and variables of the methodological scheme. The present work comprises the unfolding of a national-scale project whose objective was to construct indicators of vulnerability for some Brazilian municipalities, in the context of climate change, in order to support future adaptive actions. Financed by public resources (Ministry of Environment), a Municipal Vulnerability Indicator was built for six Brazilian states and their municipalities, including the state of Amazonas. The methodology was designed to be easily-executed by local managers, and so that the constructed indices could be promptly interpreted and updated, thus allowing the monitoring of the vulnerability profile of the municipalities of Amazonas over time. In the end, the focus of the study was to estimate how climate change may influence the vulnerability of the state of Amazonas considering its current socio-environmental conditions.
The variables chosen to compose the methodological scheme possessed the following criteria: 1) should be mainly of free access; and 2) should be systematically updated. Most of these variables were selected based on what has been established by other studies developed for Brazil, whereas other variables were specific to this study [bib_ref] Social, environmental and health vulnerability to climate change in the Brazilian Northeastern..., Confalonieri [/bib_ref] [bib_ref] Public health vulnerability to climate change in Brazil, Confalonieri [/bib_ref]. All variables passed through validation by a panel of experts comprising managers, decision makers, non-governmental organizations and researchers from various parts of the country who had expertise in the thematic areas studied-disasters, social indicators, adaptation, climatology and health, among others. After this first evaluation, successive meetings were held in the state of Amazonas, as well as in the other states that were part of the national project, to discuss the reliability and adequacy of the indicators relative to local reality.
According to Adger's definition for exposure(the nature and degree to which a system experiences environmental or socio-political stress systems), two factors were considered in the present evaluation. The first, essentially environmental, considers the relevance of the Amazonian ecosystem to the resident population (ecosystem services) and to the regional dynamics of the terrestrial-atmosphere energy flux, using as proxy the extension of vegetation cover and the area of deforestation in recent years. The assumption here is that the maintenance of the natural environment makes the municipality less exposed to the effects of climate change due to the conservation of associated ecosystem services (e.g. temperature control, air quality, clean water, among others). Stress or destruction of ecosystems can increase the physical vulnerability of human settlements as well as reduce the possibilities for traditional populations (e.g. riverine) to adequately exploit livelihoods, including alternative food sources. The second important factor evaluated was exposure to risk of hydrometeorological disasters, a phenomenon provided by both social and environmental conditions and related to climate variability. This exposure was assessed through the susceptibility to disasters and also by historical records of occurrence of such events. Exposure indicators were positively related to vulnerability.
For sensitivity (the degree to which a system is affected, either adversely or beneficially, by climate-related stimuli, some conditions intrinsic to the municipalities that may make them more susceptible to climate impacts were considered. The focus was on the susceptibility of the population, hence the variables to be chosen to compose the sensitivity index should reflect inequities in the process of socioeconomic development, since this engenders conditions of social marginalization and generates some social groups that are more vulnerable to climate and its impacts than others. The factors considered were those related to climate-sensitive diseases, which may be affected by expected changes in temperature and precipitation; those related to human poverty, in their monetary and non-monetary dimensions; and, finally, those related to the sociodemographic characteristics of the population. All of the indicators selected to comprise sensitivity have been associated with increased vulnerability and have been related with the indirect effects of climate resulting from possible damage that may occur to specific groups of populations (e.g elderly, people with disabilities, women lacking education).
For adaptive capacity (the ability of systems, institutions, people, and other organisms to adjust to potential damage, to take advantage of opportunities or to respond to consequences, the main focus was governance and social capital. Governance and local institutions are considered essential to reducing vulnerability and enhancing adaptation at the local level, since they help to shape the risks associated with climate impacts and to define the degree to which individuals will respond to the impact, including the collective scope. This aspect was evaluated by 1) the municipal competence to promote economic development in key areas such as health, education and employment, with regards to quality and access, and whose performance has been measured since 2008 by the FIRJAN Municipal Development Index; and 2) the presence of public institutions of security, risk management and health that could provide support and actions to reduce harm in the face of the expected negative impacts of climate change. As for social capital, its ability to increase the resilience of populations is well known, either through the proximity of different groups, closer ties between individuals who share a social identity or through networks of trust that are established between the different levels of authority [bib_ref] Social and ecological resilience: are they related?, Adger [/bib_ref] [bib_ref] Social capital, individual responses to heat waves and climate change adaptation: An..., Wolf [/bib_ref]. For this, a proxy measure for the possibility of citizen participation in the political process was used, namely the representation on councils or consortia. The variables used here are inversely related to vulnerability, since municipalities with better levels of governance and socio-institutional arrangements are better able to deal with climate impacts, thus reducing vulnerability.
A future climate component was associated with the other basic elements of vulnerability to represent the extent to which the municipalities could be impacted by the predicted climate change in the state of Amazonas. Thus, the present study evaluated possible future climate change (2041-2070) in relation to the present , as projected by the model ETA-HadGem-ES considering climatic parameters related to temperature and precipitation. These data were municipalized for the entire state of Amazonas (see "Construction of the Climate Scenario" section) to serve as a predictor of the occurrence of climatic extremes in the near future. The assumption was that areas with the greatest predicted changes in temperature and precipitation would be those most exposed to climatic hazards. [fig_ref] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship... [/fig_ref] describes the indicators used and their relation to vulnerability. The raw data for each indicator is available in the supplementary S3-S6 Tables.
# Material and methods
## Study area
The state of Amazonas is located in the Northern Region of Brazil and is the largest state of the country, occupying about 18% of its territory [fig_ref] SSI + Future SSI / 2 •: Current SSI [/fig_ref]. Although it has just 62 municipalities, the state capital of Manaus has a rich history, especially as a central harbor of the region, and contains 52% of the state's population. For that reason, Manaus is considered the most populous city of the Brazilian Amazon. The state has 4,001,667 inhabitants, of which most are urban residents, although there is a considerable rural contingent of traditional groups: indigenous peoples who mainly live in legal reserves, and riverine peoples who reside along lakes and rivers [bib_ref] Terras tradicionalmente ocupadas: Processos de territorialização e movimentos sociais, Almeida [/bib_ref]. Most of the population is male (50.3%), pardo (68.8%), receives at most half of the minimum wage (56.1%) and lives in houses lacking appropriate sanitation. - VCI = (Score of vegetation cover + Score of deforestation) / 2 Native vegetation cover: percentage of the area of the municipality covered by native vegetation in 2014 divided by total municipal land area. Proxy of ecosystem services.
The higher the vegetation cover, the less exposed/ vulnerable. The higher the accumulated deforestation for the time series, the more exposed/vulnerable.
## - index of natural disasters (ind)
- IDN = (NDSI + NDOI) / 2
## - natural disasters susceptibility index (ndsi)
- NDSI = (Score of population at risk + Score of CDD) / 2
## - natural disasters occurrence index (ndoi)
- NDOI = (Score of disaster occurrence + Score of deaths related to disasters) / 2 Population at risk: the percentage of the total population living in areas of high and very high risk for landslides and hydrological events in relation to the total population.
Suggestive of human losses that may result from natural disasters.
The higher the percentage of people at risk, the more exposed/vulnerable.
## Consecutive dry days (cdd):
climate parameter that indicates greater propensity for dry periods.
The higher the average CDD, the more exposed / vulnerable.
Disaster occurrence between 1991-2012: percentage of events that occurred in the municipality in relation to all events that occurred in the state. Used as an indication of the disasters' burden on each municipality.
The higher the proportion of events in relation to the state, the greater the sensitivity/vulnerability.
Deaths related to natural disasters: the proportion of deaths reported in the municipality in relation to the events in the municipality. Used as an indication of the fatality of recorded disasters.
The higher proportion of deaths, the higher sensitivity/ vulnerability.
## Sensitivity index - diseases associated with climate index (daci): dengue; malaria; american
Cutaneous Leishmaniasis; Accidents with poisonous animals (spider, snake, and scorpion)
- DACI = (Score of the proportion of cases + Score of the incidence of the disease + Score of the tendency of the incidence rate) / 3
Proportion of cases of the disease: number of cases in the municipality in relation to the total number of cases in the state, in the time series.
The higher the proportion of cases in the city, the more sensitive/vulnerable.
Incidence rate: the average incidence rate per 100,000 in the time series was used to measure the risk of the disease during the time series. For Malaria, the Annual Parasite Index was used, which considers the incidence per 1,000 inhabitants.
Higher average incidence rates, higher sensitivity/ vulnerability.
Tendency of the disease's incidence rate: the incidence rate was used to determine how cases of the disease behaved (increase, decrease, stability) in the time series.
If the incidence showed a declining trend, the city was considered less sensitive; if the trend was an increase, the city was considered more sensitive.
## - poverty index (poi)
- PoI = (Score of the probability of dying before age 40 +
## Score of illiterate population +
## Score of households whith inadequte sanitation +
Score of the probability of dying before age 5 + Score of income bellow the poverty line) / 5
Probability of dying before age 40: reflects the living conditions and mortality pattern of the population.
The higher the likelihood of dying before the age 40, the greater the sensitivity/vulnerability.
Percentage of the population aged over 25 that is illiterate: ratio between the population aged 25 years or older who can not read or write and the total number of people in the same age group. The illiteracy rate was considered a proxy of education levels and population skills.
The higher the illiteracy rate, the higher the sensitivity.
Percentage of households with inadequate sanitation: percentage of households with sanitation considered unsuitable including drinking water, sewage and garbage collection. Considered a basic need, it is related to the health conditions of the population.
Highest percentage of households without sanitation, increased vulnerability.
Probability of dying before the age of 5 per 1,000 live births: reflects the living and health standards of the population.
More likely to die by the age of 5, higher the sensitivity/ vulnerability.
## Percentage of households with per capita income below the poverty line: percentage
of total households whose nominal per capita monthly income was up to ½ minimum wage. Monetary measure of poverty.
The higher the percentage of households with up to ½ minimum wage income, the higher the sensitivity/ vulnerability.
## - sociodemographic sensitivity index (ssi)
- SSI = (Current The higher the proportion of households headed by women, the greater the vulnerability.
Percentage of young householders: percentage of total households in the total population whose head is between 10 and 29 years old
The higher the proportion of households headed by young individuals, the greater the vulnerability.
Percentage of the population under 5 years old: percentage of the total population.
Social group most vulnerable in disaster situations and who require permanent care.
The higher the percentage of infants in the population, the more sensitive/vulnerable.
## Percentage of the population that is elderly (60 years old or older):
percentage of the total population. Social group most vulnerable in disaster situations and who require permanent care.
The higher the percentage of elderly in the population, the more sensitive/vulnerable.
## Percentage of the population with disabilities: percentage of the total population
with some form of disability (visual, hearing, motor or intellectual). Most vulnerable social group in disaster situation and who may require specialized care.
The higher the percentage of the population with some form of disability, the more sensitive/vulnerable.
Riverine population: percentage of the total population. Social group very dependent on natural resources for subsistence.
The higher the percentage of the population considered riverine, the more sensitive/vulnerable.
## Projection of children aged 0 to 4 years old for the year 2040: percentage of infant population in 2040.
The higher the percentage of infants expected in the population for 2040, the more sensitive/vulnerable. . It follows the achievements and socioeconomic challenges of Brazil through the prism of municipal competence on a regular basis since 2008, and makes it possible to determine, with precision, whether the relative improvement experienced by a given municipality is the result of the adoption of specific policies. The FMDI varies between 0 and 1, where 0 represents the least developed and 1 the most developed municipality. This reasoning is opposite to that applied to vulnerability logic. Thus, for the construction of the Socioeconomic Structures Index (SEI), the reverse of FMDI was used, since the attributed scores followed the reasoning of vulnerability-if the city had high FMDI, it was considered more adapted and less vulnerable.
## Projection
It reflects governance and encompasses the following: 1) structures for employment and income generation; 2) quality of education; and 3) quality of health care services (primary care).
The higher the score (closer to 1), the more sensitive/ vulnerable.
## - institutions, services, and infrastructure for adaptation index (adapi)
- AdapI = (Score of secutiry institutions + Score of risk management instruments +
## Score of health care services) / 3
Existence of security institutions: municipal civil defense, fire department and municipal guard. It reflects the structural capacity of the municipality to cope with climate impacts.
The greater the number of security institutions, the less vulnerable.
Existence of risk management instruments for landslides and floods: mapping of risk areas, housing program, supervision control of risk areas, contingency planning, engineering projects, warning systems and risk register. It reflects the structural capacity of the municipality to cope with climate impacts and mitigate future impacts.
The larger the number of management instruments, the less vulnerable.
Health care services (indicative of structural capacity of the municipality to provide basic services to the population): the information used were: 1) the number of hospital beds per 1,000 inhabitants; and 2) primary care coverage.
The higher the number of hospital beds and the coverage of basic care, the less vulnerable.
## - sociopolitical organization index (soi)
Existence of municipal councils and consortia related to adaptation to climate (proxy of the community social capital): environment, welfare and social development, basic sanitation, employment and labor, housing, transportation, urban development, and human rights. It reflects the social capital of the community.
The greater the number of councils and consortia, the less vulnerable.
## Climate scenario index
- Temperature Index = (TMAXmean +
[formula] TMINmean) / 2 - Precipitation Index = (Rx5day + R95p + CDD + PRCPTOT) / 4 [/formula]
Temperature anomalies: average annual maximum temperature (TMAXmean) and average annual minimum temperature (TMINmean).
The higher the anomaly, the more vulnerable.
Precipitation anomalies: Rx5day (monthly maximum consecutive 5-day precipitation); R95p (annual total precipitation when daily precipitation amount on day was higher than 95 percentile); CDD (maximum number of consecutive days with daily precipitation amount lower than 1mm); PRCPTOT (annual total precipitation on wet days).
The higher the anomaly, the more vulnerable.
Environmentally, the state of Amazonas is considered only slightly deforested since 23.5% of its natural ecosystems are protected within conservation units and about 98% of the territory remains preserved. However, some regions, mostly in the southern portion, are historically linked to deforestation [bib_ref] Os processos de desenvolvimento e desmatamento da Amazônia, Prates [/bib_ref] [bib_ref] Malaria in the State of Amazonas: a typical Brazilian tropical disease influenced..., Sampaio [/bib_ref]. As part of the world's largest hydrographic basin, the state of Amazonas is essentially fluvial. The mobility of goods and people occurs mainly via rivers; since access by land is not common, an estimated 20,000 km of waterways connect distant communities of the region. To better contextualize the state of Amazonas, a brief description is provided that encompasses some environmental, economic and infrastructural conditions of the 13 microregions of the state (S1 Appendix). Vulnerability to climate change in the Brazilian Amazon
## Construction of the climate scenario
Regarding the emission scenarios, Representative Concentration Pathways coming from the last IPCC assessment report-AR5 were used (IPCC 2013). The RCP refers to the number of radiative forcing measured in watts per square meter per year by 2100. The scenarios generated by IPCC are named according to their radiative forcing as follows: RPC2.6 (2.6 Wm-2), RCP4.5 (4.5 Wm-2), RCP6.0 (6.0 Wm-2) and RCP8.5 (8.5 Wm-2). The RCP8.5 combines, among other factors, high population growth, high energy demand, and high GHG emissions in the absence of climate change policies, corresponding to the RCP with the highest greenhouse gas emissions. This scenario includes radiative forcing beyond 8.5 W/m 2 and CO 2 concentrations of 1,370 ppm up to the year 2100. Considering the RCP8.5 as a pessimistic scenario and that shorter time periods are more appropriate for proposing and articulating public policies, the outputs of the Eta-HadGem-ES model considering the RCP8.5 were used for the period of 2041-2070.
Extreme climate indicators were constructed considering the absolute difference between the climatic parameters generated by Eta-HadGem-ES for the future climate slice (2041-2070) and the current slice . These indicators were generated with the assistance of the FClimDex program, and were the following: total annual rainfall for rainy days (i.e., days when rainfall is greater than or equal to 1 mm; PRCPTOT); total annual rainfall on days when rainfall is greater than that of the 95th percentile of the rainy days (R95p); monthly maximum consecutive 5-day precipitation (Rx5day); maximum number of consecutive dry days for the year, (i.e., days on which rainfall is less than 1 mm; CDD) and annual mean maximum (TMAXmean) and minimum (TMINmean) temperatures, in degrees Celsius.
These digital spatialized data bases were processed to generate maps of climatic anomalies for all the municipalities of the state of Amazonas. The spatial analysis and municipalization of climatic indicators were obtained using geostatistical modeling performed with the Geostatistical Analyst tool of ArcGIS Desktop software, version 10.2.2, and Surfer, version 11. The data were then interpolated to estimate the value of the climatic variables for the locations not available in the database.
## Source of data
Socioeconomic, environmental, infrastructural and climatic projections data for the 62 municipalities of the state of Amazonas were collected. Information about populations and infrastructure were obtained from the Brazilian Institute of Geography and Statistics (IBGE), FIRJAN Index of Municipal Development and the Department of Informatics of the Brazilian Health System (DataSus). The environmental data were collected from the PRODES project, linked to the INPE. The information related to natural disasters was obtained from the Brazilian Atlas of Natural Disasters, the National Water Agency (ANA) and the Mineral Resources Research Company (CPRM). Finally, for the climatic parameters related to precipitation and temperature to be used in the construction of the climate scenario, data were obtained from INPE.
# Scale of analysis
The scale of municipality was chosen because this is the smallest political-administrative unit in Brazil. Likewise, most of the data used in the study is available only on this scale of analysis. Focusing on municipality as a unit helps to identify local weaknesses and potentialities, as well as to compare vulnerabilities observed in each component-exposure, sensitivity, adaptive capacity and climate. For decision makers or state managers this is of particular relevance because these are the spheres from which financial resources are distributed to municipalities. Vulnerability mapping, therefore, allows the state to understand by which aspects the regions and their municipalities are most vulnerable, and thus allocate the resources as needed.
## Calculation of the municipal vulnerability index
A synthetic indices approach was used to measure vulnerability, which is based on the selection of some indices from a potential set of variables, that are then systematically combined to generate levels of vulnerability. This approach was chosen over evaluation modeling because 1) the results achieved are quite similar, and 2) for many developing countries, such as Brazil, the technical requirements, the availability of data and the financial resources needed to perform evaluation modeling are scarce or nonexistent. For calculation of vulnerability, all the indicators were assumed to have equal importance, thus they have not received a priori weights [bib_ref] Revealing the vulnerability of people and places: a case study of Georgetown..., Cutter [/bib_ref]. The scale of analysis was that of the municipality.
The present study constructed a Municipal Vulnerability Index (MVI) for the municipalities of the state of Amazonas, Brazil, from the successive aggregations of indices and sub-indices related to exposure, sensitivity, adaptive capacity and climate conditions of the territories. MVI represents, synthetically, the relationship between the current vulnerability, represented by the Vulnerability Index (VInd), and the future climate change, represented by the Climate Scenario Index (CSI), in a gradient between 0 and 1. Thus, 0 represents the lowest and 1 the highest vulnerability observed among the evaluated municipalities for all the indices. Calculation of MVI involved three distinct steps: 1) attribution of gradual scores to the variables presented in [fig_ref] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship... [/fig_ref] ; 2) aggregation of the scores using arithmetic mean and subsequent standardization to form the indices shown in the orange boxes of in [fig_ref] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship... [/fig_ref] ; and 3) combination of the indices using the same procedures used for arithmetic mean and standardization. [fig_ref] Fig 4: Diagram illustrating the steps of calculating all indicators and indices [/fig_ref] the steps of calculating all indicators and indices used in the present work, as well as the successive aggregation of these indices to generate the MVI.
The first step was to attribute gradual scores, which represent vulnerability to the indicators shown in [fig_ref] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship... [/fig_ref]. Municipal variables were divided into groups for assigning scores so that the municipalities could receive a range of vulnerability scores. Each variable was analyzed individually and divided into percentiles-p5, p25, p75, p95 were used in most cases, but specificities as to the percentiles used can be seen in the S7 For each interval a score ranging from 0 to 4 was assigned, where 0 was attributed to the least vulnerable group and 4 was attributed to most vulnerable group of municipalities, according to the rationale explained in [fig_ref] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship... [/fig_ref]. The division into classes for assignment of scores is done to allow the aggregation of variables that are in different units of measure. After the assignment of scores, all variables possess the same scale unit. For qualitative variables (present in some adaptive capacity indicators) values were arbitrarily applied as shown in S8 Scoring was done considering the values for each variable in all 62 municipalities.
In vulnerability studies, exposure and sensitivity are considered to have the potential to increase the vulnerability of a system, while adaptive capacity the potential to decrease it. This logic was adopted in the present study. However, in view of the need to make information comprehensible to managers and decision makers, it was decided that the assignment of scores to variables would follow the same rationale for all indicators of exposure, sensitivity and adaptive capacity. In such a manner, having to interpret the indices of adaptive capacity in an Step 1 is representing the assignment of scores and Step 2 is illustrating the procedures of arithmetic mean and standardization to generate the indicators ranging from 0 (least vulnerable) to 1 (most vulnerable).
Step 3 illustrates the aggregation of the indices to generate the final index, the Municipal Vulnerability Index.
https://doi.org/10.1371/journal.pone.0190808.g004
Vulnerability to climate change in the Brazilian Amazon inverse way was avoided, thereby simplifying the understanding of this component by decision makers and managers. Thus, for all indicators, scores were assigned considering whether the assessed characteristic increased or decreased vulnerability of the population. Therefore, in the adaptive capacity indices shown in S1 and S2 Tables, the highest values represented the most vulnerable (least adapted) municipalities, while the lowest values represented the least vulnerable (most adapted) municipalities.
The assignment of scores allows the aggregation of the variables that comprise each index through the arithmetic mean, which is the second step. These indices can be observed in the column "calculation of the indices" shown in [fig_ref] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship... [/fig_ref] and in the orange boxes of the methodological scheme . However, the final value of the arithmetic mean does not vary between 0 and 1, as defined to represent the indices, so values of arithmetic means are standardized such that they range from 0 to 1 [fig_ref] Fig 4: Diagram illustrating the steps of calculating all indicators and indices [/fig_ref]. Standardization is done in relation to the values of the other municipalities, using the following formula, The same arithmetic mean and standardization procedures were used to calculate and aggregate all the indices of the methodological scheme in the final step. Accordingly, the MVI was constructed by the combination of its main indices-Vind and CSI. Likewise, VInd was formed by the combination of its three components-exposure, sensitivity and adaptive capacity-and the CSI was constructed by the aggregation of its climatic indices of temperature and precipitation anomalies [fig_ref] Fig 4: Diagram illustrating the steps of calculating all indicators and indices [/fig_ref]. Thus, MVI represents, specifically, the current vulnerability to future climate change across the municipalities of the state of Amazonas.
This measure has the primary role of allowing the comparison among municipalities over time and space, since it allows both the observation of changes in current vulnerability according to future climate change, as well as the disaggregation of the main indices to explore which conditions most influenced their vulnerability. The method also enables the ranking of the municipalities according to their observed vulnerabilities, thereby providing a consistent and transparent methodology for the comparative assessment of the vulnerability of human populations to regional environmental changes. At the end, the indices developed (MVI, VInd and CSI) resulted in a relative measure of municipal vulnerability ranging between 0 and 1. For all indices, the intervals between 0 and 1 corresponded to: 0 to 0.2 (low); 0.201 to 0.400 (mediumlow); 0.401 to 0.6 (medium); 0.601 to 0.8 (medium-high) and 0.801 to 1 (high).
# Results
The municipalities of the state of Amazonas were found to have medium current vulnerability; the average value of the Vulnerability Index (VInd) was 0.406. It is clear that the VInd was very heterogeneous throughout the state, with the central portion being the least vulnerable (lower VInd), while most of the southern, northern, and eastern regions, including some municipalities of the metropolitan region of Manaus, had higher VInd scores [fig_ref] Fig 5: Map of the current Vulnerability Index [/fig_ref]. The microregions of Purus, Manaus and Boca do Acre possessed the highest (> 0.560) average values of current vulnerability.
The distribution of the exposure, sensitivity, and adaptive capacity indices for each microregion are shown in [fig_ref] Fig 6: Values of the sub-indices that compose the current Vulnerability Index [/fig_ref] The exposure index was the main factor influencing current vulnerability (VInd) in the microregions of Rio Preto da Eva, Itacoatiara, Madeira, and Manaus, when compared to the sensitivity and adaptive capacity indices. In the Rio Preto da Eva, Madeira and Manaus microregions, this finding may be attributed to the greater occurrence and susceptibility to natural disasters, while in the Itacoatiara microregion, the vegetation cover and deforestation index had greater influence (S1 . The sensitivity had the greatest role in increasing the current vulnerability of the microregions of Juruá, Tefé, and Purus, due to the high indices observed for poverty conditions (PoI). Finally, the microregions where adaptive capacity were most important were Alto Solimões, Boca do Acre, Coari, Japurá, Parintins, and Rio Negro, all of which had high vulnerability values for the Sociopolitical Organization Index (SOI) and Socioecomic Structures Index (SEI). [fig_ref] Fig 7: Values of the main indices that compose vulnerability, and each of their... [/fig_ref] the average values of the key components of VInd. The municipalities demonstrated a poor performance in the Adaptive Capacity Index (0.530) followed by the Sensitivity Index (0.465), and by the Exposure Index (0.447). [fig_ref] Fig 7: Values of the main indices that compose vulnerability, and each of their... [/fig_ref] The temperature and precipitation indices for the emission scenario RCP 8.5 were used to develop the CSI, and its average value was considered medium-high (0.630). By disaggregating the components of CSI, both temperature and precipitation anomalies could be seen to have contributed similarly to the final value of the index. However, the value of the precipitation index (0.590) was slightly greater than that of the temperature index (0.581). In general, the climate change projections related to both parameters were not uniform for the state (S1-S6 Figs). Higher anomalies of maximum temperature were concentrated precisely in some regions (northeastern, southwestern) where CSI will be more prominent (S1 A similarity was observed between the distribution of the precipitation extremes (R95p and Rx5day) and the CSI for the northern and northeastern regions (S2 and S3 Figs). There was more homogeneity for the anomalies of minimum temperature, although the increases tended to be less pronounced, with the northeastern region again having the highest average increases for minimum temperature (S4 Regarding CDD, most of the state may show an increase in the number of days when the rain is below 1mm (S5 while total annual precipitation may show a large reduction in the eastern edge of the state and the southern region (S6 The state capital, Manaus, and its metropolitan region, located in the northeastern portion of Amazonas, as well as the northern and southwestern regions, were among the most vulnerable regarding climate [fig_ref] Fig 8: Representation of the Climate Scenario Index for the municipalities of the state... [/fig_ref].
The combination of current vulnerability (VInd) with the climate scenario (CSI) resulted in the Municipal Vulnerability Index (MVI). As demonstrated in the current vulnerability profile for the population of Amazonas (VInd- [fig_ref] Fig 5: Map of the current Vulnerability Index [/fig_ref] may be exacerbated by the climate in a more preponderant way in the northern, northeastern, extreme southern and southwestern regions.
The average value of MVI was 0.586. The municipalities with the highest MVIs were Careiro da Várzea (1.0) and Iranduba (0.893), both located in the Manaus metropolitan area; and Boca do Acre (0.962) and Lábrea (0.877), both located in the southern region. The capital, Manaus, which concentrates half of the inhabitants of the state, presented a medium MVI (0.599) (S2 . the values for the indices VInd and CSI for the most vulnerable municipalities, according to MVI, and for the state capital Manaus. It was noted that the .
# Discussion
## Insights for the state of amazonas
The results of the present study demonstrate that current vulnerability, represented by the VInd, was quite heterogeneous, with the lowest values concentrated in the central region, and increasing vulnerability radiating outward towards the southern extreme, and eastern and northern regions. Some of the microregions identified as the most vulnerable by the VInd (i.e., Purus and Boca do Acre) were similar to those indicated by the Amazon Social Progress Index studyas having the lowest social progress in the state of Amazonas. Although the Amazon Social Progress Index did not consider a monetary dimension in its construction, as was the case with VInd, the overlap of microregions from the both studies is indicative of social and poverty issues as preponderant factors in determining the current vulnerability of the population of Amazonas.
Remarkably, these two aspects were those for which the municipalities of Amazonas had the worst performance, namely, the Poverty Index and the Sociopolitical Organization Index. The poverty issue, understood as deprivation of elements essential to a dignified life that go beyond the monetary issue, was addressed in this study using the Poverty Index, whose average value was the one of the highest among all indices that contributed to VInd. In addition to socioeconomic vulnerability being intrinsically linked to poverty condition, the assessment of this aspect is essential because it acts as a proxy for other issues that may increase the vulnerability of specific groups of a population, indicating, for example, those at higher risk of food insecurity or with limited abilities to adapt to future variability and climate change [bib_ref] Double exposure: assessing the impacts of climate change within the context of..., O'brien [/bib_ref]. In this regard, the Northern Region of Brazil possesses much worse indicators related to child malnutrition, for example, than the other regions of the country, and the state of Amazonas is no exception. In recent decades the state has been facing a scenario of food insecurity, as evidenced by the high prevalence of childhood malnutrition, anemia, and hypovitaminosis [bib_ref] Magnitude da desnutrição infantil no Estado do Amazonas/AM-Brasil, Alencar [/bib_ref] [bib_ref] Alimentary insecurity determinants and consequences at Amazonas: ecosystems influences, Alencar [/bib_ref] [bib_ref] Diagnóstico da realidade nutricional no estado do Amazonas, Brasil. I-Hipovitaminose A, Alencar [/bib_ref].
Another important aspect that deserves to be highlighted is that economic growth has not reflected better living conditions for the population of the State. The watershed of Purus River, located in the southern portion of Amazonas, was the area that experienced the most economic growth in the State between 2002 and 2009 -an average rate of 94% growth in the Gross Domestic Product, with impetus coming mainly from agricultural and livestock activities. However, this same region was among the most sensitive in PoI, probably due to a strong dependency on social assistance; the Bolsa Família benefit (a government aid) that is granted to around 67% of the families in the region [bib_ref] Crescimento econômico e populacional na sub-região da Calha do Purus: uma análise..., Nascimento [/bib_ref].
The other index highlighted in the present study was that reflecting the issue of sociopolitical organization. This aspect is considered to be capable of contributing to satisfying popular demands in a variety of areas that have a direct impact on well-being, health, and social protection, thus enhancing the resilience of territories. Studies point out that different processes of construction and interaction of social capital are capable of increasing the resilience of a population [bib_ref] Social and ecological resilience: are they related?, Adger [/bib_ref] [bib_ref] Social capital, individual responses to heat waves and climate change adaptation: An..., Wolf [/bib_ref]. However, the sociopolitical organization index of the present study demonstrated that the state of Amazonas lacks an organizational structure that allows its population to discuss the problems that affect communities in matters related to climate, environment, housing, and basic sanitation. In general, the presence of councils and consortia related to these and other sectors was observed to be precarious, including even the state capital, Manaus.
In this sense, it is necessary to value the internal capacity that traditional populations possess to organize themselves and to overcome adversities posed by climatic events, which have been characteristically imprinted by the seasonality of the rivers in the riverine way of life. Studying the 1999 flood in the Reserva de Desenvolvimento Sustentável de Mamirauá (Mamirauá Sustainable Development Unit), a protected area along the Solimões River, Moura and Peres [bib_ref] Aspectos demográficos, sócio-econômicos e de saúde da população ribeirinha durante a enchente..., Moura [/bib_ref] observed that the problems faced were solved based on a strong level of solidarity established among the residents of the community to work around the needs unmet by the state. Although only 44% of the existing communities in the reserve and their surroundings received some kind of governmental aid, most of the families (70%) decided to continue living in their residences, which varied according to the proximity of urban centers. These facts show that whatever initiative is aimed at mitigating and reducing the impacts of climate on these populations, it should focus on the sense of belonging observed between traditional populations and their socio-productive space, without which adaptive actions can be undermined for lack of adhesion and recognition of local peculiarities.
The above-mentioned facts demonstrate that the key components of vulnerability-sensitivity and adaptive capacity, which are the components to which the poverty and socio-political organization indices are related-are the most important factors in need of improvement in order to reduce the vulnerability of the Amazonas population to future climate variability and change. Although the Amazon region has experienced increased frequency of extreme events over the last few decades, this was not reflected in higher exposure rates in the present study, possibly because the environmental aspect (vegetation cover index and deforestation) has helped to modulate the effects of disasters in the state.
Natural disasters, included in the exposure component, was found to have a medium value, although other studies have demonstrated the susceptibility of the Amazon system to these phenomena, especially metropolitan areas [bib_ref] The droughts of 1997 and 2005 in Amazonia: floodplain hydrology and its..., Tomasella [/bib_ref] [bib_ref] Future change of temperature and precipitation extremes in South America as derived..., Marengo [/bib_ref] [bib_ref] Avaliação de risco de desastres na Bacia Hidrográfica do Rio Purus (Brasil)..., Pereira [/bib_ref] [bib_ref] Climate change impact on precipitation for the Amazon and La Plata basins, Llopart [/bib_ref] [bib_ref] Management of natural disasters in the Brazilian Amazon region, Szlafsztein [/bib_ref]. The more frequent and intense extreme natural events reported in recent years has resulted in disasters of great magnitude due to the scarcity of safe water and food and the displacement of people throughout the region [bib_ref] The drought of 2010 in the context of historical droughts in the..., Marengo [/bib_ref] [bib_ref] Extreme climatic events in the Amazon basin: climatological and hydrological context of..., Marengo [/bib_ref] [bib_ref] Segregação socioespacial, dinâmica populacional e rede urbana na cidade de Parintins/AM, Marinho [/bib_ref] [bib_ref] Avaliação de risco de desastres na Bacia Hidrográfica do Rio Purus (Brasil)..., Pereira [/bib_ref] [bib_ref] Recent Extremes of Drought and Flooding in Amazonia: Vulnerabilities and Human Adaptation, Marengo [/bib_ref]. A recent study on the vulnerability of Brazilian municipalities to the increase in flood and landslide events showed that the western portion of Amazonas can anticipate increased flood-related vulnerability ranging from 4% to more than 30%, considering the HADGEM-ES climatic model in a pessimistic emission scenario (RCP8.5) [bib_ref] An index of Brazil's vulnerability to expected increases in natural flash flooding..., Debortoli [/bib_ref]. As for landslides, starting from the same scenario and climatic model, the study showed that the entire state of Amazonas, except for small extensions in the extreme north and east, could experience increased vulnerability until the end of the century of between 3% and more of 30%.
Analyzing these data from the perspective of the results found in the Natural Disaster Index of the present study, found that most of the state may, in the near future, suffer doubly the impacts of both flood and landslide disasters, given the position of vulnerability to disasters already present in the southern and eastern portions of the state, which will thus tend to be increased. Pereira et al. [bib_ref] Avaliação de risco de desastres na Bacia Hidrográfica do Rio Purus (Brasil)..., Pereira [/bib_ref] , studying the risk of disasters in the basin of Purus River, observed that the municipality of Boca do Acre was an area of high threat for disasters associated with increased precipitation. Simultaneously, this same municipality, together with Canutama, also proved to be highly threatened by the occurrence of disasters associated with the intense reduction of precipitation. Boca do Acre was also one of the municipalities most vulnerable to the occurrence of disasters according to Natural Disaster Index, revealing that this municipality needs timely preventive actions to be implemented soon in order to avoid material and human damage in the coming decades.
Regarding climate, the CSI showed the northern and northeastern regions and the southwest portion of the state to be more vulnerable. In general, the projections of climate change for the region are inconsistent due to complex thermodynamic ocean-atmosphere interactions and the different results presented by different climate models [bib_ref] Regional climate modeling over south america: A review, Solman [/bib_ref] [bib_ref] Climate projections for South America: RegCM3 driven by HadCM3 and ECHAM5, Reboita [/bib_ref]. Some models have projected rainier climates whereas others have projected drier climates for the Amazon Basin, though, the consensus is of an increase in mean temperature and reduction of rainfall for most of the Amazon territory-the same was found in the present survey by CSI [bib_ref] Socio-climatic hotspots in Brazil, Torres [/bib_ref] [bib_ref] Climate change impact on precipitation for the Amazon and La Plata basins, Llopart [/bib_ref] [bib_ref] Assessment of climate change over South America under RCP 4.5 and 8.5..., Chou [/bib_ref]. The impacts may be severe since climate change might turn extreme events, like the droughts of 2005 and 2010, into the rule more than an exception. The close relationship and dependence of the populations of Amazonas to the hydrological regime has raised concerns about food security, mobility, and water potability, among others. It is noteworthy that the regions of the state where climate change has been most prominent are also those associated with higher disaster rates (southwest and northeastern regions), the poorest social indicators (northern and southwestern) and the highest population density in the state (metropolitan area).
The long-term consequences of maintaining this socio-environmental pattern might be the loss of economic, human, and natural assets.
The climatic issue also poses a challenge to the conservation of the tropical rainforest of the Amazon. Recent estimates suggest that the combination of deforestation and global climate change could cause an increase in the occurrence of forest fires in the Amazon of up to 50% by 2050, creating a cycle of degradation and biodiversity loss [bib_ref] Simulating fire regimes in the Amazon in response to climate change and..., Silvestrini [/bib_ref]. Studies report thresholds that should not be exceeded in order to ensure the maintenance of the Amazon rainforest-up to 40% deforestation and a temperature rise of 3˚C to 4˚C [bib_ref] Regional climate change over eastern Amazonia caused by pasture and soybean cropland..., Sampaio [/bib_ref] [bib_ref] Tipping elements in the Earth's climate system, Lenton [/bib_ref] [bib_ref] Tipping points" for the Amazon forest, Nobre [/bib_ref] [bib_ref] Climate change and thresholds of biome shifts in Amazonia, Salazar [/bib_ref]. In the Amazon, there exists a nonlinear interaction between several drivers that has culminated in the environmental changes that have been observed-the region has warmed 1˚C in the last 60 years and lost about 20% of its original vegetation cover [bib_ref] Land-use and climate change risks in the Amazon and the need of..., Nobre [/bib_ref]. Among these drivers are climate change and land use due to global warming and deforestation, which in turn induce a greater frequency of extreme climatic events and forest fires, increasing the vulnerability of the systems of the Amazon [bib_ref] Land-use and climate change risks in the Amazon and the need of..., Nobre [/bib_ref]. These studies show how vegetation suppression and environmental change interact synergistically in tropical areas, which places the northeast and southern regions of the Amazonas on highest alert. The northeast portion because: 1) it was the one region that possessed many microregions-Manaus, Itacoatiara and Rio Preto da Eva-with the highest values for the indicator of vegetation cover and deforestation; and 2) it could experience the greatest climate change according to the CSI. The southern portion, comprising the microregions of Madeira and Purus, deserves special mention because: 1) according to reports of the Instituto do Homem e Meio Ambiente da Amazônia (IMAZON), it contains a concentration of municipalities in which deforestation increased the most in 2016, in addition to suffering intense pressure from the agricultural frontier, which is expanding from neighboring states; and 2) it is one of the places where climate change may be more prominent, according to the CSI.
Ultimately, the MVI demonstrated the northern, northeastern, and south-southwestern regions of the state to be the most vulnerable to future climate change, considering current socio-environmental conditions. However, the spatial distribution of vulnerability was quite different between MVI and CSI, suggesting that the areas most impacted by climate are not, necessarily, more vulnerable from the socio-environmental perspective. These findings are corroborated by other studies about vulnerability to climate in Brazil [bib_ref] Socio-climatic hotspots in Brazil, Torres [/bib_ref] [bib_ref] Social, Environmental, and Health Vulnerability to Climate Change: The Case of the..., Quintão [/bib_ref]. The microregions of Purus and Boca do Acre, in the south-southwestern region, had similar values for vulnerability and climate indices, whereas other regions, such as Rio Preto da Eva, Itacoatiara, and Manaus (northeastern), showed the climate index to be more important for the definition of MVI. Regarding Manaus, Torres et al. [bib_ref] Socio-climatic hotspots in Brazil, Torres [/bib_ref] identified it as a socio-climatic hotspot, mainly due to the high values observed in the climate index and the low human development of the peripheries, which may translate into greater incidence of diseases and/or more frequent landslides and floods related to high population density.
It is important to highlight that the reduced vulnerability values observed for the municipalities in the central region of the state, as well as for the Madeira microregion, does not mean that these territories do not require actions to enhance their adaptive capacity and resilience. Thus, for microregions that have municipalities in the central parts of the state-Coari, Tefé, Japurá and Juruá -investment is essential to reduce poverty together with the improvement of socio-political articulations, since the climatic aspect was not decisive in determining the vulnerability of these human populations to the climate change.
## Broader implications
Although the conditions that affect people's vulnerability profiles are determined by local interactions, which is reflected in dramatically different responses throughout the world to the same climate stimulus, some conditions are considered "generic" determinants of vulnerability and are closely related to developmental issues (e.g., poverty, governance, health) [bib_ref] The determinants of vulnerability and adaptive capacity at the national level and..., Brooks [/bib_ref]. Interestingly, findings from the present study show that some generic issues, namely poverty (sensitivity index) and sociopolitical organization (adaptive capacity index), as well as the climate scenario, were the main aspects influencing vulnerability of the municipalities of Amazonas (MVI). These generic aspects demonstrate that, although vulnerability assessments are often based on intricate methods of analysis, due in part to the complexity of interactions, shortterm actions to reduce vulnerability in developing countries should focus on improving quality of life and coping skills of the population, along with tackling problems with risk on a localbasis. This aspect is reviewed by Brooks et al [bib_ref] The determinants of vulnerability and adaptive capacity at the national level and..., Brooks [/bib_ref] , who state that addressing issues of health, education and governance would improve adaptation, specific measures and technologies should also be promoted among specific localities or population groups.
The aspects of governance and institutional capacity, for example, are considered a challenge in vulnerability assessments and for the implementation of effective strategies for adaptation at the local level. On the one hand, governance and social capital are difficult to measure because they are highly variable factors among populations, and present structural dimensions that are still barely visible or poorly understood [bib_ref] Unpacking governance: building adaptive capacity to climate change of river basins in..., Engle [/bib_ref]. On the other hand, the most vulnerable population groups are often excluded from decision-making processes, and marginalization itself is a trigger for situations of vulnerability. In Brazil, there is evidence of positive impacts from the participation of civil society in municipal councils and the cooperation between local governments through consortia for provisioning basic services to the population [bib_ref] Do participatory governance institutions matter?: Municipal councils and social housing programs in..., Donaghy [/bib_ref] [bib_ref] Local government cooperation for joint provision: the experiences of Brazil and Spain..., De Mello [/bib_ref] [bib_ref] Participatory publics: civil society and new institutions in democratic Brazil, Wampler [/bib_ref]. On a local-basis, these agencies represent possible channels of communication between the population and the public power, established by the law, which may assist in voice and accountability issues. This shows the key role of those institutions in meeting popular demands and better allocating public resources. Moreover, these findings reiterate that the existence of these institutions is a viable step towards articulating the best policy of the population to the local government. However, there is no doubt that the present method does not allow a thorough evaluation of the quality of the institutional articulation promoted between these bodies and the population, which calls for new ways of thinking about the evaluation of aspects of governance as capable of increasing the adaptive capacity in local contexts.
Regarding climate, the results presented here highlight that the incorporation of CSI substantially rose the vulnerability scores from the VInd to the MVI, showing that the climatic issue needs to be specifically addressed and is of crucial importance in differentiating the relative vulnerability to climate change presented by the municipalities. O'brien et al [bib_ref] Mapping vulnerability to multiple stressors: climate change and globalization in India, O'brien [/bib_ref] have shown that the districts with greater sensitivity to climate in India were not those considered more vulnerable, mainly due to varying levels of adaptive capacity. Using the Socio-climatic Vulnerability Index, Torres et al [bib_ref] Socio-climatic hotspots in Brazil, Torres [/bib_ref] reported similar findings for some metropolitan areas in Brazil. These authors showed that high population densities were an important factor in making cities with moderate expected climate change highly vulnerable to future climate changes.
As demonstrated by Engle & Lemos [bib_ref] Unpacking governance: building adaptive capacity to climate change of river basins in..., Engle [/bib_ref] , from a policy perspective, decision makers are interested in identifying specific characteristics of the system on which they can work. Thus, the analysis of the vulnerability indicators proposed herein, and in other studies, lead to policy recommendations that depend directly on the local perspective [bib_ref] Climate change vulnerability assessments: An evolution of conceptual thinking, Füssel [/bib_ref] [bib_ref] Social, Environmental, and Health Vulnerability to Climate Change: The Case of the..., Quintão [/bib_ref] [bib_ref] Recent Extremes of Drought and Flooding in Amazonia: Vulnerabilities and Human Adaptation, Marengo [/bib_ref]. These relativizations are important to ensuring that decision makers can use the information produced in a relevant and practical way. In the present work, the disaggregation of the indices allows for a detailed analysis to identify specific actions and priority areas for intervention, mainly through the mapping of the results, since stakeholders and decision makers can systematically identify and evaluate the spatial distribution of the likely vulnerabilities. The disaggregation of the indices allows for a detailed analysis to identify specific actions and priority areas for intervention.
For example, the results for the microregion of Boca do Acre showed that it possessed the highest current vulnerability status due to poor performance in adaptive capacity, particularly due to low socio-political organization (S1 . On the other hand, the microregion of Tefé presented the lowest value for the present vulnerability, but with a worse or similar performance in the components of the sensitivity index when compared to the microregion of Boca do Acre, especially regarding the diseases associated with climate index. This possibility of disaggregating the indices and analyzing them individually, either by microregion or by municipality, makes the MVI a useful tool for planning actions in the medium and long term that are adapted to the local reality. In addition, the use of systematic information allows constantly feeding the database and to evaluate the modification of the conditions of vulnerability and/or actions implemented over time.
These remarks show that broad comparisons of the results of different vulnerability studies are a risky task in the face of the profusion of concepts and complexity surrounding the theme, which favors the proliferation of approaches without methodological and/or conceptual uniqueness [bib_ref] What's in a word?, O'brien [/bib_ref]. However, Cannon et aldemonstrated the applicability of a method called the analysis of capacities and vulnerabilities (CVA), which was proposed by a non-governmental institution in 1980. The CVA has since then been absorbed by other methodological approaches, mainly in the Philippines, where it was progressively revised and applied for more than a decade to the theme of disasters. In addition, these authors also showed that although the focus and availability of data in each study using CVA varied greatly, the method was able to assess the vulnerability and capabilities of the populations studied with considerable breadth and depth. Likewise, in Brazil, there are studies applying and improving the same conceptual model of vulnerability proposed, initially, to evaluate the vulnerability of the municipalities of the state of Rio de Janeiro to climate, to other regions of the country, such as the municipalities of the state of Minas Gerais [bib_ref] Social, Environmental, and Health Vulnerability to Climate Change: The Case of the..., Quintão [/bib_ref] and the Paraguay River Basin. Although several methodologies are available to carry out vulnerability assessments (i.e. qualitative diagnostics, future model simulations, statistical analysis), indicators-based approach have been presenting satisfactory results in evaluating the vulnerability of specific sectors (i.e. agriculture) and populations to climate change. In a review of 35 vulnerability case studies carried out across the globe at different scales using the indictors-based approach, Sabellidemonstrated that this kind of evaluation is useful in countries with significantly lack of data, and technical and financial resources to undertake more complex modeling simulations, mainly at small scales, showing the practical application of this approach.
This demonstrates that although the MVI methodology was developed for the Amazon, its adaptation to other regions of Brazil is possible because it encompasses both the main dimensions of vulnerability and the interactions between its components-exposure, sensitivity and adaptive capacity-with the perspective of future climate change. This demonstrate its reproducibility and simple approach comparing to other methods of vulnerability evaluations, allowing to include abstract concepts in a quantitative analysis. Its applicability is possible as long as local conditions are considered, the variables or indicators are adapted to regional peculiarities, and the specificity of scale is respected, which, methodologically, can limit its use.
Thus, it is possible that MVI can be used simultaneously as a diagnostic and planning tool, in the same way that Cannon et aldemonstrated for CVA. As a diagnostic tool, the indices created can help to (i) understand the problems and underlying the causes of vulnerability, and to (ii) highlight priority areas for reducing vulnerability, as well as what actions should be taken (e.g. reducing deforestation, improving social indicators). As a planning tool, the MVI methodology may, in addition to helping prioritize actions, allow the dynamic monitoring of proposed future adaptation actions, since the constructed indices can be systematically updated.
# Conclusions
The results presented here are a pioneering attempt to evaluate the social-environmental and health vulnerability of the population of the state of Amazonas to the impacts of climate change. The major contributions of this study are the development of a set of indicators adapted to the reality of Brazil and the mapping of the vulnerabilities of the municipalities of the state of Amazonas using a climatic perspective. The results have shown that a first step towards making the population of Amazonas better prepared to cope with climate impacts would be the improvement of their life and health conditions, as poverty was one of the factors that most influenced the current vulnerability of the municipalities.
Another important step towards improving adaptive capacity is to invest in the infrastructure of municipalities, such as health care networks and disaster preparedness, since drought and flood events for the Amazon region may become more frequent and intense. Although this does not represent a greater risk of death to the local population, which is already adapted in many ways, a greater severity of climate events implies a threat to food and the nutritional security of the region. The dependency of the smallholder on the river flood regime, which determines not only the appropriate time for farming and the viable crops, but also the flow of goods between the small cities and Manaus, is evidence of how harmful a change in rainfall and hydrological patterns of the Amazon Basin could be.
The methodology applied was developed to be simple and easy to implement so that it could be appropriated by decision-makers at municipal and state levels. The methodology allowed the interpretation of vulnerability in a synthetic manner and the targeting of resources and actions according to regional peculiarities. In the future, this method is likely to be applied to other Amazonian states of Brazil that share the same socio-spatial characteristics as the municipalities of the state of Amazonas. projections. We thank the Instituto Brasileiro de Geografia e Estatística of Manaus for its assistance in obtaining the riverine population data, the Companhia de Recursos Minerais for providing the population at risk data, Pedro and Ricardo from the Universidade Federal do Rio de Janeiro for the CDD modeling data, and Juliana Vasconcelos de Souza Barros for the demographic projections.
## Supporting information
# Author contributions
Conceptualization: Júlia Alves Menezes, Ulisses Confalonieri.
[fig] SSI + Future SSI / 2 •: Current SSI: • Current SSI = (Score of female householder + Score of young householder + Score of the infant population + Score of the elderly + Score of the population with disabilities + Score of the riverine population) / 6 • Future SSI: • Future SSI = (Score of the infant population + Score of the elderly population) / 2 Percentage of female householders with incomplete primary school or no education: percentage of total households in the total population. Indicative of inequities related to gender and economics. [/fig]
[fig] Fig 2: Microregions of the state of Amazonas, Brazil, and the location of the state capital, Manaus. https://doi.org/10.1371/journal.pone.0190808.g002 [/fig]
[fig] Fig 4: Diagram illustrating the steps of calculating all indicators and indices. Steps 1 and 2 comprise transforming the raw variables into indicators. [/fig]
[fig] Fig 5: Map of the current Vulnerability Index (VInd) for the municipalities of the state of Amazonas. https://doi.org/10.1371/journal.pone.0190808.g005 [/fig]
[fig] Fig 6: Values of the sub-indices that compose the current Vulnerability Index (VInd) of the microregions of Amazonas. Distribution of average values of exposure, sensitivity, and adaptive capacity indices for each microregion of the state of Amazonas. https://doi.org/10.1371/journal.pone.0190808.g006 [/fig]
[fig] Fig 7: Values of the main indices that compose vulnerability, and each of their components. (A) Radar chart of the average values of the 62 municipalities in the core indices interact in different ways in determining the final MVI of each municipality and microregion. However, when analyzing the values of vulnerability and climate scenario aggregated by microregion, it is clear that climate change projections represent the component with the greatest influence on the final vulnerability of the municipalities of Amazonas, represented here by MVI [/fig]
[fig] Fig 8: Representation of the Climate Scenario Index for the municipalities of the state of Amazonas, Brazil. https://doi.org/10.1371/journal.pone.0190808.g008Fig 9. Municipal Vulnerability Index (MVI) of the state of Amazonas, Brazil. Representation of distribution of MVI values, considering the IPCC's emission scenario RCP8.5. https://doi.org/10.1371/journal.pone.0190808.g009 Vulnerability to climate change in the Brazilian Amazon Fig 10. Distribution of the current Vulnerability Index, Climate Scenario Index, and Municipal Vulnerability Index. (A) Values of the indices referring to the most vulnerable municipalities and the Amazonas state capital, Manaus, Brazil, according to MVI. (B) Distribution of the indices, in average values, for the microregions of the state of Amazonas, Brazil. [/fig]
[fig] S1: Fig. Representation of the average anomaly of maximum temperature for the state of Amazonas, Brazil. The anomalies (˚C) were calculated considering the current slice as 1961-1990 and the future slice as 2041-2070 from a pessimistic emission scenario, the IPCC's RCP8.5. (TIF) S2 Fig. Representation of the R95p anomaly, which represents extreme precipitation above 95 percentile, for the state of Amazonas, Brazil. The anomaly was calculated considering the percentage difference between the current slice (1961-1990) and the future slice (2041-2070) from a pessimistic emission scenario, the IPCC's RCP8.5. (TIF) S3 Fig. Representation of the Rx5day anomaly, which represents the maximum precipitation accumulated in five days, for the state of Amazonas, Brazil. The anomaly was calculated considering the percentage difference between the current slice (1961-1990) and the future slice (2041-2070) from a pessimistic emission scenario, the IPCC's RCP8.5. (TIF) S4 Fig. Representation of the average anomaly of minimum temperature for the state of Amazonas, Brazil. The anomalies (˚C) were calculated considering the current slice as 1961-1990 and the future slice as 2041-2070 from a pessimistic emission scenario, the IPCC's RCP8.5. (TIF) S5 Fig. Representation of the CDD anomaly-consecutive dry days-for the state of Amazonas, Brazil. The anomaly was calculated considering the percentage difference between the current slice (1961-1990) and the future slice (2041-2070) from a pessimistic emission scenario, the IPCC's RCP8.5. (TIF) S6 Fig. Representation of the total precipitation anomaly for the state of Amazonas, Brazil. The anomaly was calculated considering the percentage difference between the current slice (1961-1990) and the future slice (2041-2070) from a pessimistic emission scenario, the IPCC's RCP8.5. (TIF) Table. Average values of the main indices and sub-indices that composed the Municipal Vulnerability Index for the microregions of the state of Amazonas, Brazil. (DOCX) S2 Table. Values of the main indices and sub-indices, by municipality, that composed the Municipal Vulnerability Index of the state of Amazonas, Brazil. (DOCX) S3 Table. Raw values of the variables used to compose the exposure index of the municipalities of the state of Amazonas, Brazil. (DOCX) S4 Table. Raw values of the variables used to compose the sensitivity index of the municipalities of the state of Amazonas, Brazil. (DOCX) S5 Table. Raw values and information on the variables that composed the Adaptive Capacity Index of the municipalities of the state of Amazonas, Brazil. (DOCX) S6 Table. Raw values of the climatic parameters used to compose the climatic scenario index of the municipalities of the state of Amazonas, Brazil. (DOCX) S7 Table. Percentiles used to assign scores to the quantitative variables. (DOCX) S8 Table. Assignment of values to the qualitative variables used to perform the institutions, services, and Infrastructure for Adaptation Index (AdapI). (DOCX) Appendix. A brief description of the microregions of the state of Amazonas. (DOCX) [/fig]
[fig] Formal analysis: Ana Paula Madureira. Funding acquisition: Ulisses Confalonieri. Investigation: Júlia Alves Menezes, Isabela de Brito Duval, Rhavena Barbosa dos Santos, Carina Margonari. [/fig]
[table] Table 1: Indices that compose the vulnerability, their calculation, short description, and their relationship to the vulnerability. [/table]
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Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase
Benzyl 2-O-(2-O-acetyl-3,4,5-tri-O-benzyl-α-mannopyranosyl)-( 1→2)-(4-cyano-4-deoxy-β-Darabinopyranoside) (17)TfOH (4.4 µL, 0.050 mmol) was added to a mixture of acceptor 16 (125 mg, 0.50 mmol), 2-O-acetyl-3,4,5tri-O-benzyl-α-mannopyranosyl trichloroacetimidate 6 15 (372 mg, 0.625 mmol) and 4 Å mol. sieves in CH 2 Cl 2 at -40 ˚C. The mixture was stirred for 15 min, warmed to 0 ˚C and quenched with Et 3 N (7.0 µL, 5.07 mmol then concentrated under reduced pressure. Flash chromatography (EtOAc/pet. spirits 40:60) gave the disaccharide 17 (168 mg, 46%) as a colourless oil; [α] D 23 -44, (c 0.9, CHCl 3 ); 1 H NMR (500 MHz, CDCl 3 ): δ 1.21 (3 H, s, Ac), 3.17-3.18 (1 H, m, H4), 3.32 (1 H, s, OH), 3.5 (1 H, dd, J 5',6a' = 6.5, J 6a',6b' = 10.5 Hz, H6a'), 3.60 (1 H, dd, J 5',6b' = 1.5, J 6a',6b' = 10 Hz, H6b'), 3.67-3.96 (6 H, m, H2,3',4',5a,5b,5'), 4.18-4.22 (1 H, m, H3),
## Contents
2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→3)-1,2,4,6-tetra-O-acetyl-α,β-D-mannopyranose (11) S11 -(1→3)- 2D SOFAST-HMQC NMR spectra of 15 N-labelled BtGH99 (left) or BxGH99 (right) in the absence (blue) or presence of an excess of ManGlucal (7; red). The arrows highlight the chemical shift perturbation observed for the signal corresponding to Nε-Hε of R291 (BtGH99) and R295 (BxGH99). . Raw NMR spectra for titration curves reported in for binding of binding of ManddMan and ManGlucal to 15 N-labelled BtGH99 and BxGH99.
## Supporting information
## S5
Nε-Hε of R295 (BxGH99) and R291 (BtGH99) were used as binding reporters. Arrows denote the signals used for monitoring the titration.
## Figure s4. isothermal titration calorimetry of mannoe binding to (left) btgh99 and (right)
BxGH99.
## Figure s5. conformational free-energy landscape for protonated noe.
Conformational free-energy landscape contoured at 1 kcal mol -1 . The FEL for protonated NOE broadly exhibits the same topology as for neutral NOE, but differs in relative energies of the global and local minima. For protonated NOE the 1 C 4 conformation is the global minimum, and the 1 S 5 conformation is stabilized relative to that for the neutral NOE FEL. The extracted structures (shown at right) for the 4 C 1 , 1 S 5 and 1 C 4 conformations of protonated NOE that suggest the main reason for these changes are the presence of transannular intramolecular hydrogen bonds between the charged group and the 3-or 6-OH groups in the last two conformers that are absent in the first.
## Figure s6. error surfaces for all inhibitors.
Plots were calculated according to the time-independent free energy estimator described by Tiwary and Parrinello.The energy surface for NOE is for the neutral molecule.
## Table s1. data collection and refinement statistics for bxgh99 complexes e333q -glcchex
## Bxgh99-manddman
## Bxgh99-
## Manddman-1,2α-mannobiose
## Bxgh99-manglucal
## Bxgh99-manglucal-2α-mannobiose
## Bxgh99-mannoe
## Protein expression, purification, and crystallization
Gene expression and protein purification E. coli BL21 (DE3) cells were transformed by plasmids reported previously 2 containing BtGH99 (pETYSBLic), BxGH99 wild-type, E333Q and E336Q (pET28a). Single clones were shaken at 180 rpm and 37 °C in 50 ml tubes (Falcon) containing 5 ml LB media with 50 µg ml -1 kanamycin. Large-scale selective LB cultures in 2 l conical flasks were inoculated 1:1000 with starter cultures and shaken at 180 rpm and 37 °C until the OD 600 was higher than 1, induced using then switched to 16 °C for 24 h. Cell pellets were separated from the media by centrifugation, resuspended in 25 mM HEPES pH 7.0, 300 mM NaCl, 20 mM imidazole and lysed by sonication. The lysate was centrifuged twice and the supernatant was applied to a pre-equilibrated 5 ml HisTrap FF or FF crude column (GE). Protein bound to the column was eluted using imidazole gradient (up to 500 mM). Fractions containing purest protein were combined and concentrated using Amicon Ultra 30 kDa concentrator (Millipore). Further purification was done using a Sepharose S75 16/60 or 16/600 column equilibrated in 25 mM HEPES pH 7.0, 100 mM NaCl, 1 mM DTT, which was also used as the buffer for storage. Fractions containing pure GH99 proteins were combined, concentrated as previously, aliquoted, flash frozen in liquid nitrogen and stored at -80 °C.
## Crystallization and data collection
BxGH99 wild-type and E333Q variant were crystallized in darkness at 19 °C using 24-well plates (Greiner Bio-One) using hanging drop vapour diffusion method. Each 0.5 ml reservoir solution contained 3 M sodium acetate at pH 6.4-7.4. The hanging drop was created by mixing 1 volume of protein solution (30 mg ml -1 ) with 1 volume of reservoir solution. Soaking was performed by adding 0.5-1 droplet volume of 20 mM ligand stock to the droplet with grown BxGH99 crystals. Initial cryoprotection with 20% ethylene glycol before flash freezing in liquid nitrogen was omitted for ManNOE soaks because the diffraction of X-rays by the crystals was not adversely affected. , 90.8 (1 C, C1'), 99.2 (1 C, C1), 168.0-170.8 ppm (8 C, 8 × C=O).
## (2,3,4,6-tetra-o-acetyl-α-d-mannopyranosyl)-(1→3)-(2,4,6-tri-o-acetyl-α-d-mannopyranosyl) bromide (12)
The octaacetate 11 (96.9 mg, 0.142 mmol) was dissolved in 33% w/v HBr/AcOH (0.1 mL, 0.507 mmol) and stirred for 3 h. The mixture was diluted with EtOAc (20 mL), poured into ice water and quickly washed with NaHCO 3 (3 × 10 mL), and brine (2 × 10 mL), then dried (MgSO 4 ) and co-evaporated with toluene under reduced pressure to afford the crude glycosyl bromide 12 as a viscous brown oil, which was used directly in the next step; 1 H NMR (400 MHz, CDCl 3 ), partial spectrum:
## 2,3,4,6-tetra-o-acetyl-α-d-mannopyranosyl-(1→3)-4,6-di-o-acetyl-1,5-anhydro-2-deoxy-d-arabinohex-1-enitol (13)
Activated Zn (46.7 mg, 0.714 mmol), NH 4 Cl (38.2 mg, 0.714 mmol), and VO(salen) 5 (0.478 mg, 7.10 µmol) were added to the crude bromide 12 (99.9 mg, 0.143 mmol) in methanol (1.5 mL) and the mixture was stirred for 30 min. The mixture was filtered through Celite and concentrated to give a light-brown residue that was diluted with EtOAc (20 mL) and washed with water (2 × 20 mL), aq. sat. NaHCO 3 (2 × 10 mL), and brine ( A mixture of the glucal 13 (5.60 mg, 9.99 µmol) and 5% Pd/C (8.5 mg) in methanol (1.5 mL) was stirred under a hydrogen atmosphere for 2 h. The mixture was filtered through Celite and concentrated, followed by acetylation with acetic anhydride (0.5 mL) in pyridine (0.5 mL). Flash chromatography (EtOAc/pet. spirits 30:70) afforded the dideoxy compound 14 (4.50 mg, 80%) as a colourless oil; [α] D 23 +16 (c 0.42, CHCl 3 ); 1 H
[fig] S4, Figure S3 S5, Figure S4 S6, Figure S5 S7, Figure S6: S1. NMR titration curves for binding of ManddMan and ManGlucal to 15 N-labelled BtGH99 and BxGH99. ............................................................................................................................................................ S3 Figure S2. 2D SOFAST-HMQC spectra for binding of ManGlucal to 15 N-labelled BtGH99 and BxGH99. .......... Raw NMR spectra for titration curves reported in Figure S1 for binding of binding of ManddMan and ManGlucal to 15 N-labelled BtGH99 and BxGH99. ...................................................................................... Isothermal titration calorimetry of ManNOE binding to BtGH99 and BxGH99. .............................. Conformational free-energy landscape for protonated NOE. ......................................................... Error surfaces for all inhibitors. ....................................................................................................... S8 Table S1. Data collection and refinement statistics for BxGH99 complexes ................................................... S9 Protein expression, purification, and crystallization ...................................................................................... S10 Synthetic chemistry ........................................................................................................................................ S11 [/fig]
[fig] S24, Figure S1: -O-acetyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-acetyl-α-D-mannopyranosyl) bromide (12).............................................................................................................................................................. S15 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→3)-4,6-di-O-acetyl-1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol (13) ................................................................................................................................................ S16 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→3)-(4,6-di-O-acetyl-1,2-dideoxy-D-mannopyranose (14) .................................................................................................................................................................... S17 α-D-Mannopyranosyl-(1→3)-1,2-dideoxy-D-mannopyranose (ManddMan; 5) ......................................... S18 α-D-Mannopyranosyl-( 1→3)-1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol (ManGlucal; 7) ................... S19 Benzyl 2-O-(2-O-acetyl-3,4,5-tri-O-benzyl-α-mannopyranosyl)-( 1→2)-(4-cyano-4-deoxy-β-Darabinopyranoside) (17) ............................................................................................................................. S20 Benzyl 2-O-(2-O-acetyl-3,4,5-tri-O-benzyl-α-mannopyranosyl)-( 1→2)-(3-O-acetyl-4-cyano-4-deoxy-β-Darabinopyranoside) .................................................................................................................................... S21 Benzyl 2-O-(3,4,5-tri-O-benzyl-α-mannopyranosyl)-(1→2)-(4-cyano-4-deoxy-β-D-arabinopyranoside) (18) .................................................................................................................................................................... S22 Benzyl 2-O-(2-O-acetyl-2,4,5-tri-O-benzyl-α-mannopyranosyl)-( 1→2)-(4-C-[(tertbutoxycarbonyl)amino]methyl-4-deoxy-β-D-arabinopyranoside) (19) ...................................................... S23 α-D-Mannopyranosyl-(1→3)-noeuromycin hydrochloride (ManNOE.HCl; 9) ............................................ NMR titration curves for binding of ManddMan and ManGlucal to 15 N-labelled BtGH99 and BxGH99.Binding of (A) ManddMan to BxGH99, (B) ManGlucal to BxGH99, (C) ManddMan to BtGH99, and (D) ManGlucal to BtGH99. Nε-Hε of R295 (BxGH99) and R291 (BtGH99) were used as binding reporters. Duplicate experimental points at three different ligand concentrations were used for error-bar estimation. [/fig]
[fig] Figure S2: 2D SOFAST-HMQC spectra for binding of ManGlucal to 15 N-labelled BtGH99 and BxGH99. [/fig]
[fig] 2, 3 ,4, 6 -,: Tetra-O-acetyl-α-D-mannopyranosyl-(1→3)-1,2,4,6-tetra-O-acetyl-α,β-D-mannopyranose (11) Acetic anhydride (0.61 mL) was added slowly to a solution of 1,3-α-mannobiose 10 (50.8 mg, 0.148 mmol) and DMAP(18.1 mg, 0.148 mmol) in pyridine (1.22 mL) at 0 °C. The mixture was warmed to rt and stirred for 16 h. The mixture was diluted with EtOAc (20 mL) and washed with water (2 × 20 mL), aq. sat. NaHCO 3 (4 × 10 mL), aq. 1 M HCl (4 × 10 mL), and brine(2 × 10 mL), then dried (MgSO 4 ) and concentrated to afford the crude octaacetate 11 (97.7 mg, 97%; α/β 1.0:0.4) as a pale yellow oil, α anomer; J 3',4' = 10.0 Hz, H3'), 5.23 (1 H, dd, J 1,2 = 2.0, J 2,3 = 3.5 Hz, H2), 5.27 (1 H, t, J 3',4' = J 4',5' = 10.0 Hz, H4'), 5.36 (1 H, t, J 3,4 = J 4,5 = 10.0 Hz, H4), 6.08 ppm (1 H, d, J 1,2 = 2.0 Hz, H1); 13 C NMR (100 MHz, CDCl 3 ) δ 20.6-21.0 (8 C, 8 × Me), 62. [/fig]
[fig] δ 4 60: (1 H, dd, J 1,2 = 12.0, J 2,3 = 4.0 Hz, H2), 5.38 (1 H, t, J 3,4 = J 4,5 = 8.0 Hz, H4), 6.34 ppm (1 H, d, J 1,2 = 1.4 Hz, H1). [/fig]
[table] 2: × 10 mL), then dried (MgSO 4 ) and concentrated to give a pale-yellow residue. Flash chromatography (EtOAc/pet. spirits/Et 3 N 50:49.5:0.5) afforded the protected glucal 13 (18.1 mg, 23% over two steps) as a golden-yellow oil; [α] D dd, J 5,6a = 2.5, J 6a,6b = 12.2 Hz, H6a), 4.18 (1 H, dd, J 5',6a' = 4.0, J 6a',6b' = 12.0 Hz, H6a'), 4.21 (1 H, m, H3), 4.25 (1 H, m, H5), 4.27 (1 H, dd, J 5,6b = 5.0, J 6a,6b = 15.0 Hz, H6b), 4.43 (1 H, dd, J 5',6b' = 6.2, J 6a',6b' = 11.9 Hz, H6b'), 4.87 (1 H, dd, J 1,2 = 6.2, J 2,3 = 3.6 Hz, H2), 5.05 (1 H, d, J 1',2' = 1.7 Hz, H1'), 5.12 (1 H, dd, J 1',2' = 2.8, [/table]
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The Epidermis in Microgravity and Unloading Conditions and Their Effects on Wound Healing
The future objectives of human space flight are changing from low-term permanence in the International Space Station to missions beyond low Earth orbit to explore other planets. This implies that astronauts would remain exposed for long time to a micro-gravity environment with limited medical support available. This has sparkled medical research to investigate how tissues may adapt to such conditions and how wound repair may be influenced. This mini-review is focused on the effects of microgravity and unloading conditions on the epidermis and its keratinocytes. Previous studies, originally aimed at improving the in vitro protocols to generate skin substitutes for plastic surgery purposes, showed that epidermal stem cells cultured in simulated microgravity underwent enhanced proliferation and viability and reduced terminal differentiation than under normal gravity. In the meantime, microgravity also triggered epithelial-mesenchymal transition of keratinocytes, promoting a migratory behavior. The molecular mechanisms, only partially understood, involve mechano-trasduction signals and pathways whereby specific target genes are activated, i.e., those presiding to circadian rhythms, migration, and immune suppression, or inhibited, i.e., those involved in stress responses. However, despite the above in vitro studies suggest that microgravity would accelerate keratinocyte growth rate and migration, in vivo findings on animals in experimental set-ups to simulate low gravity rather suggest that prolonged mechanical unloading contributes to delayed and impaired epidermal repair. This is in keeping with the finding that microgravity interferes at multiple levels with the regulatory signals which coordinate the different cell types involved in the repair process, thereby negatively influencing skin wound healing.
# Introduction
The new millennium has initiated a new chapter of space exploration. The many data gathered upon the unmanned missions to Mars have supported the feasibility of long-range space flights to bring astronauts to explore the closer planets, a perspective that may become something more than a screenplay for a science-fiction movie. To make this possible, many scientific issues have to be considered and managed, especially those related to the physical and mental health of crews exposed for unprecedented long times to the confined environment of a spaceship under low gravity conditions, and limited availability of medical resources. Studies on astronauts who performed enduring missions in the International Space Station (ISS) have allowed to collect numerous medical data on the effects of low gravity on the human body, crucial to identify microgravityinduced diseases [bib_ref] Issues of Health Care under Weightlessness, Sekiguchi [/bib_ref] [bib_ref] Fifty Years of Human Space Travel: Implications for Bone and Calcium Research, Smith [/bib_ref] [bib_ref] Human Biomechanical and Cardiopulmonary Responses to Partial Gravity -A Systematic Review, Richter [/bib_ref] , as well as their pathogenic mechanisms at the cellular and molecular level [bib_ref] The Effects of Weightlessness on the Human Organism and Mammalian Cells, Pietsch [/bib_ref]. The best-known adverse effects of low gravity exposure, yet emerged upon short-term low-Earth-orbit permanence, consist in bone and muscle loss, reduction of cardiovascular capacity, delayed wound and bone fracture healing, and impaired immune function. Over the longterm, exposure to microgravity may impair stem cell-dependent tissue regeneration and homeostasis, adversely affecting bone formation and remodeling, and hemato/lymphopoiesis. In this context, long-term low gravity experiments on amphibians have demonstrated the loss of ability of stem cells of blastemata to regenerate the tail and ocular lens . Another reason for concern is the susceptibility of astronauts to trauma due to peculiar working needs and conditions, since observational data suggest an impaired response to wounding and injury, such as the unusual behavior of hemorrhage, microbiologic flora, and wound healing [bib_ref] Blunt Trauma and Operative Care in Microgravity: a Review of Microgravity Physiology..., Kirkpatrick [/bib_ref]. This has sparkled medical research to investigate how tissues, particularly the skin, may adapt to such conditions, and how wound repair may be influenced. This mini-review will be focused on the effects of microgravity and unloading conditions on the epidermis and its keratinocytes, viewed in the context of their contribution to the wound healing process of the skin.
## Keratinocytes, stem cells and epidermal homeostasis
Keratinocytes are directly involved in several functions of the epidermis relevant for healing of skin wounds: first, they proliferate to maintain epidermal tissue homeostasis and repair tissue losses; second, they produce an array of cytokines and growth factors involved in the autocrine regulation of keratinocyte proliferation, migration, and differentiation, as well as in paracrine effects on stromal, inflammatory and immune cells [bib_ref] Epidermal Stem Cells in Wound Healing and Regeneration, Yang [/bib_ref]. Epidermal cell homeostasis results from the continuing activity of the so-called epidermal proliferative unit (EPU), which encompasses undifferentiated epidermal stem cells (ESCs), transit amplifying (TA) cells and committed keratinocytes which, in normal conditions, evolve in terminally differentiated corneocytes within 20-30 days. Approximately, a single ESC yields by asymmetric mitosis 2 siblings, another ESC and a rapidly dividing TA from which approx. 32 terminal keratinocytes arise. Typically, ESCs are mainly harbored in stem cell niches located in hair follicle bulges, from which they can settle in the basal layers of interfollicular epidermis and sebaceous glands. Interestingly, evidence has emerged that ESCs from the different sites follow their own differentiation paths in normal homeostasis of the epidermis and its annexes, whereas all of them can synergize to give rise to any differentiated epidermal/annexal cell type in response to skin injury [bib_ref] Stem Cells in the Hair Follicle Bulge Contribute to Wound Repair but..., Ito [/bib_ref] [bib_ref] Epidermal Stem Cell Diversity and Quiescence, Watt [/bib_ref]. Obviously, these populations of ESCs and TA keratinocytes are the most susceptible to regulatory signals, including micro-mechanical stimuli: hence, their disruption in altered gravity conditions can have an impact on homeostasis of the epidermis and its ability to respond to injuries.
## Keratinocytes and microgravity: effects and possible mechanisms
How microgravity influences the morpho-functional features of keratinocytes and their contribution to skin wound healing is a poorly explored field. Most cell types are able to respond to mechanical cues that activate specific sensor molecules, chiefly integrin-extracellular matrix (ECM) pairings, intercellular adhesion molecules and junctions, ion channels, α/βcatenins, and cytoskeletal components which transduce them into molecular signals modulating cell morphology, proliferation, differentiation and migration [bib_ref] Cell Mechanics: Integrating Cell Responses to Mechanical Stimuli, Janmey [/bib_ref] [bib_ref] Microgravity and the Implications for Wound Healing, Farahani [/bib_ref]. Therefore, it is conceivable that abnormal micro-mechanical stimuli, as those operating in microgravity, may have an impact on keratinocyte behavior, especially when resting cells are aroused to the dynamic condition needed for wound healing.
Previous in vitro studies, originally aimed at improving the in vitro protocols to generate skin substitutes for plastic surgery purposes, showed that human ESCs cultured in simulated microgravity underwent enhanced proliferation and viability and reduced terminal differentiation as compared with those cultured under normal gravity condition, albeit they retained the capability to form a multi-layered epidermis-like tissue . Later investigations with human immortalized keratinocytes exposed for up to 60 h to simulated microgravity by a random-positioning machine have revealed changes in the expression and periodicity of Bmal1 gene involved in the regulation of circadian rhythm, not accompanied by substantial changes in overall cell morphology, proliferation and apoptosis rates, and at least at the explored experimental times. Concurrently, exposure of human keratinocytes to the same simulated microgravity conditions was shown to trigger epithelial-to-mesenchymal transition (EMT), mediated by over-expression of specific transcription factors and markers, such as Snail1, Snail2, ZEB2, MMPs, and ECM adhesion molecules, as well as by re-arrangement of cytoskeletal components in a pro-motile pattern [bib_ref] Simulated Microgravity Triggers Epithelial Mesenchymal Transition in Human Keratinocytes, Ranieri [/bib_ref]. As will be discussed in more detail in a following section, EMT is a first key step required to promote a migratory behavior of keratinocytes whereby they accelerate re-epithelization during wound healing.
The molecular signals and mechanisms involved in these cellular effects of microgravity are only partially understood. Since epidermal growth factor (EGF) and other molecules of the EGF family, which also include TGF-α and heparin-binding EGF, are major regulators of epithelial cell growth and differentiation and are known to play a pivotal role in reepithelization during the early steps of wound healing [bib_ref] Modifying the Wound Healing Response with Exogenous Growth Factors, Steed [/bib_ref] it appeared conceivable that these could be altered under low gravity conditions. Indeed, exposure of a human squamous cell line to low gravity was shown to decrease the expression of key genes (c-fos, c-jun) downstream EGF activation involved in cell cycle progression, likely through an interference with protein kinase C-dependent signal transduction and actin cytoskeleton [bib_ref] Chapter 7 Effects of Gravity on the Cellular Response to Epidermal Growth..., Rijken [/bib_ref] [bib_ref] Growth Factor-Induced Signal Transduction in Adherent Mammalian Cells Is Sensitive to Gravity, Boonstra [/bib_ref].
In view of a possible translation of the results of in vitro microgravity experiments to space medicine, studies were performed to ascertain whether the microgravity-induced changes of keratinocytes were reversible upon restoration of normal gravity. Of note, human keratinocytes are capable to recover a static epithelial phenotype, as assessed by reestablishment of intercellular adherent junctions and normal cytoskeletal features, mirrored by reduction of their mesenchyme-like migratory phenotype [bib_ref] Microgravity Induces Transient EMT in Human Keratinocytes by Early Down-Regulation of E-Cadherin..., Ricci [/bib_ref]. On the other hand, microarray studies on gene expression patterns by human keratinocytes exposed to microgravity for up to 10 days have shown that the longer the exposure to low gravity, the slower and less complete the return to a normal cell morphology and gene expression profile [bib_ref] Gene Expression Profiling of Human Epidermal Keratinocytes in Simulated Microgravity and Recovery..., Clement [/bib_ref] , In partial agreement with these findings, recent data from cultured endothelial cells suggested that short-term microgravity posttranscriptionally modulated the expression of several genes involved in angiogenesis and vascular patterning [bib_ref] Interactome of miRNAs and Transcriptome of Human Umbilical Cord Endothelial Cells Exposed..., Kasiviswanathan [/bib_ref]. Other studies have shown that cells in cultures recovered from spaceflight did not migrate normally, as occurs during epithelial wound closureand that their cytokine, and growth factor secretion pattern is altered [bib_ref] Feasibility, Potency, and Safety of Growing Human Mesenchymal Stem Cells in Space..., Huang [/bib_ref]. These results suggest that the migratory and paracrine ability of microgravity-exposed precursor cells is impaired and support the hypothesis that the tissue regenerative potential of stem cells, including ESCs, and may be decreased during spaceflight [bib_ref] Stem Cell Health and Tissue Regeneration in Microgravity, Blaber [/bib_ref]. The positive aspect is that chromosomal, DNA damage, and tumorigenicity assays performed upon return of cell cultures to Earth showed no signs of damage which could be related to malignant transformation [bib_ref] Feasibility, Potency, and Safety of Growing Human Mesenchymal Stem Cells in Space..., Huang [/bib_ref].
A major limitation of the above findings consists in the fact that they have been obtained by in vitro cell culture experiments, in which cells are distanced from the complex network of signals they would receive in their physiological tissue environment. This is especially true for skin wound healing, characterized by a functional co-ordination between different player cells which, besides epidermal keratinocytes, also include platelets, inflammatory cells, mesenchymal stem cells, fibroblasts, and endothelial cells [bib_ref] Cellular and Molecular Mechanisms of Repair in Acute and Chronic Wound Healing, Martin [/bib_ref] [bib_ref] Stem Cell Dynamics, Migration and Plasticity during Wound Healing, Dekoninck [/bib_ref]. Nonetheless, the above in vitro data can rise concerns about the adverse effects of long-lasting space missions on epidermal integrity. Additional background to concerns was provided by in vivo studies on skin wound healing using the tailsuspended hindlimb-unloaded rat model to induce functional disuse and intended to simulate microgravity. These studies demonstrated that keratinocyte migration and wound closure were delayed in the mechanically unloaded rats. These effects were accompanied by lower density of dermal microvessels, which also lacked the directional growth toward the epidermis typical of normal skin [bib_ref] Mechanical Unloading Impairs Keratinocyte Migration and Angiogenesis during Cutaneous Wound Healing, Radek [/bib_ref]. These results collectively indicate that both keratinocyte and endothelial cell function are impaired during wound healing in unloading conditions, likely because of alterations of the complex interplay of regulatory signals exchanged between the epidermis and dermis in the skin as a whole. However, due to the extreme complexity in tuning up reliable microgravity animal models, not to mention true spaceflight in vivo experiments [bib_ref] Hindlimb Unloading: Rodent Analog for Microgravity, Globus [/bib_ref] , the scientific data required to better understand how epidermal cells behave during healing of skin wounds in low gravity and the exact mechanisms involved are limited yet.
## Wound healing: the roles of keratinocytes and the effects of microgravity
Wound healing is the process that makes organisms resilient to injuries, allowing survival. Being of fundamental importance for life, it basic pathways and mechanisms have been substantially conserved throughout evolution [bib_ref] Wound Repair and Regeneration: Mechanisms, Signaling, and Translation, Eming [/bib_ref] , although the final outcome diverges from lower vertebrates-like fishes and amphibians-which retain throughout life the embryonic capability to regenerate the missing tissues and organs, and upper vertebrates, which rather heal by reparative scarring [bib_ref] Cellular Plasticity in Vertebrate Regeneration, Odelberg [/bib_ref] [bib_ref] New Insights into the Morphogenic Role of Stromal Cells and Their Relevance..., Bani [/bib_ref]. As mentioned above, wound healing results from an interplay of diverse cells involved and is classically divided into three phases: inflammation, proliferation, and remodeling, whose mechanisms are partially overlapped both spatially and temporally. Detailed analysis of the complex events and molecular mechanisms of wound healing is outside the scope of this mini-review; here, we will limit to recapitulate the key points. After an injury, clotting suddenly takes place due to immediate interactions among endothelial cells and platelets and activation of the coagulation cascade. Mediators released during this early process trigger an inflammatory reaction summoning neutrophils and macrophages from the bloodstream: in turn, these cells produce pro-inflammatory cytokines and growth factors, resulting in recruitment of stromal cells and their differentiation into myofibroblasts, which are responsible for wound contraction and ECM deposition, and stimulation of endothelial and epithelial cell proliferation in the wound site to induce neoangiogenesis and re-epithelialization, respectively. Clot and tissue debris are eventually removed by macrophages and extracellular hydrolases (matrix metalloproteases MMPs, elastase and plasmin) and tissue repair proceeds towards and terminates with scarring [bib_ref] Cutaneous Wound Healing, Singer [/bib_ref] [bib_ref] Wound Repair and Regeneration: Mechanisms, Signaling, and Translation, Eming [/bib_ref] [bib_ref] Cellular and Molecular Mechanisms of Repair in Acute and Chronic Wound Healing, Martin [/bib_ref] [bib_ref] Current Concepts in the Physiology of Adult Wound Healing, Thiruvoth [/bib_ref].
Many factors can impair wound healing. A crucial one is tissue ischemia, which may be caused by primary vascular diseases, diabetes and persistent local pressure. Another adverse factor is persistence of inflammation, as occurs in necrotic and chronically infected wounds and in burns, which leads to inactivation of growth factors and other molecular stimuli required for tissue repair, trapped by ECM molecules or degraded by extracellular proteases . A further factor is persistence of myofibroblast activation, leading to hypertrophic scars, or keloids [bib_ref] Cellular and Molecular Mechanisms of Repair in Acute and Chronic Wound Healing, Martin [/bib_ref] [bib_ref] Keloids and Hypertrophic Scars: Pathophysiology, Classification, and Treatment, Berman [/bib_ref]. Complicated wound healing represent a major public health issue, as it requires complex and lengthy treatments, prolonged hospitalization and an increasing burden on healthcare expenses. These problems become even more challenging when transposed to space medicine, considering the limited therapeutic options available to astronauts during long-lasting space flights.
Keratinocytes play a major role in wound healing since they are activated during the inflammatory phase to secrete several cytokines and growth factors [bib_ref] PERSPECTIVE ARTICLE: Growth Factors and Cytokines in Wound Healing, Barrientos [/bib_ref]. The activated phenotype is marked by changes in the cytoskeleton (i.e., expression of prekeratins K6 and K16) and plasma membrane receptors essential for re-epithelialization, allowing keratinocytes to migrate towards the wound to fill the defect [bib_ref] Towards a Molecular Definition of Keratinocyte Activation after Acute Injury to Stratified..., Coulombe [/bib_ref]. For successful wound healing, keratinocytes should be able to not only detach from the underlying basal lamina but also to move and migrate through fibrin and ECM of the wound, a process facilitated by MMP-1, which is expressed at high levels at the wound edges [bib_ref] Epithelialization in Wound Healing: A Comprehensive Review, Pastar [/bib_ref].
Keratinocyte migration from the free edges of the wound takes place within 24 h. As reported in Keratinocytes, Stem Cells and Epidermal Homeostasis above, the major contribution to the new cells needed to close the wound is given by ESCs located in stem niches of hair follicle bulges, whereas ESCs of the basal layers of the interfollicular epidermis likely play a minor role [bib_ref] Stem Cells in the Hair Follicle Bulge Contribute to Wound Repair but..., Ito [/bib_ref]. Migration is accompanied by morpho-functional changes of the keratinocytes: in resting phase they appear as cuboid-shaped basal cells reciprocally connected by desmosomes and fixed over their basement membrane by hemidesmosomes. A few hours after wounding, keratinocytes become flattened and elongated, lose their cell-cell and cell-matrix junctions, detach cytoskeletal intermediate filaments from the inner aspect of plasma membrane at the junctional level, show a thick network of contractile filaments in the cortical cytoplasm extending into newly formed lamellipodia, all typical features of EMT occurring during embryo development. While keratinocytes are migrating, their proliferative potential is inhibited. Migrating basal cells are thought to express specific surface markers such as CD44, at variance with resting basal cells [bib_ref] Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in..., Lü [/bib_ref] [bib_ref] Epithelialization in Wound Healing: A Comprehensive Review, Pastar [/bib_ref] [bib_ref] How Do Epidermal Matrix Metalloproteinases Support Re-epithelialization during Skin Healing?, Michopoulou [/bib_ref].
The mechanisms of wound re-epithelialization have not been completely unveiled. The most commonly accepted model is the "leap frog" theory, whereby keratinocytes migrate two or three cell lengths from their initial position and slide or roll over the similar cells previously implanted in the wound. In this way, the epidermal border progressively advances and closes the defect. Such movement depend on surface integrins interacting with fibronectin and newly formed collagen molecules in the wound bed. Keratinocytes are also capable to slide under the scab by exploiting the underlying moist environment (this may also explain the success of occlusive dressings in speeding wound healing). Among the stimuli required for re-epithelialization, TGF-α, keratinocyte growth factor (KGF), and EGF have been identified. Migrating keratinocytes can also produce MMPs to remove damaged matrix: of note, keratinocytes secrete MMP-1/ collagenase when in contact with fibrillar collagens of the wound bed, but this secretion is stopped as soon as a new basement membrane is formed and the wound is re-epithelialized. [bib_ref] Proteolytic Events of Wound-Healing -Coordinated Interactions Among Matrix Metalloproteinases (MMPs), Integrins, and..., Steffensen [/bib_ref] [bib_ref] Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in..., Lü [/bib_ref] [bib_ref] Epithelialization in Wound Healing: A Comprehensive Review, Pastar [/bib_ref] [bib_ref] How Do Epidermal Matrix Metalloproteinases Support Re-epithelialization during Skin Healing?, Michopoulou [/bib_ref]. Taken together, these data suggest that keratinocytes require proper micro-mechanical stimuli to activate their wound closure abilities, which may be altered or absent in low gravity conditions. During wound healing, keratinocytes can also modulate the functional activity of stromal cells. In vitro experiments on skinequivalent models show that keratinocyte conditioned medium downregulates the production of the profibrotic cytokines TGF-ß and connective tissue growth factor (CTGF) by dermal fibroblasts. Of note, while normal keratinocytes increase fibroblast proliferation but simultaneously reduce collagen production and increase MMP-1 expression and collagen breakdown, thereby promoting normal wound healing, keratinocytes from keloids show higher, and persistent proliferation rates and induce an abnormal, pro-fibrotic phenotype of dermal fibroblasts [bib_ref] Characterization of In Vitro Reconstructed Human Normotrophic, Hypertrophic, and Keloid Scar Models, Limandjaja [/bib_ref].
# Conclusion
The literature on skin wound healing in weightlessness is relatively poor. In vitro studies on immune cells, fibroblasts, endothelial, and epithelial cells cultured both in real and modeled micro-gravity conditions show alterations of functions involved in wound healing, such as phagocytosis, adhesion/migration, apoptosis, proliferation, intercellular crosstalking, production of inflammatory mediators, ECM molecules, and growth factors, etc. On the other hand, the studies on animal models in unloading conditions are scanty and insufficient to get definite conclusions. In astronauts, impaired immune function, signs of chronic inflammation, metabolic alterations and skin atrophy have been observed, all factors capable to jeopardize the known skin repair mechanisms [bib_ref] Microgravity and the Implications for Wound Healing, Farahani [/bib_ref]. The space biology community is aware that only a few wound healing studies have been performed in a real microgravity environment. To fill this gap, specific experiments have been scheduled for being carried out at the ISS in the next months: these have been designed by an international multi-disciplinary research team with the aim to provide further insight into the effects of real unloading conditions on surgically wounded and sutured human skin tissues [bib_ref] Tissue Repair and Regeneration in Space and on Earth, Monici [/bib_ref]. Despite this uncertain scenario, careful appraisal of the literature suggests a possible general response to microgravity across various cell, tissue, and organ experimental models, consisting of a partial inhibition of the transition of stem cells towards TA cells and terminally differentiated adult cells [bib_ref] Stem Cell Health and Tissue Regeneration in Microgravity, Blaber [/bib_ref]. Thus, gravityrelated micro-mechanical stimulation appears a fundamental need for tissues to maintain their regenerative potential and overall health and emerges as a basic feature of mammalian life on Earth under normal gravity load. This notion also suggests that long-lasting microgravity may result in compromised tissue homeostasis and wound healing capability. For this reason, management of wounds during enduring space missions represents a very challenging issue, especially considering the limited availability of diagnostic and therapeutic tools and, likely, the lack of a specialized on-board medical staff. In low-orbit missions, the management of traumatic and surgical emergencies consists of patient stabilization and rapid return to Earth. In future interplanetary missions, timely medical evacuation to Earth would not be possible, nor would telemedicine by either surgical robots or remote guidance of crew medical actions because of communication lag. Nonetheless, the rapid advancements in robotics allow to foresee that the medic/paramedic crew could be assisted by specifically designed robots, programmed to perform basic medical interventions, such as surgical sutures, anaesthesia and vital signs monitoring, as well as diagnostic procedures by ultrasound, computed tomography scan or magnetic resonance imaging, thus improving the capability of onboard assistance to severely ill or injured astronauts [bib_ref] Robot-assisted Surgery in Space: Pros and Cons. A Review from the Surgeon's..., Pantalone [/bib_ref]. This bio-engineering challenge also underscores the need for further studies on wound healing in space to better understand the problems and define adequate countermeasures.
# Author contributions
Conceptualization, resources, writing, review, and editing: SB and DB.
[fig] FIGURE 1 |: (A) Schematic diagram of epidermal keratinocyte proliferation and migration during wound closure: ESC, epidermal stem cells; TA transit amplifying cells; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix. (B) Wound healing in rat skin: a thin, flattened layer of newly formed epidermis (E) lies over the wound bed and under the clot; the inset shows a detail of an ESC undergoing mitosis (arrowhead). Hematoxylin, picrosirius red, scale bar = 100 μm (courtesy Dr. P. Nardini).Frontiers in Bioengineering and Biotechnology | www.frontiersin.orgMarch 2022 | Volume 10 | Article 666434 [/fig]
[fig] FUNDING: This research was funded by the Italian Space Agency (Wound Healing and Sutures in Unloading Conditions ASI N. 2018-14-U.0). [/fig]
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Evaluating the effectiveness and cost-effectiveness of health facility-based and community-based index-linked HIV testing strategies for children: protocol for the B-GAP study in Zimbabwe
# Abstract
Introduction The number of new paediatric infections per year has declined in sub-Saharan Africa due to preventionof-mother-to-child HIV transmission programmes; many children and adolescents living with HIV remain undiagnosed. In this protocol paper, we describe the methodology for evaluating an index-linked HIV testing approach for children aged 2-18 years in health facility and community settings in Zimbabwe. Methods and analysis Individuals attending for HIV care at selected primary healthcare clinics (PHCs) will be asked if they have any children aged 2-18 years in their households who have not been tested for HIV. Three options for HIV testing for these children will be offered: testing at the PHC; home-based testing performed by community workers; or an oral mucosal HIV test given to the caregiver to test the children at home. All eligible children will be followed-up to ascertain whether HIV testing occurred. For those who did not test, reasons will be determined, and for those who tested, the HIV test result will be recorded. The primary outcome will be uptake of HIV testing. The secondary outcomes will be preferred HIV testing method, HIV yield, prevalence and proportion of those testing positive linking to care and having an undetectable viral load at 12 months. HIV test results will be stratified by sex and age group, and factors associated with uptake of HIV testing and choice of HIV testing method will be investigated. Ethics and dissemination Ethical approval for this study was granted by the Medical Research Council of Zimbabwe, the London School of Hygiene and Tropical Medicine and the Institutional Review Board of the Biomedical Research and Training Institute. Study results will be presented at national policy meetings and national and international research conferences. Results will also be published in international peer-reviewed scientific journals and disseminated to study communities at the end of study.
# Introduction
HIV testing is the critical first step to accessing life-saving antiretroviral therapy (ART).Despite this, many children living with HIV in sub-Saharan Africa, which was home to approximately 92% of the 3.2 million children below 15 years with HIV globally in 2014, remain undiagnosed.Children infected through mother-tochild transmission who are not diagnosed in infancy often remain undiagnosed until strengths and limitations of this study ► Our study will provide evidence for the effectiveness of index-linked HIV testing in facilities and communities. The strategy has the potential to be a cost-effective and efficient strategy for HIV testing in children, given the relatively low prevalence in this age group. ► Our intervention is relevant to policy questions for the implementation of HIV testing for children. This study will provide evidence for use of lower level cadres to offer and perform HIV testing as advocated by the WHO in both rural and urban settings. ► Other HIV testing interventions will be implemented by other stakeholders in the study areas during the study period. This may affect the impact assessment of this study. ► Potential challenges include low uptake of the intervention, lost-to-follow-up of clients once they have agreed to have their child tested and finding clients who have opted for home-based testing. ► Due to relatively low HIV prevalence (<4%), analysis of linkage to care will be limited to a purely descriptive analysis of the proportion of children linking to care and virologically suppressed at 12 months.
Open access they present with symptomatic HIV infection in later childhood.Coverage of early infant diagnosis is suboptimal and therefore a substantial proportion of children infected through mother-to-child transmission are not diagnosed timely in infancy and are only identified in later childhood when they develop advanced disease.In 2017, only 63% of HIV-exposed infants in eastern and southern Africa received an HIV test within 8 weeks of age as recommended.In a study that implemented provider-initiated HIV testing and counselling (PITC) among children aged 6-16 years in primary care clinics in Zimbabwe from 2013 to 2015, the median age of HIV diagnosis was 11 years and those aged >13 years were less likely than younger children to be diagnosed. The high proportion of undiagnosed HIV among children in sub-Saharan Africa, together with low paediatric treatment coverage, continues to be key in sustaining the epidemic.
Barriers to HIV testing of children and adolescents include the need for guardian consent in most countries in sub-Saharan Africa for children younger than 16 years, negative attitudes and lack of training among healthcare workers, stigma and long distances between homes and healthcare facilities where testing is provided, inconvenient opening hours of facilities and long waiting times.A 2011 study from Zambia assessing reasons for non-uptake of HIV testing among children aged below 15 years found that the majority (76%) with confirmed or suspected HIV infection had a primary caregiver who was also living with HIV.A study from Zimbabwe testing children aged 6-16 years conducted in 2014 found that 95% of those who tested HIV positive were perinatally infected. More importantly, 65% had a caregiver or sibling known to be HIV positive or taking ART, and 20% of the accompanying caregivers also tested HIV positive.Similarly, in a Malawian study including patients (15-49 years) on ART at a large ART clinic in 2006-2007, 81% of their children (0-16 years) had reportedly not been tested.These studies show that children living with HIV-infected adults are at high risk for being HIV positive themselves; however, in most instances, these children have not been provided with HIV testing.
The WHO has recommended index-linked HIV testing, whereby household members, sexual contacts or children of a known HIV-infected person are offered an HIV test, as one of the approaches for addressing the gap in HIV testing.Two studies that have implemented such an approach among children in Malawi and Kenya found that more than 40% of adults in HIV care were living in households with children of unknown HIV status and the prevalence of HIV in children tested through this approach was significantly higher than from other 'unselective' HIV testing approaches within Africa. The Bridging the GAP in HIV testing and care for Children in Zimbabwe (B-GAP) study aims to investigate the effectiveness of index-linked HIV testing among children aged 2-18 years in Zimbabwe. Testing will use both facility-based and community-based approaches to address the barriers described above. Notably, as well as offering facility-based and home-based HIV testing for untested children of index adults with HIV, this study will also investigate a novel approach whereby caregivers can test their children using an oral mucosal HIV test. We elaborate on the formative research that informed the approaches to index-linked HIV testing, study design and methodology, analysis and intended study impact. In addition, the study will also evaluate the cost-effectiveness and conduct a process evaluation to inform factors that will influence scalability of this approach in programmatic settings. This study speaks strongly to the WHO recommendations to develop innovative feasible strategies to reduce the burden of undiagnosed HIV among children.
# Methods study objectives
The aim of the study was to evaluate the effectiveness of index-linked HIV testing in identifying children with HIV aged 2-18 years in Zimbabwe. The objectives are to 1. Evaluate the acceptability, uptake and yield of an index-linked HIV testing strategy for children and adolescents in facility-based and community-based settings. 2. Investigate factors associated with uptake of index-linked HIV testing and with choice of a certain method (clinic-based, community-based provider-delivered and community-based caregiver-delivered testing). 3. Investigate linkage to care, retention in care and virological suppression among children living with HIV who are offered community health worker (CHW) delivered support in addition to clinic-based care (the standard of care). 4. Estimate the cost and calculate the cost-effectiveness of index-linked HIV testing for children and adolescents, compared with current standard of care. 5. Conduct a process evaluation of the intervention's implementation, mechanisms of impact and local context to inform the components required for sustainability and scalability.
## Patient and public involvement
Formative research and key considerations for study design Between May and December 2017, we conducted extensive stakeholder engagement and a situational analysis to inform the intervention design with regard to the testing strategy. The specific objectives were first to understand HIV testing pathways within public healthcare facilities and their partnerships with non-governmental stakeholders; to inform selection of study sites and healthcare worker cadres to implement the intervention; and second to elicit key stakeholder views on preferences, potential limitations and foreseeable obstacles for delivery of indexlinked HIV testing. Results were used to inform the intervention design and preparedness for implementation, as well as development of standard operating procedures.
## Open access
Interviews were conducted with Ministry of Health and Child Care (MoHCC) officials and key informants from MoHCC partner organisations that were implementing HIV programmes in Zimbabwe. In total, 25 field observations, 19 site assessments at healthcare facilities across 2 provinces and 53 in-depth interviews (IDIs) with healthcare providers, community-based organisations, adults and adolescents living with HIV and CHWs were undertaken. These findings will be reported in detail elsewhere; however, a summary of how findings informed design is shown in table 1.
Formative work was also conducted to inform the community-based support intervention for children living with HIV delivered by CHW. The research findings, development and design of the community-based support intervention will be presented in a separate manuscript.
## Study sites
Zimbabwe has the sixth highest adult HIV prevalence globally and approximately 1.3 million people were living with HIV in 2017. The study will be conducted in six primary healthcare clinics (PHCs) in Bulawayo (urban) and three PHCs in Mangwe district (rural) in Zimbabwe. In 2016, Bulawayo had the highest adult HIV prevalence (18.7%) in an urban setting, while Mangwe in Matabeleland South Province has the highest national HIV prevalence (22.3%).Clinics were purposively selected based on (1) the size of the facility so that the target sample size could be reached, and (2) their geographical locations to allow good accessibility by the study team in rural sites (figure 1). HIV clinics in hospitals were excluded due to their larger and less well-defined catchment area which would make community-based testing and follow-up difficult.
## Participant recruitment: inclusion and exclusion criteria
All individuals with HIV attending for care at study clinics will be screened daily to identify attendees with children aged 2-18 living in their household (index). If the index attendee is <18 years, they will need to be accompanied by a parent or caregiver aged >18 years to provide consent. Indices who have children with unknown status or children who previously tested HIV negative more than 6 months ago will be offered HIV testing for their children. Being tested more than 6 months ago was included as a criterion for offering testing to account for older children who may have been horizontally infected. If the index consents to having the child or children in the household tested, three testing options will be offered: (1) testing at the clinic by a healthcare provider, (2) home-based testing performed by a healthcare provider or (3) at home by the caregiver using a self-test kit. Study sites were chosen based on an anticipated high prevalence of undiagnosed HIV. Index-linked testing has not been fully implemented due to staff shortage at health facilities.
Research staff were hired to support implementation of intervention in facilities.
HIV testing services are provided by multiple stakeholders across the country with poor coordination between stakeholders. This results in duplication of services and failure to link individuals accessing services through community partners with health facilities.
A collaborative agreement between the research team and the MoHCC and its implementing partners conducting HIV testing in facilities and communities was established. HIV test kits will be provided by the MoHCC and all HIV testing data reported to the MoHCC.
User fees are in place for health service provision at the urban facilities including HIV testing (US$5 and US$3 depending on age of child).
For the purpose of this study, user fees will be dropped for all children and adolescents undergoing HIV testing in selected healthcare facilities Individuals have to travel long distances to access healthcare facilities particularly in rural settings.
A novel HIV testing strategy will be introduced whereby caregivers will be given the option to test their children at home using an HIV self-test kit, thus eliminating the requirement for caregivers to bring children to the healthcare facility for testing.
## Open access
Demographic details of the index and the children will be recorded electronically on tablets by the research assistants using Open Data Kit.Locator details of the household including mobile phone numbers and physical addresses will be collected for all consenting index cases and particularly for community-based HIV testing and to ascertain HIV test outcomes among those who opt to test their children using a self-test kit. All participants will be given a study helpline number to call for further information, counselling referrals or support.
hIV testing procedures HIV testing will be carried out according to national guidelines using a serial testing algorithm.Clinic-based HIV testing Caregivers who opt for clinic-based HIV testing of their children will be given referral cards on the day of screening by a research assistant. HIV testing will be performed by routine clinic staff. Research assistants will use locator information to follow-up participants who have consented to have their child(ren) tested in the clinic but have not presented to the clinic within 7 days of recruitment. They will make up to three attempts to locate the index via telephone or home visit at day 7, 14 and 21 postscreening.
Community-based HIV testing by a healthcare provider Caregivers who opt for community-based testing will indicate a suitable date for HIV testing on the day of screening. A MoHCC implementing partner for community-based testing will visit the household to conduct an HIV test. If no contact is made on the scheduled visit date, the provider will conduct a further two visits to perform HIV testing. A test outcome will be recorded once three attempts have been made within 30 days of screening.
Community-based HIV testing by the caregiver Caregivers who opt to use an HIV oral mucosal test (OMT) to test their children at home will undergo an assessment of guardianship status for each child. Only indices who are parents or legal guardians of eligible children will be eligible to take an OMT kit. Detailed instructions on performing an oral HIV test and interpreting the test result will be given by research assistants in the clinic using an OMT, an instruction pamphlet from the manufacturer translated to local languages and demonstration videos. The research assistant will complete a brief competency assessment including a demonstration of how to perform the test and how to interpret a set of results displayed on pictures by the caregiver. If the caregiver fails the competency assessment, they will be asked to take up either of the other two testing options. Caregivers who successfully complete the competency assessment will be provided with a test kit for each eligible child and will be instructed to conduct the test within 5 days of screening and to keep the used OMT kit. They will be instructed to bring each child who tests OMT reactive for HIV to the clinic for confirmatory HIV testing as recommended by WHO guidelines.Written information about confirmatory HIV testing services will be provided. Caregivers will be followed up to ascertain HIV test outcome either by telephone call or home visit within 7 days and their used/ unused OMT test kits will be collected within 21 days of screening.
The first 10-15 index cases who select caregiver testing in each clinic will undergo supervised caregiver selftesting to evaluate if there are any problems with caregivers' understanding. The parent/guardian will conduct the test while a research assistant is present at the home on the selected testing date and in parallel the research assistant will conduct a rapid blood test as per the national HIV testing algorithm.During the assessment, the research assistant will note any challenges the caregiver had with completing the test and whether or not the caregiver requested assistance from the research assistant. Appropriate changes will be made to the training and demonstration practices if necessary.
## Linkage to care if hiv positive
Children and adolescents will be told their HIV status according to their level of understanding and maturity as described by the national HIV testing and counselling guidelines.All caregivers and children who test HIV positive will receive post-test counselling and a written referral to their nearest healthcare facility for confirmatory HIV testing and linkage to HIV care. For clinic-based testing, referrals will be made to the clinic staff responsible for initiating care on the day of testing. For community-based testing and caregiver testing, written referrals will be made to the nearest/preferred healthcare facility and all participants will be followed up by study staff to ascertain linkage to HIV care. Study staff will share all HIV testing records with the relevant healthcare facility. Caregivers of and children who test HIV positive (or are identified as known HIV positive but not linked to care) will be offered support visits from a CHW at home or at a community-based location of their choice conducted at 1, 3 and 6 months postdiagnosis.
In addition to the above-described testing procedures, partners of index cases will be offered HIV testing. This will not contribute to study outcomes but will be implemented to ensure the intervention complies with current standard of care and WHO recommendations.
## Outcomes
The primary outcome will be uptake of HIV testing, defined as an eligible child having completed an HIV test and the caregiver knowing the test result within 30 days of the HIV test being offered. The HIV test result or reasons why a test is not done will be recorded. Secondary outcomes for the study will include the preferred HIV testing method, HIV yield and prevalence. HIV prevalence and yield will be stratified by age group (0-5, 6-10 and 10-18 years). Additional secondary outcomes will be linkage to HIV care and viral load suppression at 12 months postdiagnosis.
## Open access
Cost-effectiveness analysis The cost-effectiveness analysis aims to determine whether index-linked testing in children is a cost-effective strategy compared with current standard of care in Zimbabwe; passive provider-initiated testing at healthcare facilities. Both the full costs of delivering the intervention and the incremental costs will be estimated using a provider perspective through mixed methods involving a combination of primarily bottom-up micro-costing, with the use of top-down costing when necessary. Costs will be presented as follows: (1) Cost per child tested via each of the indexlinked testing modalities; (2) Cost per HIV-positive child detected through each of the index-linked testing modalities; and (3) cost per new HIV initiate. The base case Cost-Effectiveness Analysis (CEA) will be conducted over a lifetime time horizon, annually discounted at the standard rate of 3%,to reflect future costs and effects at current value, to estimate the incremental cost effectiveness ratio, as cost per disability-adjusted life year (DALY) averted. A dynamic transmission model will be used to estimate the DALYs averted, derived from a combination of accessing treatment and the HIV infections averted, using parameters extracted from the literature. A sensitivity analysis will be conducted to explore the impact of key variables on the results including HIV prevalence and geographical location. As there is much debate and uncertainty surrounding recommended C-E threshold values for different settings, 20 a cost-effectiveness acceptability curve will be constructed. The resulting Incremental Cost-Effectiveness Ratio (ICER) will be compared against different threshold values that are likely to be appropriate to the Zimbabwe setting. This analysis will help inform MoHCC policy around alternate forms of HIV testing and counselling.
## Process evaluation
A detailed mixed-methods process evaluation will be conducted alongside the delivery of the intervention. The process evaluation will be based on the Medical Research Council Process Evaluation Framework and will explore three core evaluation functions: implementation, mechanisms of impact and context to evaluate the delivery and receipt of the intervention, in order to better understand what components of the intervention work, for whom and under what circumstances.The process evaluation data will be analysed and written up in order to contribute to understanding the study results and to provide guidance for sustainable and scalable implementation of the intervention by local health authorities should it prove successful. Quantitative indicators for the process evaluation will include staff turnover, record of shortage of test kits and supplies and incident reports about facility-based and community-based testing from research assistants and the project coordinator. Descriptive statistics of these indicators will be used to help assess aspects of the intervention's fidelity, feasibility and acceptability, and to identify lessons important in informing the replication and scale-up of this approach to testing. Focus group discussions (FGDs) with research assistants and FGDs and IDIs with a purposively selected sample of indices will be conducted to elicit provider and caregiver perceptions and experiences of index-linked HIV testing for children at baseline and end line. Sampling for FGDs will include a group of caregivers representing index age (older vs younger) and sex, HIV testing option, HIV test uptake (accepted vs did not accept testing; and whether child tested or not) and study site (rural vs urban). We will also conduct IDIs with caregivers who had a child test HIV positive through index-linked HIV testing at end line.
As testing by caregivers is a novel intervention, the acceptability of this method will be explored from providers' and caregivers' perspectives. IDIs will be held with caregivers who selected this option to understand how messaging and training for caregiver testing used in the competency and accuracy assessments can be improved, as well as how testing is performed by caregivers on their children in their homes. Interviews will also be held with index participants who did not select his method to understand their concerns.
sample size estimates Sample size estimations are based on precision of estimates of uptake of index-linked HIV testing. We will recruit participants from nine clinics, with an average daily adult attendance of 32 per clinic (excluding repeat attenders). Over 12 weeks per clinic, we expect to screen a total of 5184 HIV-positive index cases at nine clinics with children aged 2-18 years (assuming 5 working days a week and 30% of adults with HIV have children and adolescents living in the household). Assuming an index refusal rate of 35%, and an average of 2 children per household, we would screen approximately 6739 children and adolescents. This sample size provides ±1% precision for an estimate of 80% of screened children and adolescents taking up HIV testing, with a 95% CI of 79.0%-81.0%. Sample sizes for qualitative work will be determined on an ongoing basis by study staff until thematic saturation has been reached.
Assuming HIV prevalence of 4%, this would yield 215 children living with HIV eligible for linkage-to-care. This sample size provides good precision of ±6% around an estimated 80% linking-to-care (n=172) and precision of ±9% around an estimated 70% with viral suppression at 12 months out of 85% retained in care (n=102/146).
# Data analysis
A study flowchart showing total number of index cases screened, eligibility of index cases and children in their household, proportions of index cases providing consent for collection of additional data about themselves and the children living in their households will be presented (figure 2). Demographic characteristics of index participants and children and adolescents living in their households will be summarised using proportions, means and SD. The uptake of HIV testing and HIV prevalence stratified by age group (0-5, 6-10 and 10-18 years) and sex will Open access be calculated, and factors associated with uptake of indexlinked HIV testing will be assessed using univariate analysis. A multivariate logistic regression will be conducted to assess factors predictive of uptake/non-uptake of HIV testing adjusted for clustering by index case. We will evaluate factors associated with the uptake of the preferred HIV testing method using multinomial logistic regression. The proportion of children who tested positive and linked to HIV care and the proportion with virological suppression will be calculated at 12 months.
## Study status
The study began recruitment of study participants in January 2018. Recruitment is ongoing.
# Discussion
There has been a remarkable scale-up of prevention of mother-to-child HIV transmission (PMTCT) programmes resulting in a decline in numbers of perinatally acquired HIV infections.However, many children and adolescents with HIV, acquired before PMTCT programmes were scaled-up, remain undiagnosed, with diagnosis only occurring when they present with advanced disease.In addition, suboptimal coverage of early infant diagnosis within PMTCT programmes results in many HIV-exposed children not being identified. Those who acquire HIV postnatally through breastfeeding may also present later in childhood. HIV testing is the critical step to accessing HIV treatment and high rates of undiagnosed HIV partly explain the lower coverage of HIV treatment in children and adolescents compared with adults.PITC has been recommended by the WHO since 2007.However, this relies on an individual attending a health facility. Also, PITC may not be as cost-effective a strategy in children as in adults given the lower HIV prevalence among children compared with adults. Indexlinked testing is a targeted strategy focusing on testing of children at higher risk of HIV infection. If effective, this strategy has the potential to be a more cost-effective and efficient HIV testing strategy in resource-limited settings. Our study will implement both community-based and health facility-based HIV testing for children and investigate a novel approach of caregivers testing their own children to increase accessibility and uptake of HIV testing.
We believe that community-based strategies in our study have the potential to eliminate many of the barriers to HIV testing for children and adolescents. 14 In a study conducted in Malawi, index-linked testing offered to children and young people (1-24 years) had higher uptake for community (95.5%) when compared with facility-based (4.5%) testing. 14 Enabling caregivers to test their children may reduce the burden of client-associated costs such as time spent in and getting to facilities for testing by clients, provider time, and maintenance and upkeep of health facilities. However, most importantly, it allows the caregivers to perform the test in the comfort and privacy of their homes and empowers them to take responsibility for the testing. Another innovation is the use of CHWs to perform HIV testing in the community. While a similar cadre termed 'primary care counsellor' is widely used to provide HIV testing in health facilities, HIV testing in communities is largely provided by nurses. The CHWs will complete a 2-week training in ethics and rapid HIV testing as well as on study procedures. If effective, our study will provide evidence for use of lower level cadres such as community health workers to offer and perform HIV testing as advocated by the WHO.There is evidence to show that the use of lower level cadres can reduce cost of staffing for the government and may improve cost-effectiveness of community-based approaches when compared with traditional facility-based HIV testing.Furthermore, the study also addresses linkage to care, initiation of ART and adherence through a community-based support intervention. It is crucial that any HIV testing intervention addresses these steps in the continuum of HIV care to ensure maximum benefits (ie, reduced morbidity and onward transmission).
Potential challenges include low uptake of the intervention, lost-to-follow-up of clients once they have agreed to have their child tested and finding clients who have opted for home-based testing. Within our study, user fees for HIV testing were dropped. This has implications for the generalisability of our findings in settings where user fees are in place and may act as a barrier to uptake of HIV testing services. Similarly, our study findings may not be generalisable to other settings as contextual factors may affect uptake of the intervention. It is important to note that in parallel to evaluation of uptake and yield of indexlinked HIV testing, we will conduct a detailed process evaluation to explore the implementation, mechanisms of impact and contextual factors affecting the delivery and acceptability of this intervention, which will help Open access inform sustainability and scale-up should it prove effective. Another limitation is that other HIV testing interventions will be implemented in our study settings at the same time. However, detailed recording of other HIV testing interventions will be part of the process evaluation. This will help with understanding how other HIV testing interventions interact with each other and indexlinked testing.
## Ethics and dissemination
Ethical approval was sought and granted by the Medical Research Council of Zimbabwe (MRCZ), the London School of Hygiene and Tropical Medicine ethics committee and the Institutional Review Board of the Biomedical Research and Training Institute.
Study progress and findings will be reported annually to MRCZ and feedback meetings will be held quarterly with the health directorates of the two study sites. Results of interim data analysis will be presented at national and international research meetings and conferences. Results will also be prepared for publication in international peer-reviewed scientific journals and disseminated to study communities at the end of study. A detailed dissemination plan to inform scale-up will be developed for implementation. Map disclaimer The depiction of boundarieson the map(s) in this article do not imply the expression of any opinionwhatsoever on the part of BMJ (or any member of its group) concerning the legalstatus of any country, territory, jurisdiction or area or of its authorities.The map(s) are provided without any warranty of any kind, either express orimplied.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
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## References
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Infected Leg Hematoma: A Rare Cause of Recurrent Leg Cellulitis
# Introduction
While traumatic muscular hematomas can occur in patients of all demographics, spontaneous muscle hematoma (SMH) is more commonly associated with the elderly population receiving treatment with anticoagulants; it is reported to occur in approximately 5% of such patients with an annual mortality rate of 0.65%. SMH is an uncommon condition that is often overlooked or misdiagnosed, and it is potentially life-threatening, particularly in elderly and frail patients. Several predisposing or contributing factors have been described, and the most frequent ones include minor trauma, increased abdominal pressure, anticoagulation medications, hypertension, and iatrogenic causes. SMH has a significantly increased incidence in the elderly and frail patients receiving treatment with anticoagulants. In the majority of cases, muscle hematomas affect the abdominal rectus sheath or the gluteal muscles. Lower extremities like the calf are an uncommon site of hematoma. Therefore, a high degree of suspicion is always needed when patients present with pain, edema, and ecchymosis in a lower extremity muscle region. Infected spontaneous hematoma of the lower extremities is very rare and currently, there is no data in the literature on this mysterious entity. In this report, we present the case of a chronically ill elderly patient who developed a non-traumatic intramuscular hematoma of the leg muscle associated with apixaban therapy.
## Case presentation
A 64-year-old male with a past medical history of atrial fibrillation on apixaban presented to the ER in a febrile condition complaining of right leg pain with swelling and serosanguineous drainage. He had initially visited the urgent care facility a few weeks ago for the management of leg pain with erythema and had been treated with antibiotics. His symptoms had reoccurred after two weeks, which had led him to present back to the ED due to the recurrent cellulitis associated with fever, chills, and wound abscess.
A review of systems showed fever, drowsiness, genialized weakness, mild confusion, nausea, vomiting, and bilateral lower extremity skin rash. There was no associated joint pain or swelling, dysuria, chest pain, or abdominal pain. Examination showed dry skin with hyperkeratotic plaques with peeling and bilateral lower extremity non-pitting edema. Right medial calf open wound was noted and was draining minimal serous fluid with some erythematous streaking, and granulation tissue .
## Figure 1: a picture of the cellulitis and associated open wound
The patient was admitted on account of recurrent cellulitis for further evaluation. On admission, he had a temperature of 98.1 °F, a heart rate of 102 beats per minute, a blood pressure of 140/61 mmHg, and an elevated respiratory rate (18). Laboratory workup was done, which showed a white blood cell count of 3.26 x 10 9 /L, hemoglobin (Hb) of 10.8 g/dL, c-reactive protein (CRP) of 10.2 mg/L, and procalcitonin of 0.13 ng/ml. The culture of the wound swab showed no growth, and the same was the case with blood culture, acid-fast bacilli (AFB) wound culture, as well as urine culture and reflex culture. Methicillin-resistant Staphylococcus aureus (MRSA) nasal was negative, and so were respiratory panel and C. diff toxin. The liver function test (LFT) was within the normal range. Chest X-ray showed no acute infectious process. Transthoracic echo was negative for obvious vegetations. The patient was empirically started on IV vancomycin and Zosyn. A series of imaging was done to find the source of the infection. CT angiography (CTA) of the chest was ordered and came back negative for pulmonary embolism (PE) or consolidation. CT of the abdomen and pelvis revealed ileus enteritis but no abscess. CT tibia and fibula right with IV contrastshowed findings consistent with right lower extremity cellulitis, with a lentiform-shaped collection situated between the soleus and gastrocnemius muscles, which are most suggestive of a hematoma. An incision and drainage were done to drain the abscess and the hematoma. Repeat culture of blood came out positive for Serratia marcescens resistant to amoxicillin/clavulanate and sensitive to piperacillin/tazobactam. He was started on a four-week course of IV ertapenem and was discharged home with a peripherally inserted central catheter (PICC) line to continue the antibiotics and advised to follow up with outpatient wound care. The patient reported that he was doing well until the discontinuation of ertapenem, following which he had recurrent pain and swelling in the right lower leg accompanied by fever and chills. His condition significantly worsened within a few days. He returned to the ED and was admitted on account of recurrent cellulitis. Laboratory workup showed leukocytosis and blood culture grew Serratia marcescens. A repeat CT of the leg with IV contrast showed a persistent hematoma, similar to the previous CT. The hematoma was suspected as the nidus for bacteremia and ongoing infection. Transesophageal echo showed no vegetation. He was referred to another facility where he had previously received care for incision and drainage of the abscess with some of the hematoma evacuated. During the incision and drainage, a 15-cc sanguinopurulent drainage was drained and culture showed growth for Serratia marcescens. He was continued on IV ertapenem during the hospitalization, with a plan to continue wound care and repeat blood culture to ensure the eradication of bacteremia before discharge. The blood culture came back with no growth and he was discharged home on Bactrim and advised to follow up with cardiology for his anticoagulation.
However, after one month, the patient presented back to the ED with sudden onset of fever and altered mental status with the right lower extremity cellulitis. He was admitted and an extensive workup for the source of bacteremia was done. No evidence of infection was noted. The likely source was assumed to be the intramuscular hematoma in the right lower extremity that measured 5.6 x 3.5 cm on a repeat CT scan. He underwent an incision and drainage of the right leg abscess and subsequent evacuation of the hematoma. The postoperative report showed intramuscular chronic abscess with thick pseudo-capsule and cheese-curdlike material in the location of the hematoma. The culture of the hematoma yielded growth for Serratia marcescens. Postoperatively, he remained hemodynamically stable. He was monitored overnight and subsequently discharged home on IV ertapenem via PICC line for four weeks. The patient was advised to follow up with weekly labs, and a follow-up visit with infectious disease upon completion of antibiotics was also recommended.
# Discussion
The present case shows that recurrent lower extremity cellulitis secondary to infected intramuscular hematoma in elderly patients could be easily missed under a low level of suspicion.
We discussed the case of an elderly patient with lower extremity cellulitis who presented to the ED and was discharged home on antibiotics with a recommendation to revisit the ED upon the onset of any symptoms. The patient developed a non-healing wound within two weeks after hospital discharge, necessitating a re-admission for further evaluation.
This case is the first to report an infected non-expanding intramuscular hematoma with resultant recurrent leg cellulitis following atraumatic leg pain and swelling. In the literature, only a few cases of lower extremity hematoma have been associated with localized infection. However, most cases reported are of chronically expanding hematoma secondary to trauma or anticoagulant therapy.
In addition, we reported the unique finding of intramuscular hematomas, leading to multiple incisions and drainage and recurrent sepsis. Isolated non-expanding hematoma between muscle groups in the leg is a rare occurrence and poses a serious threat to the limb when infected. For a quick diagnosis and to choose the best treatment, a high degree of suspicion is needed especially in patients with acquired coagulopathy. As also reported by Baek and Kim, morbidity due to infected hematoma in the lower extremity can also be avoided if a high level of suspicion is maintained.
Our patient took apixaban for atrial fibrillation prophylaxis, which may have contributed to his hematoma formation in the lower extremity. On the culture of the hematoma, after incision and drainage in the OR with subsequent hematoma evacuation, a growth of Serratia marcescens was noticed. This organism is a gram-negative bacillus that occurs naturally in soil and water and produces a red pigment at room temperature. It is associated with urinary and respiratory infections, endocarditis, osteomyelitis, septicemia, wound infections, eye infections, and meningitis. However, it is not one of the most common causes of hematoma infections.
Our case illustrated the difficulties in diagnosing and treating recurrent cellulitis in a geriatric patient. Although our patient eventually started recuperating well, it is important to realize that an infected intramuscular hematoma is a potential cause of recurrent sepsis. Therefore, we recommend having a high level of suspicion when an older patient on anticoagulant presents to the ED with non-healing cellulitis, followed by recurrent sepsis. It is pertinent to always consider an infected intramuscular hematoma especially in a patient on anticoagulation therapy.
# Conclusions
We discussed a rare case of recurrent cellulitis due to an infected intramuscular hematoma. This report highlights the importance of including suspected hematoma on the differential list, especially in elderly patients on anticoagulation. Physicians need to be vigilant of atypical presentation of infected hematoma and consider a multidisciplinary approach in any patient having recurrent cellulitis and bacteremia of an unknown source.
# Additional information disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. |
ROCK2 Inhibition With Belumosudil (KD025) for the Treatment of Chronic Graft-Versus-Host Disease
PURPOSE The rho-associated coiled-coil-containing protein kinase-2 (ROCK2) signaling pathway regulates the Th17/regulatory T cells balance and controls profibrotic pathways. Selective ROCK2 inhibition with belumosudil (KD025) may offer a novel approach to the management of chronic graft-versus-host disease (cGVHD).PATIENTS AND METHODS A phaseIIa, open-label, dose-finding study of belumosudil enrolled 54 patients with cGVHD who had received one to three prior lines of therapy (LOTs). The primary end point was overall response rate (ORR).RESULTSThe median time from cGVHD diagnosis to enrollment was 20 months. Seventy-eight percent of patients had severe cGVHD, 50% had $ 4 organs involved, 73% had cGVHD refractory to their last LOT, and 50% had received $ 3 prior LOTs. With an overall median follow-up of 29 months, the ORR (95% CI) with belumosudil 200 mg once daily, 200 mg twice daily, and 400 mg once daily was 65% (38% to 86%), 69% (41% to 89%), and 62% (38% to 82%), respectively. Responses were clinically meaningful, with a median duration of response of 35 weeks, and were associated with quality-of-life improvements and corticosteroid (CS) dose reductions. CS treatment was discontinued in 19% of patients. The failure-free survival rate was 76% (62% to 85%) and 47% (33% to 60%) at 6 and 12 months, respectively. The 2-year overall survival rate was 82% (69% to 90%). Belumosudil was well-tolerated, with low rates of cytopenia. There were no unexpected adverse events and no apparent increased risk of infection, including cytomegalovirus infection and reactivation.CONCLUSION Belumosudil treatment resulted in a high ORR and overall survival rate and demonstrated qualityof-life improvements, CS dose reductions, and limited toxicity. Data from the study indicated that belumosudil may prove to be an effective therapy for patients with treatment-refractory cGVHD.
# Introduction
Chronic graft-versus-host disease (cGVHD) is an immune-mediated inflammatory and fibrotic disorder. [bib_ref] Chronic graft-versus-host disease: Biological insights from preclinical and clinical studies, Macdonald [/bib_ref] It is a leading cause of morbidity, 2 mortality, [bib_ref] Late acute and chronic graft-versus-host disease after allogeneic hematopoietic cell transplantation, Arora [/bib_ref] [bib_ref] Long-term survival and late deaths after allogeneic hematopoietic cell transplantation, Wingard [/bib_ref] and impaired quality of life (QOL) [bib_ref] Long-term recovery after hematopoietic cell transplantation: Predictors of quality-of-life concerns, Wong [/bib_ref] after an allogeneic hematopoietic cell transplant (alloHCT). [bib_ref] Late acute and chronic graft-versus-host disease after allogeneic hematopoietic cell transplantation, Arora [/bib_ref] [bib_ref] Long-term survival and late deaths after allogeneic hematopoietic cell transplantation, Wingard [/bib_ref] [bib_ref] Long-term recovery after hematopoietic cell transplantation: Predictors of quality-of-life concerns, Wong [/bib_ref] cGVHD affects up to 70% of all alloHCT recipients, 2,5-10 with an incidence of 20%-50% in children, [bib_ref] Chronic graft versus host disease (GVHD) in children, Baird [/bib_ref] who survive more than 100 days after alloHCT. [bib_ref] Chronic graft-versus-host disease, Lee [/bib_ref] It is the leading cause of nonrelapse mortality beyond 2 years after alloHCT. [bib_ref] Chronic graft-versus-host disease, Lee [/bib_ref] The estimated prevalence of cGVHD is 14,000 patients in the United States (as of 2016).Of those patients, approximately 40% have severe disease. [bib_ref] Success of immunosuppressive treatments in patients with chronic graft-versus-host disease, Lee [/bib_ref] [bib_ref] National Institutes of Health classification for chronic graft-versus-host disease predicts outcome of..., Saillard [/bib_ref] In addition, 42% of patients have $ 4 organs involved at the time of diagnosis.Patients with cGVHD have substantial impairment in QOL, [bib_ref] Long-term recovery after hematopoietic cell transplantation: Predictors of quality-of-life concerns, Wong [/bib_ref] [bib_ref] Patient-reported quality of life is associated with severity of chronic graft-versus-host disease..., Pidala [/bib_ref] [bib_ref] Development and validation of a scale to measure symptoms of chronic graft-versus-host..., Lee [/bib_ref] as assessed by the Lee Symptom Scale (LSS), which measures the effect of cGVHD on patients' functioning and well-being. [bib_ref] Development and validation of a scale to measure symptoms of chronic graft-versus-host..., Lee [/bib_ref] Only one third of patients who have cGVHD and start systemic treatment will be alive, in remission and off immunosuppressive therapy by 5 years. [bib_ref] Success of immunosuppressive treatments in patients with chronic graft-versus-host disease, Lee [/bib_ref] The pathophysiology of cGVHD can be separated into three phases: early inflammation because of tissue injury, a dysregulated adaptive immune system, and chronic inflammation and aberrant tissue repair with fibrosis. [bib_ref] Pathophysiology of chronic graft-versus-host disease and therapeutic targets, Zeiser [/bib_ref] First-line therapy for National Institutes of Health (NIH)-defined moderate to severe cGVHD is corticosteroids (CSs) alone or in combination with sirolimus or a calcineurin inhibitor. [bib_ref] A phase II/III randomized, multicenter trial of prednisone/sirolimus versus prednisone/sirolimus/calcineurin inhibitor for..., Carpenter [/bib_ref] However, up to 70% of patients require additional lines of therapy (LOTs). [bib_ref] Success of immunosuppressive treatments in patients with chronic graft-versus-host disease, Lee [/bib_ref] [bib_ref] How we treat chronic graft-versus-host disease, Flowers [/bib_ref] Furthermore, the long-term use of CS is associated with significant side effects. [bib_ref] How we treat chronic graft-versus-host disease, Flowers [/bib_ref] [bib_ref] Ancillary therapy and supportive care of chronic graft-versus-host disease: National Institutes of..., Couriel [/bib_ref] Management of cGVHD continues to evolve with the advent of targeted therapies. In 2017, the US Food and Drug Administration approved ibrutinib, a Bruton's Tyr kinase inhibitor, for the treatment of adults with cGVHD after failure of $ 1 systemic LOTs.In patients with cGVHD who were required to have either . 25% body surface area erythematous rash or an NIH mouth score of . 4, 21 a study with ibrutinib reported an overall response rate (ORR) of 67% and a discontinuation rate because of treatment-emergent adverse events (TEAEs) of 43%. [bib_ref] Ibrutinib for chronic graft-versus-host disease after failure of prior therapy: 1-Year update..., Waller [/bib_ref] There remains an opportunity to study other treatment options for patients who have failed $ 1 LOTs.
Belumosudil is an oral selective rho-associated coiled-coilcontaining protein kinase-2 (ROCK2) inhibitor. ROCK2 has been shown to be activated in a Th17-skewed milieu, leading to the upregulation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and the consequent increased expression of Th17-specific transcription factors, such as RAR-related orphan receptor and interferon regulatory factor 4. [bib_ref] Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T..., Zanin-Zhorov [/bib_ref] Moreover, selective ROCK2 inhibition restores immune homeostasis and shifts the Th17/regulatory T cells (Tregs) balance toward Tregs via an STAT5-dependent mechanism. [bib_ref] Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T..., Zanin-Zhorov [/bib_ref] [bib_ref] Targeted rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through..., Flynn [/bib_ref] Belumosudil demonstrated a significant reduction of lung and skin fibrosis in animal models of bronchiolitis obliterans syndrome and sclerodermatous cGVHD, respectively, 24 which was consistent with the central role of ROCK in facilitating multiple fibrotic pathways. [bib_ref] Promising therapeutic targets to ameliorate pulmonary fibrosis, Riches [/bib_ref] Based on the biologic rationale and compelling preclinical data, a phase IIa study was conducted to evaluate the safety and efficacy of belumosudil in patients with cGVHD.
# Patients and methods
## Patient eligibility
This phase IIa, dose-finding, open-label study was conducted at seven centers in the United States. Eligible patients were allogeneic bone marrow transplant or alloHCT recipients of age $ 18 years with persistent cGVHD manifestations after having received one to three prior systemic LOTs and who were receiving CS treatment with or without a calcineurin inhibitor and/or concurrent extracorporeal photopheresis. Belumosudil was continued until cGVHD progression or unacceptable toxicity.
## Study design and treatment
Patients were enrolled into three sequential cohorts: cohort one received belumosudil 200 mg once daily, cohort two received belumosudil 200 mg twice daily (twice a day), and cohort three received belumosudil 400 mg once daily. Sixteen patients were planned for each of the cohorts. Before enrollment of the subsequent cohort, safety data in each previous cohort were analyzed after eight patients reached 2 months of treatment to assure that there was no safety signal. The 2-month timeframe was selected because all clinically significant belumosudil-related adverse events (AEs) to date had occurred in # 36 days of starting belumosudil. No safety concerns were identified, allowing for planned dose escalation.
Belumosudil was administered orally in 28-day cycles until disease progression or unacceptable toxicity. Progression was defined per the 2014 NIH cGVHD Consensus Criteria. [bib_ref] National Institutes of Health consensus development project on criteria for clinical trials..., Jagasia [/bib_ref] Long-term follow-up was conducted via telephone every 8 weeks until study closeout. After 4 weeks of belumosudil therapy, CS could be tapered at the investigators' discretion.
Screening was conducted within 28 days of the first study dose. Response was initially assessed after two cycles; however, this was amended to evaluate response on day 1 of each cycle, starting at cycle 2 day 1.
The study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The institutional review board/independent ethics committee at each center approved the study. All patients provided written informed consent.
The study was registered with ClinicalTrials.gov identifier (NCT02841995) and sponsored by Kadmon Corporation, LLC. All authors reviewed and approved the submitted manuscript.
## Study end points
The primary efficacy end point was ORR, defined as the proportion of patients who achieved either a complete response (CR) or partial response (PR), per the 2014 NIH cGVHD Consensus Criteria, at any time point. [bib_ref] National Institutes of Health consensus development project on criteria for clinical trials..., Jagasia [/bib_ref] Only response assessments before the next LOT after belumosudil were counted toward ORR. All responses were assessed by the investigators.
Secondary end points included the number and the percentage of patients with steroid-dependent cGVHD who had a best response of PR or CR, duration of response (DOR), response rate by organ system, LSS score, CS dose reductions, time to next treatment (TTNT), failure-free survival (FFS), and overall survival (OS). The safety and tolerability of belumosudil were evaluated via AE assessments, physical examinations, vital sign measurements, laboratory tests, and electrocardiograms throughout the study. Predose samples were collected for pharmacodynamic (PD) evaluation, which included the assessment of immune cell subtypes in peripheral blood.
# Statistical analysis
With a sample size of 16 patients per cohort, the study had a . 90% probability of $ 1 study participants experiencing an AE with an underlying rate of $ 14%, which was derived from the probability calculations of the assumed sample size. Assuming a best ORR of 25%, which was determined to be clinically meaningful, the study was expected to have approximately 90% probability to show a response in $ 2 patients per cohort. This study was not powered to show significant differences between cohorts with respect to efficacy, AEs, or PD analyses. The primary analysis was conducted using the safety population, defined as enrolled patients who received $ 1 dose of study medication. The Clopper-Pearson (exact) method was used to construct the two-sided 95% CI for ORR. The Kaplan-Meier (K-M) method was used to calculate estimates of FFS and OS.
# Results
## Patients
Fifty-four patients in total were enrolled in sequential cohorts between September 2016 and March 2018: 17 patients in cohort 1, 16 patients in cohort 2, and 21 patients in cohort 3. As of the data cutoff for this analysis, February 19, 2020, the median duration of follow-up was 36 months in cohort 1, 32 months in cohort 2, and 24 months in cohort 3. The overall median duration of follow-up was 29 months (range, 1-39 months).
Demographics and baseline characteristics were overall comparable across cohorts [fig_ref] TABLE 1: Baseline Demographics and Clinical Characteristics [/fig_ref] , online only). The median age at baseline was 52 years (range, 20-75 years). The median time from cGVHD diagnosis to treatment was longest in cohort 1 at 26 months (compared with 18 months and 16 months in cohorts 2 and 3, respectively). Seventy-eight percent of patients had severe cGVHD per investigator assessment. Half of the patients had involvement of $ 4 organs, and more patients in cohort 3 had lung involvement (48%) compared with those in cohorts 1 (24%) and 2 (19%). The baseline median CS dose (mg/kg/d prednisone equivalent) was 0.22, 0.19, and 0.17 across cohorts, respectively. Patients in cohort 1 had received a median of three prior LOTs, whereas patients in cohorts 2 and 3 had received a median of two prior LOTs. Seventy-three percent (35 of 48, data not available for six patients) of patients were refractory to their last LOT before study enrollment.
The CONSORT diagram [fig_ref] FIG 1: CONSORT diagram [/fig_ref] shows patient disposition. The median duration of treatment was 8.5 months (range, 2-39 months) in cohort 1, 7.5 months (range, 1-35 months) in cohort 2, and 9 months (range, 1-29 months) in cohort 3. Twenty-eight percent of patients have received . 18 months of belumosudil.
Seven patients (13%) remained on belumosudil at the time of this analysis. Reasons for discontinuing belumosudil included cGVHD progression (n 5 22), voluntary withdrawal by patients (n 5 8), relapse of underlying disease (n 5 7), investigator decision (n 5 3), AEs considered to be possibly treatment related (n 5 3), and death (n 5 2).
## Efficacy
Overall response rate. In the safety population (N 5 54), the ORR (95% CI) was 65% (51% to 77%). The ORR (95% CI) was similar across cohorts: 65% (38% to 86%) in cohort 1, 69% (41% to 89%) in cohort 2, and 62% (38% to 82%) in cohort 3 [fig_ref] TABLE 2: Efficacy and CS Reduction Abbreviations [/fig_ref]. Efficacy data for subgroups and secondary end points are presented as pooled data across cohorts.
Responses were achieved across key subgroups, with ORRs of 60% (25 of 42) in patients with severe cGVHD, 66% (23 of 35) in patients who had received $ 2 prior Disease severity was determined using the Physician-reported Global cGVHD Activity Assessment Form.
## Journal of clinical oncology
systemic LOTs, 63% (22 of 35) in patients who were refractory to their last LOT before enrollment, and 70% in patients with $ 4 organs involved [fig_ref] FIG 2: Forest plot for subgroup analyses of ORR in the safety population [/fig_ref]. All responses at the patient level were PR; however, organspecific analyses showed that CR was achieved across all affected organs, with the exception of the lungs, where PR was the best response achieved (Appendix Figs A1 and A2, online only).
Responses were generally rapid, with . 75% of all responses achieved by the first response assessment at week 8 (Appendix [fig_ref] FIG 3: Durability of response to belumosudil in patients with cGVHD [/fig_ref]. Four of 35 responses occurred after 24 weeks of belumosudil treatment, with late organ responses observed in the lungs, joints and/or fascia, and eyes (Appendix . Among responders, the K-M median DOR across cohorts was 35 weeks [fig_ref] FIG 3: Durability of response to belumosudil in patients with cGVHD [/fig_ref]. The K-M median DOR was 38 weeks for patients who had received $ 2 prior systemic LOTs.
Time to next treatment. The K-M median TTNT was 14 months [fig_ref] FIG 3: Durability of response to belumosudil in patients with cGVHD [/fig_ref]. Subsequent systemic cGVHD therapies included tacrolimus, sirolimus, ibrutinib, ruxolitinib, extracorporeal photopheresis, and mycophenolate mofetil.
FFS and OS. The FFS rate (95% CI) was 76% (62% to 85%), 47% (33% to 60%), and 33% (21% to 46%) at 6, 12, and 24 months, respectively [fig_ref] FIG 3: Durability of response to belumosudil in patients with cGVHD [/fig_ref]. FFS was defined as the time from the first dose of belumosudil to a failure event. [bib_ref] Failure-free survival after initial systemic treatment of chronic graft-versus-host disease, Inamoto [/bib_ref] Reasons for failure included initiation of a new systemic therapy (n 5 27), relapse of the underlying disease (n 5 7), and death (n 5 2). An important end point is the percentage of patients achieving FFS with response (CR/PR) at 12 months, [bib_ref] An endpoint associated with clinical benefit after initial treatment for chronic graft-versus-host..., Martin [/bib_ref] which was 24% in this study. The OS rate (95% CI) was 91% (79% to 96%) and 82% (69% to 90%) at 12 and 24 months, respectively [fig_ref] FIG 3: Durability of response to belumosudil in patients with cGVHD [/fig_ref].
QOL assessment. Clinically meaningful improvement in LSS score, defined as a decrease of $ 7 points in the LSS summary score, [bib_ref] Development and validation of a scale to measure symptoms of chronic graft-versus-host..., Lee [/bib_ref] during belumosudil treatment was observed in 50% of patients. Thirty-five percent of all patients (37% of responders and 32% of nonresponders) reported a Changes in cGVHD symptom burden were measured by the LSS. Clinically meaningful improvement in symptom burden was defined as a decrease of at least seven points in LSS summary score. clinically meaningful improvement in LSS score on consecutive assessments.
## Orr (%)
CS sparing. During belumosudil treatment, 67% of patients reduced CS dose and 19% completely discontinued CS. The mean CS dose was reduced by 45%. The median time to CS discontinuation was 29 weeks (range, 8-77 weeks). The mean CS dose reduction was 55% in responders and 26% in nonresponders [fig_ref] TABLE 2: Efficacy and CS Reduction Abbreviations [/fig_ref].
## Safety
Belumosudil was well-tolerated, with . 56 patient-years of belumosudil exposure. The median relative dose intensity was 98% overall. The percentage of patients with a relative dose intensity . 95% was 77%, 63%, and 71% across cohorts, respectively. Dose reductions occurred in 9% of patients, and the median duration of reduction was 97 days (range, 21-859 days). Dose interruptions occurred in 41% of patients, and the median duration of interruption was 10 days (range, 2-39 days).
AEs were consistent with those expected in a population of patients with advanced cGVHD receiving CS. AEs reported in $ 20% of patients were upper respiratory infection (46%), diarrhea (33%), fatigue (33%), nausea (33%), increased liver function tests (33%), dyspnea (30%), headache (24%), peripheral edema (24%), cough (22%), and hypertension (20%) [fig_ref] TABLE 3: Safety Overview [/fig_ref]. Serious AEs were reported in 43% of patients, and serious AEs reported in . 1 patient were dyspnea (7%), lung infection (6%), hypoxia (4%), and influenza-like illness (4%). Sixty-one percent of patients had a grade $ 3 AE, with the most common being dyspnea (13%), increased liver function tests (7%), hyperglycemia (7%), and hypoxia (7%) [fig_ref] TABLE 3: Safety Overview [/fig_ref].
Grade $ 3 cytopenias were reported in two patients (4%). These occurred at relapse of underlying malignancy in patients who had otherwise maintained normal blood counts during their belumosudil treatment.
No cases of cytomegalovirus (CMV) infection or reactivation were reported with belumosudil.
Three patients discontinued belumosudil because of potentially drug-related AEs (cohort 1: diarrhea and headache; cohort 3: fatigue). Four patients, all in cohort 3, died during the study-secondary to relapse of leukemia, pneumonia (unknown pathogen), cardiac arrest, and cGVHD progression-with none of the deaths attributed to belumosudil. There was no dose response with respect to the observed AEs.
## Pd analyses
In exploratory PD analyses of peripheral blood mononuclear cells across cohorts, the percentage of CD4 1 Tregs demonstrated an increasing trend early on by cycle 2 day 1 of belumosudil treatment. A simultaneous decrease in Th17 cells was also observed. The Th17 cells continued to decrease through C4D1 and C6D25. The percentage of CD4 1 Tregs continued to increase through C4D1 and C7D1, as shown in . Because of the small sample size, correlative data with steroid dose were limited for any statistical analysis.
# Discussion
This study was the first to evaluate belumosudil treatment in patients with cGVHD. All phenotypes of cGVHD, without requirements for inflammatory or fibrotic manifestations, were included. Patients with advanced multiorgan cGVHD treated with belumosudil achieved an ORR of 65%, with QOL improvements, CS dose reductions, and limited toxicity. With relatively small sample sizes, there was no difference in the ORR across cohorts.
Belumosudil achieved response rates that were meaningful and consistent across subgroups, including patients with severe cGVHD, patients who had received $ 2 prior systemic LOTs, patients who were refractory to their last LOT before enrollment, and patients with $ 4 organs involved. The ORR among patients with nonsevere cGVHD was 83%, suggesting that further studies of how belumosudil may benefit patients earlier in their disease are indicated. All responses at the patient level were PR; no CR was achieved. However, given the severity and extent of fibrotic cGVHD manifestations in this patient population, achieving CR in all organs was not expected, as some advanced fibrotic changes in the eyes, mouth, lungs, or joints and/or fascia can be irreversible. CR was observed in all organs except the lungs, where PR was achieved. Belumosudil response kinetics suggest that most responders achieved responses rapidly within 8 weeks after receiving belumosudil.
Belumosudil was well-tolerated, with a median DOR of 35 weeks across all responders. The ability to stay on therapy is dependent on the safety and long-term tolerability profile of the intervention. The median treatment duration was 8 months (range, 1-39 months). Twenty-eight percent of patients remained on belumosudil for . 18 months, with 11 patients remaining on treatment at the time of this analysis. There was no reported CMV infection or reactivation, despite 57% of patients being CMV seropositive. The incidence of TEAEs and grade $ 3 TEAEs was similar across cohorts.
The combination of well-tolerated therapy and efficacy in inducing responses translated into a 2-year OS rate of 82%, a median TTNT of 14 months, and FFS rates of 76% and 47% at 6 and 12 months, respectively.
In a prospective study conducted by the cGVHD Consortium, the 12-month FFS rate with response (CR/PR) after first-line therapy was 12% to 15%. [bib_ref] An endpoint associated with clinical benefit after initial treatment for chronic graft-versus-host..., Martin [/bib_ref] In our study (after 1-3 prior LOTs), the 12-month FFS rate with response was 24%.
Belumosudil therapy was associated with a CS-sparing effect. The current treatment paradigm relies on CS as the mainstay of therapy; however, the related long-term toxicities mandate the use of the lowest possible dose or discontinuation whenever possible. The use of CS is tied to QOL, as the side effect profile of CS contributes to patient symptom burden. [bib_ref] How we treat chronic graft-versus-host disease, Flowers [/bib_ref] [bib_ref] Ancillary therapy and supportive care of chronic graft-versus-host disease: National Institutes of..., Couriel [/bib_ref] [bib_ref] Diagnosis and management of chronic graft-versus-host disease, Dignan [/bib_ref] CS dose reduction was observed across both responders and nonresponders to belumosudil. Approximately 20% of patients were able to discontinue CS during belumosudil treatment. Even in the absence of an NIH-defined response, patients experienced clinical benefit, as evidenced by improvements in LSS score or reductions in CS doses.
The pathophysiology and clinical phenotype of cGVHD reflect both immune and fibrotic components. [bib_ref] Pathophysiology of chronic graft-versus-host disease and therapeutic targets, Zeiser [/bib_ref] An ideal intervention would target both aspects. Fibrotic cGVHD manifestations, including fasciitis, ocular fibrosis, cutaneous sclerosis, and bronchiolitis obliterans syndrome, are notoriously difficult to treat. 18,31-33 As a ROCK2 inhibitor, belumosudil has been shown to decrease inflammation, restore immune homeostasis, and decrease fibrosis. [bib_ref] Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T..., Zanin-Zhorov [/bib_ref] [bib_ref] Targeted rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through..., Flynn [/bib_ref] In our study, responses were achieved in patients with fibrotic manifestations in the lungs, joints and/or fascia, and eyes. These responses were observed in some cases after . 24 weeks of treatment,further highlighting the need to sustain effective therapy to achieve clinical benefit, particularly in patients with difficult-to-treat disease.
In summary, belumosudil is a selective ROCK2 inhibitor with a novel mechanism of action that targets both inflammation and fibrosis in cGVHD. [bib_ref] Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T..., Zanin-Zhorov [/bib_ref] Belumosudil was welltolerated and achieved clinically meaningful responses in patients with cGVHD across all dose regimens evaluated. [bib_ref] KD025-208: A phase 2a study of KD025 for patients with chronic graft..., Jagasia [/bib_ref] Responses have been durable and associated with QOL improvements and CS dose reductions or discontinuations. These clinical benefits were also seen in patients who did not achieve NIH-defined responses. [bib_ref] KD025-208: A phase 2a study of KD025 for patients with chronic graft..., Jagasia [/bib_ref] Data from this study are very encouraging, given the unmet needs of patients with cGVHD. These data have led to belumosudil being granted Breakthrough Therapy Designation by the US Food and Drug Administration. Because the lower belumosudil 200-mg once daily dose was equally safe and effective, it has been further compared in a subsequent randomized registration study (ClinicalTrials.gov identifier: NCT03640481) against the 200-mg twice a day dose for final dose recommendation.
[formula] Y Y Y Y Y Y Y Y Y Y Y N N N N N N Y Y Y Y Y Y Y Y Y Y Y N N N N N Y Y Y Y Y Y Y Y Y Y [/formula]
[fig] FIG 1: CONSORT diagram. cGVHD, chronic graft-versus-host disease; LTFU, long-term follow-up. [/fig]
[fig] FIG 2: Forest plot for subgroup analyses of ORR in the safety population. Note that subgroups were defined based on baseline assessment. cGVHD, chronic graft-versus-host disease; ORR, overall response rate. [/fig]
[fig] FIG 3: Durability of response to belumosudil in patients with cGVHD. Kaplan-Meier curves of estimated (A) DOR for responders, (B) time to next cGVHD systemic treatment (TTNT) in the safety population, (C) FFS in the safety population (including reasons for failure), and (D) overall survival in the safety population. DOR was defined as the time from response until documented progression. FFS was defined as the time from first dose of study drug to the start of another cGVHD systemic treatment, relapse of the underlying disease, or death. 28,29 cGVHD, chronic graft-versus-host disease; DOR, duration of response; FFS, failure-free survival; TTNT, time to next treatment. [/fig]
[fig] FIG 4 FIG A1: AFFILIATIONS 1 Prior affiliation: Vanderbilt-Ingram Cancer Center, Nashville, TN 2 Moffitt Cancer Center, Tampa, FL 3 TriStar Centennial Medical Center, Nashville, TN 4 City of Hope Comprehensive Cancer Center, Duarte, CA 5 Masonic Cancer Center, University of Minnesota, Minneapolis, Changes in percentage of CD4 1 Tregs following treatment with belumosudil compared with baseline. (A) Tregs (all), (B) Tregs (responders)Best individual response by organ system among responders. n 5 number of responder population for global severity rating and number of specific organs involved at baseline. The percentages are calculated based on the corresponding n. CR, complete response; PR, partial response. [/fig]
[fig] FIG A2: Response and progression heat map for all patients in the safety population. (A) Best response by organ. (B) Organ responses at time of progression or end of study. Of 11 patients with progression in joints, seven had a reduction in P-ROM of just one unit. cGVHD, chronic graftversus-host disease; CR, complete response; GSR, Global Severity Rating; PR, partial response; P-ROM, photographic range of motion. [/fig]
[table] TABLE 1: Baseline Demographics and Clinical Characteristics [/table]
[table] TABLE 2: Efficacy and CS Reduction Abbreviations: cGVHD, chronic graft-versus-host disease; CS, corticosteroid; LOT, line of therapy; LSS, Lee Symptom Scale; ORR, overall response rate.Disease severity was determined using the Physician-reported Global cGVHD Activity Assessment Form. [/table]
[table] TABLE 3: Safety Overview [/table]
[table] TABLE A1: Additional Baseline Demographics [/table]
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Across a significant body of research, psychopathy has often been conceptualized as a biologically based malady. In this research, genetic and neurobiological differences have been conceptualized to underlie psychopathy, while affected individuals' life experiences only influence expressed psychopathic features and their severity. Psychopathy research has largely ignored developmental evidence demonstrating significant influences of environment on both biological and behavioral processes, resulting in several prominent criticisms(Edens & Vincent, 2008;Loeber, Byrd, & Farrington, 2015). The current review was conducted with two main aims: (a) to collect and consider etiological evidence from the extant body of research on genetic and neurobiological factors in psychopathy; and (b) to evaluate findings from genetic, neurotransmitter, brain structure, and brain function studies in the context of relevant evidence from developmental research. Examples from research on adversity and traumatic stress, a common correlate of psychopathy, were used to highlight current research gaps and future directions to aid in the integration of developmental and neurobiological research agendas. While some promising evidence exists regarding possible underlying neurobiological processes of psychopathic traits, this evidence is insufficient to suggest a largely biological etiology for the disorder. Further, information from developmental and epigenetic research may suggest complex, multidimensional trajectories for individuals experiencing psychopathy. Based on these observations, the authors make several recommendations for future research, as well as for current clinical application and practice.
Psychopathy is a clinical term originating in early 19th-century psychiatry, first comprehensively described inseminal book, The Mask of Sanity. In recent years psychopathy has been used extensively in clinical, legal, and forensic settings [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref] [bib_ref] Psychopathy and antisocial personality disorder: A case of diagnostic confusion, Hare [/bib_ref]. Psychopathy is often described as an untreatable personality disorder consisting of an apparent absence of empathy and remorse, along with superficial charm, shallow relationships, and rational, cold-blooded self-gratification, which often occurs at the expense of others [bib_ref] Psychopathy and antisocial personality disorder: A case of diagnostic confusion, Hare [/bib_ref] [bib_ref] Severe (psychopathic) personality disorder: A review, Moss [/bib_ref]. Today, the label can produce significant consequences within the legal realm, such as increased sentence severity and likelihood of execution [bib_ref] Juror perceptions of the interpersonal-affective traits of psychopathy predict sentence severity in..., Cox [/bib_ref] [bib_ref] No sympathy for the devil: Attributing psychopathic traits to capital murderers also..., Edens [/bib_ref] [bib_ref] Psychopathy and antisocial personality disorder: A case of diagnostic confusion, Hare [/bib_ref] and harsher, less engaged treatment in community settings [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref] [bib_ref] Psychopathy: A confusing clinical construct, Kirkman [/bib_ref] [bib_ref] Treatment of psychopathy: A review and brief introduction to the mental model..., Salekin [/bib_ref] [bib_ref] Effect of psychopathy, abuse, and ethnicity on juvenile probation officers' decision-making and..., Vidal [/bib_ref].
In his 1999 book Without Conscience, developer of the Psychopathy Checklist (PCL), Robert Hare, explained that while the terms psychopath and sociopath are used interchangeably, the distinction lies in how one interprets the origins and determinants of each label. Generally, the former is used by those who ascribe the label a perdominantly biological etiology, whereas environmental factors are highlighted by those using the latter. A similar distinction is often made in considering primary and secondary psychopathy, where the former is thought to be a result of biological deficits, while the latter is attributed to various forms of social disadvantage [bib_ref] Validating a distinction between primary and secondary psychopathy with measures of Gray's..., Newman [/bib_ref] [bib_ref] An investigation of primary and secondary psychopathy in a statewide sample of..., Vaughn [/bib_ref].
In the following decades, Hare's implied positionthat psychopathy was much more a biological or genetic maladyhas gained largely uncritical acceptance in both research and practice [bib_ref] Neurobiological basis of psychopathy, Blair [/bib_ref] [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref] [bib_ref] Psychopathy as a clinical and empirical construct, Hare [/bib_ref]. Researchers have invested heavily in exploring the various neurological and genetic factors suspected to cause or underlie psychopathic traits. The premise of such research has been that genes influence brain structures that are associated with psychopathic tendencies [bib_ref] Neurobiological basis of psychopathy, Blair [/bib_ref] [bib_ref] Psychopathy as a clinical and empirical construct, Hare [/bib_ref]. Environmental influences merely act to shape psychopathic characteristics, which manifest in childhood as callous unemotional (CU) traits, and are then stable throughout the lifespan [bib_ref] Neurobiological basis of psychopathy, Blair [/bib_ref] Hare & Neumann,
## Developmental parallels
Prior to the advent of medicalized theories of its etiology, psychopathy was often regarded as environmentally derived [bib_ref] Neurobiological basis of psychopathy, Blair [/bib_ref] [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref] [bib_ref] Psychopathy as a clinical and empirical construct, Hare [/bib_ref]. In a parallel toconceptualization of psychopathy, [bib_ref] Forty-four juvenile thieves: Their character and home-life, Bowlby [/bib_ref] affectionless psychopaths," who would meet today's standard for diagnosis of reactive attachment disorder, given their documented behavior, affect, and histories of early and prolonged deprivation of care. Bowlby hypothesized that deficits in early bonding with a consistent caregiver had caused these young people to perceive others as unworthy of trust, empathy, and concern [bib_ref] Psychopathy in juvenile offenders: Can temperament and attachment be considered as robust..., Saltaris [/bib_ref]. Subsequently, insecure attachment patterns [bib_ref] Current attachment representations of incarcerated offenders varying in degree of psychopathy, Frodi [/bib_ref] and neglectful and maltreating family environments [bib_ref] Early traumatization and psychopathy in female and male juvenile offenders, Krischer [/bib_ref] [bib_ref] The childhood experiences of psychopaths: A retrospective study of familial and societal..., Marshall [/bib_ref] [bib_ref] Childhood risk, offending trajectories, and psychopathy at age 48 years in the..., Piquero [/bib_ref] have been reported for individuals high in psychopathic traits.
Developmental research points toward the critical influence of early caregiving in healthy emotional development. Specifically, caregiver attunement and responsiveness to an infant's emotional and physical needs plays a critical role in many psychological and physiological development processes. For instance, [bib_ref] Ordinary variations in maternal caregiving influence human infants' stress reactivity, Hane [/bib_ref] reported that even typical variations in maternal sensitivity and intrusiveness affected infants' social interaction, with infants whose mothers were less sensitive and/or more intrusive showing significantly less interest in social interaction and significantly more negative affect than those whose mothers were more sensitive and less intrusive. More importantly, even in their sample of low-risk, non-maltreated children, parenting differences were associated with neurological differences among the children [bib_ref] Ordinary variations in maternal caregiving influence human infants' stress reactivity, Hane [/bib_ref]. These neurological differences [bib_ref] Ordinary variations in maternal caregiving influence human infants' stress reactivity, Hane [/bib_ref] appear to be the product of neurobiological changes during a critical developmental period, caused by the infant's interaction with his or her social environment [bib_ref] Attachment, affect regulation, and the developing right brain: Linking developmental neuroscience to..., Schore [/bib_ref]. For example, [bib_ref] Maternal neglect: Oxytocin, dopamine and the neurobiology of attachment, Strathearn [/bib_ref] pointed out numerous biological influences of neglect. Observations of reductions in dopamine transporter binding in the ventral striatum, which cause elevated baseline dopamine levels, increased dopamine release in response to acute stress in adulthood, and increased sensitivity to psychostimulants were noted [bib_ref] Maternal neglect: Oxytocin, dopamine and the neurobiology of attachment, Strathearn [/bib_ref]. In a retrospective analysis, [bib_ref] Dopamine release in response to a psychological stress in humans and its..., Pruessner [/bib_ref] reported increased dopamine and cortisol release during stressful situations in individuals who reported low-quality relationships with caregivers in childhood.
Noteworthy is a timeline for the aforementioned neurobiological changes. Infants' brain development begins with a growth spurt of the right hemisphere during the first 2 years of life [bib_ref] Attachment, affect regulation, and the developing right brain: Linking developmental neuroscience to..., Schore [/bib_ref]. The right hemisphereinvolved in emotional and social processingmaintains dominance for the first 3 years after birth, until the left hemisphere's development catches up [bib_ref] Attachment, affect regulation, and the developing right brain: Linking developmental neuroscience to..., Schore [/bib_ref]. Across longitudinal and cross-sectional brain imaging studies, chronic stress, deprivation, or maltreatment in the first 3 years of life has been shown to cause brain volume reductions and significant brain development abnormalities in affected 3-year-olds [bib_ref] Altered brain development following global neglect in early childhood, Perry [/bib_ref] [bib_ref] Annual research review: Enduring neurobiological effects of childhood abuse and neglect, Teicher [/bib_ref]. Yet children are highly unlikely to remember or have the ability to report on these early environments due to infantile amnesia [bib_ref] Infantile amnesia across the years: A 2-year follow-up of children's earliest memories, Peterson [/bib_ref]. Consequently, the impact of adversity in infancy and early childhood cannot be captured by common traumatic exposure measures.
## Exploring current research on psychopathy
Currently, despite the absence of conclusive evidence of a specific innate cause for psychopathy and despite indications of significant environmental influences on its development, research continues to focus principally on the search for its biological determinants, often to the exclusion of social, developmental, and environmental factors. Such efforts have also been extended to children, with both a modified version of the Psychopathy Checklist (PCL-Youth Version) and research targeting children with CU traits, which are argued to precede psychopathy in adulthood [bib_ref] The importance of callous-unemotional traits for extending the concept of psychopathy to..., Barry [/bib_ref] [bib_ref] Integrating research on temperament and childhood psychopathology: Its pitfalls and promise, Frick [/bib_ref] [bib_ref] Evidence for substantial genetic risk for psychopathy in 7-year-olds, Viding [/bib_ref]. In these research lines, researchers hope to identify psychopathic traits when they are more malleable, or find environmental triggers that activate the expression of genetically based psychopathic traits [bib_ref] The importance of callous-unemotional traits for extending the concept of psychopathy to..., Barry [/bib_ref] [bib_ref] Integrating research on temperament and childhood psychopathology: Its pitfalls and promise, Frick [/bib_ref] [bib_ref] Evidence for substantial genetic risk for psychopathy in 7-year-olds, Viding [/bib_ref].
The application of psychopathy to children's underdeveloped personalities has drawn the criticism of developmental psychologists and life-course criminologists alike [bib_ref] No sympathy for the devil: Attributing psychopathic traits to capital murderers also..., Edens [/bib_ref] [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref]. In their criticism of the practice, [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref] note that along with the increase in research evaluating psychopathic traits in youth, the applied (clinical and forensic) use of these measures has been on the rise. Yet, in adult populations, such applications lead to inherently negative, lifealtering outcomes [bib_ref] No sympathy for the devil: Attributing psychopathic traits to capital murderers also..., Edens [/bib_ref]. Given limited evidence of temporal stability in personality characteristics, such applications can pose serious consequences on children whose current behavior and personality may be nothing beyond a mere state [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref]. Notably, heterotypic continuity, the differential manifestation of symptoms over time [bib_ref] Equifinality and multifinality in developmental psychopathology, Cicchetti [/bib_ref] [bib_ref] Understanding the downward extension of psychopathy to youth: Implications for risk assessment..., Vitacco [/bib_ref] , and typical developmental shifts in personality have been observed repeatedly in research [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref]. For instance, when assessing the stability of psychopathic characteristics during the transition to adulthood, [bib_ref] Structural coherence and temporal stability of psychopathic personality features during emerging adulthood, Hawes [/bib_ref] noted a decrease in these characteristics over time. The Diagnostic and Statistical Manual 5 , and previous editions, specifically excludes children from its diagnosis of antisocial personality disorder (APD), based on similar concerns, while allowing diagnosis with oppositional-defiant disorder and conduct disorder in youth.
These research trends have been more broadly criticized for failing to adequately integrate forensic and developmental research. For instance,argued that criminological and clinical research is largely based on male inmate or patient samples, and is rarely longitudinal. Consequently, neurological differences identified in this research cannot be shown to represent life-course stable abnormalities rather than normal or temporary variants. These critiques implicate the need to integrate clinical and developmental approaches to further develop the knowledge base on the etiology of antisociality [bib_ref] Juvenile psychopathy: A clinical construct in need of restraint?, Edens [/bib_ref]. However, few comprehensive efforts for such an integration have been made. The purpose of this article is to offer one such effort, by (a) evaluating the degree of integration of environmental and developmental correlates in biological research and (b) reviewing neurobiological research findings in the context of their developmental correlates. Conclusions regarding both the current state of knowledge on the etiology, treatment, and prognosis of psychopathy are also discussed.
# Method
## Inclusion criteria
Since 1999, when Hare's book was published, a multitude of studies have investigated psychopathic traits in biological terms. However, much of this literature has been affected by two areas of debate 2 A Frazier et al.
among scholars and practitioners: (a) whether the psychopathy construct is driven by violent or rule-breaking behavior [bib_ref] Neurocognitive models of aggression, the antisocial personality disorders, and psychopathy, Blair [/bib_ref] [bib_ref] Psychopathy and aggression, Porter [/bib_ref] [bib_ref] One measure does not a construct make: Directions toward reinvigorating psychopathy research..., Skeem [/bib_ref] and (b) whether APD and psychopathy represent two distinct constructs [bib_ref] Psychopathy/antisocial personality disorder conundrum, Ogloff [/bib_ref] [bib_ref] Antisocial personality disorder/psychopathy, Patrick [/bib_ref]. Within this body of literature, violence and aggression, CU traits, and antisocial personality and behavior are often discussed as proxies to (or precursers of) psychopathy [bib_ref] The importance of callous-unemotional traits for extending the concept of psychopathy to..., Barry [/bib_ref] [bib_ref] Exploring the association between the 2-repeat allele of the MAOA gene promoter..., Beaver [/bib_ref] [bib_ref] Neurocognitive models of aggression, the antisocial personality disorders, and psychopathy, Blair [/bib_ref] [bib_ref] Behavioral genetics in antisocial spectrum disorders and psychopathy: A review of the..., Gunter [/bib_ref] [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] [bib_ref] Amygdala reactivity and negative emotionality: Divergent correlates of antisocial personality and psychopathy..., Hyde [/bib_ref] [bib_ref] Corpus callosum abnormalities in psychopathic antisocial individuals, Raine [/bib_ref]. The authors therefore elected to consider studies that measured these psychopathy traits using established measures of the construct (e.g., PCL and PCL-R), but also theoretically related traits and behaviors used in lieu of psychopathy measures due to the age of the subjects (e.g., CU traits) or their role as a protopsychopathy indicator (e.g., antisocial behavior). Within the current review, authors sought to identify studies that (a) were published between 1999 and 2017; (b) described quantitative analyses that included some measure of psychopathic tendency (broadly construed to include antisocial personality and behavior); and (c) included some neurobiological (i.e., genetic, neurotransmitter, brain imaging) measures with a human sample.
## Search procedure
Based on the aforementioned criteria, the authors conducted a literature search on Google Scholar, using keyword combinations to account for the largest possible body of research in psychopathic and antisocial traits. For example, keywords included "psychopathyþgene," as well as "antisocialþpersonalityþneurotransmitter." Abstracts of studies identified by this search (>1500) were then reviewed for relevance to the current review. Studies that (a) did not include a neurobiological measure, (b) where no human sample was used, (c) in which only traits or behaviors indirectly related to psychopathy were assessed (e.g., criminal or delinquent behavior; sexual aggression), or (d) where no empirical data collection was reported (i.e., theoretical papers and literature reviews) were excluded from this review. This process resulted in the inclusion of 54 studies in this review. Upon detailed review, four additional studies were excluded because they used the same dataset for multiple similar analyses (National Longitudinal Study of Adolescent Health; sample from employment temp agencies) or because characteristics of the sample were not described, ultimately resulting in a review of 50 studies.
## Initial evaluation of studies
Each study was then assessed to determine whether at least one environmental factor (e.g., socioeconomic status, maltreatment, or trauma) was analyzed. The frequency with which environmental/ developmental factors were reported was counted. Of the 50 studies, 17 (34%) included at least one environmental covariate or control. The list of studies included, and factors evaluated in them, is presented in [fig_ref] Table 1: (Continued ) [/fig_ref]. Similarly, studies were evaluated for their use of PCL type instruments and use of forensic and clinical samples, adults and children, and male and female participants.
## Sampling and measurement trends
Several general trends were observed in reviewed studies. First and foremost, environmental and developmental factors were not adequately integrated with biological research. Sixty-six percent of the studies reviewed did not consider any influences of environment or relevant developmental processes. In the remaining studies, the most common environmental variables included socioeconomic status (10% of studies), family environment (8% of studies), and maltreatment (8% of studies). In these studies, divergent measures and definitions made comparisons across findings difficult to make.
Second, the use of PCL-type measurese.g., PCL-Revised (PCL-R,and PCL-Screening Version (PCL-SV,decreased over time, corresponding with an increase in the use of child samples (16% of studies), although adults remained the primary subjects of this research. Overall, 46% of studies used PCL instruments, while 54% gauged psychopathic tendencies in other ways. The latter studies predominantly used the Structured Clinical Interview for DSM (SCID) diagnosis (12% of studies) along with some measure(s) of criminality or aggressionthough others used versions of the Antisocial Process Screening Device (8% of studies), the Psychopathic Personality Inventory (6% of studies), and the NEO Five-Factor Inventory (4% of studies). While the majority of these instruments correlate with one another, they measure psychopathy differently (e.g., informal interview compared to self-report), and many consider criminality, which may be related tobut not essential tothe psychopathy construct. Studies that utilized PCL-type measures were similar to studies using other instruments across demographic characteristics (i.e., inclusion of female participants), except that 83% used adult forensic or prisoner samples. Given that the PCL-R requires a record review as part of accurate administration, reliance on forensic samples in which these records exist for research using the PCL-R is unsurprising.
Further, neurobiological studies consistently favored male participants over females, and both forensic and clinical samples over community samples (36% of studies). Most frequently, adult participants were recruited from prisons and forensic institutional settings (26% of studies). Yet the influence of residing in the institution [bib_ref] Exposure to violent crime during incarceration: Effects on psychological adjustment following release, Boxer [/bib_ref] [bib_ref] Psychological effects of imprisonment on confined individuals, Bukstel [/bib_ref] [bib_ref] Inmate experiences and psychological wellbeing, Wooldredge [/bib_ref] was not itself considered in the research. Female participants were included in 44% of studies, but in these studies, 50 male participants were recruited for every 27 female participants, on average. None of the studies used a female-only sample, despite concerns regarding the capacity of psychopathy measures to adequately capture the construct in females [bib_ref] The reliability and validity of the Psychopathy Checklist-Revised in a sample of..., Vitale [/bib_ref]. Relatedly, only 30% of studies considered nonwhite racial and ethnic groups. While all humans share basic biological characteristics, genetic and biomedical differences (and thus risk factors) within different ethnic groups have been noted [bib_ref] The importance of race and ethnic background in biomedical research and clinical..., Burchard [/bib_ref] , including different genetic correlates of antisociality [bib_ref] Neither antisocial personality disorder nor antisocial alcoholism is associated with the MAO-A..., Lu [/bib_ref]. Importantly, these shortages in participant diversity pose significant limitations to the generalizability of psychopathy research findings to the population.
## Genetic etiology
Several authors have argued for a genetic or hereditary pathway toward psychopathy [bib_ref] Intergenerational transmission of psychopathy and mediation via psychosocial risk factors, Auty [/bib_ref] [bib_ref] Neurobiological basis of psychopathy, Blair [/bib_ref] [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref] [bib_ref] Evidence for substantial genetic risk for psychopathy in 7-year-olds, Viding [/bib_ref]. The notion is that psychopathic individuals inherit a genetic makeup that manifests in altered brain functioning and physiological reactivity, and along with some environmental triggers or influences, shapes their behavior in childhood [bib_ref] Neurobiological basis of psychopathy, Blair [/bib_ref] [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref] [bib_ref] Evidence for substantial genetic risk for psychopathy in 7-year-olds, Viding [/bib_ref]. For instance, [bib_ref] Evidence for substantial genetic risk for psychopathy in 7-year-olds, Viding [/bib_ref] reported finding strong evidence of heredity and no evidence of shared environmental influences in their study (2015) found strong evidence of transmission of psychopathy from fathers to their children, though this was mediated by environmental factors. Given the strength of genetic influence reported in these and similar findings, examining the evidence for specific genetic factors is important.
## Mao-a
The strongest cumulative evidence base for a genetic pathway toward psychopathy is associated with the low-expression variant of the Monoamine Oxidase-A (MAO-A) gene, which encodes an enzyme that degrades mono-amine neurotransmittersthat is, dopamine, norepinephrine, and serotonin [bib_ref] Role of genotype in the cycle of violence in maltreated children, Caspi [/bib_ref] [bib_ref] The effects of child maltreatment on early signs of antisocial behavior: Genetic..., Cicchetti [/bib_ref]. The low-expression variant of the MAO-A gene is linked to the X chromosome. Possessing only a single X chromosome, males are more likely to be influenced by a low-expression variant [bib_ref] The psycho gene, Hunter [/bib_ref]. In all eight studies investigating MAO-A polymorphisms [bib_ref] Exploring the association between the 2-repeat allele of the MAOA gene promoter..., Beaver [/bib_ref] [bib_ref] Role of genotype in the cycle of violence in maltreated children, Caspi [/bib_ref] [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] [bib_ref] Lower monoamine oxidase-a total distribution volume in impulsive and violent male offenders..., Kolla [/bib_ref] [bib_ref] Personality traits and platelet monoamine oxidase activity in a Swedish male criminal..., Longato-Stadler [/bib_ref] [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] [bib_ref] A polymorphism of the MAOA gene is associated with emotional brain markers..., Williams [/bib_ref] [bib_ref] Interaction between MAO-A genotype and maltreatment in the risk for conduct disorder:..., Young [/bib_ref] , statistically significant correlations were identified between the short allele and psychopathic and/or antisocial traits. The use of differing measures of psychopathy and antisocial behavior, along with inadequate delineation of participants' histories of criminality and delinquency from the psychopathy construct, posed a significant limitation for many studies. Several authors found associations between subjects' criminality and a lower-expression variant of the MAO-A gene [bib_ref] Exploring the association between the 2-repeat allele of the MAOA gene promoter..., Beaver [/bib_ref] [bib_ref] Lower monoamine oxidase-a total distribution volume in impulsive and violent male offenders..., Kolla [/bib_ref] [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] , or specifically investigated APD diagnosis [bib_ref] Personality traits and platelet monoamine oxidase activity in a Swedish male criminal..., Longato-Stadler [/bib_ref] [bib_ref] Interaction between MAO-A genotype and maltreatment in the risk for conduct disorder:..., Young [/bib_ref] , which placed significantly more weight on rule-breaking behavior [bib_ref] Psychopathy and antisocial personality disorder: A case of diagnostic confusion, Hare [/bib_ref]. Focusing specifically on core psychopathic traits, only two studies [bib_ref] Lower monoamine oxidase-a total distribution volume in impulsive and violent male offenders..., Kolla [/bib_ref] [bib_ref] A polymorphism of the MAOA gene is associated with emotional brain markers..., Williams [/bib_ref] identified significant differences between carriers of lower-and higher-expression MAO-A variants (i.e., increased psychopathic traits in short allele carriers). [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] found lower-expression MAO-A variants to be significantly related to emotional dysfunction scores in youth diagnosed with attention deficit hyperactivity disorder (ADHD), but the cumulative effect size for the same relation with total psychopathy-related scores was medium-sized (f 2 = 0.16).
In 2002, Caspi et al. observed an interaction between childhood adversity and the MAO-A risk allele, demonstrating that for those who carried this allele, adversity was positively related with psychopathy. Building on findings from [bib_ref] Role of genotype in the cycle of violence in maltreated children, Caspi [/bib_ref] , several subsequent MAO-A studies [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] [bib_ref] A polymorphism of the MAOA gene is associated with emotional brain markers..., Williams [/bib_ref] [bib_ref] Interaction between MAO-A genotype and maltreatment in the risk for conduct disorder:..., Young [/bib_ref] included measures of childhood adversity or maltreatment. However, whereas some studies found associations between maltreatment, adversity, and rule-breaking behavior [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] [bib_ref] Interaction between MAO-A genotype and maltreatment in the risk for conduct disorder:..., Young [/bib_ref] or between the risk allele and antisocial behavior in participants who experienced severe family adversity [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] , others did not . Notably, these studies varied not only in their measures of psychopathic traits but also in their measurement of adversity and maltreatment. For instance, [bib_ref] A polymorphism of the MAOA gene is associated with emotional brain markers..., Williams [/bib_ref] analysis used only a dichotomous adversity score, comparing participants who experienced more than three adverse events to those who experienced three or fewer. Nonetheless, the interaction between MAO-A and maltreatment observed by [bib_ref] Role of genotype in the cycle of violence in maltreated children, Caspi [/bib_ref] was not replicated in these subsequent studies.
## 5-htt
Based on observations of reduced serotonin in aggressive and impulsive individuals [bib_ref] Natural born killers: The genetic origins of extreme violence, Ferguson [/bib_ref] [bib_ref] Impulsive aggression in borderline personality disorder, Glenn [/bib_ref] [bib_ref] Impulsivity, aggression, and serotonin: A molecular psychobiological perspective, Lesch [/bib_ref] , multiple studies have sought to associate 5-HTT with psychopathy. Findings in the seven studies that examined this relationship [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] [bib_ref] Incremental effect for antisocial personality disorder genetic risk combining 5-HTTLPR and 5-HTTVNTR..., Garcia [/bib_ref] [bib_ref] Association between low activity serotonin transporter promoter genotype and early onset alcoholism..., Hallikainen [/bib_ref] [bib_ref] Serotonin transporter gene associations with psychopathic traits in youth vary as a..., Sadeh [/bib_ref] , experiments 1 and 2; [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] [bib_ref] Serotonin transporter availability in impulsive aggressive personality disordered patients: A PET study..., Van De Giessen [/bib_ref] were inconsistent. [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] , for instance, found that PCL:SV Factor 2 scores were significantly related to carrying the long allele of 5-HTT and to Childhood Trauma Questionnaire scores, though no interaction was identified. Similarly, in , CU traits increased in long/long allele carriers, but only as socioeconomic resources decreased. In contrast, aggression, impulsivity, and antisocial behavior was found to be related to carrying the short allele of 5-HTT [bib_ref] Incremental effect for antisocial personality disorder genetic risk combining 5-HTTLPR and 5-HTTVNTR..., Garcia [/bib_ref]. No relation between aggression and 5-HTT availability was identified in [bib_ref] Serotonin transporter availability in impulsive aggressive personality disordered patients: A PET study..., Van De Giessen [/bib_ref] , but the authors observed a positive relation with callousness traits.
## Dopamine receptor genes
Because of their role in the pleasure/reward system in humans, dopaminergic system genes have been a source of considerable research relating to violence, aggression, and antisocial behavior [bib_ref] Natural born killers: The genetic origins of extreme violence, Ferguson [/bib_ref] [bib_ref] Genetic contributions to antisocial personality and behavior: A meta-analytic review from an..., Ferguson [/bib_ref]. Nonetheless, only three of the studies investigated dopaminergic gene systems [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] [bib_ref] DRD2 and ANKK1 genotype in alcohol-dependent patients with psychopathic traits: Association and..., Ponce [/bib_ref] [bib_ref] Two dopamine receptor genes (DRD2 and DRD4) predict psychopathic personality traits in..., Wu [/bib_ref]. An additional study [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] investigated the COMT gene, which regulates the production of the dopamine degrading enzyme, and is therefore discussed here as well.
All four studies reported relatively small impact of dopaminergic system genes. For instance, [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] reported the high-activity COMT genotype was statistically associated with significantly higher emotional dysfunction scores, with a small effect size (f 2 = 0.08), and without significant impact on total psychopathy scores. [bib_ref] Two dopamine receptor genes (DRD2 and DRD4) predict psychopathic personality traits in..., Wu [/bib_ref] found that only DRD4 (and not DAT1 or DRD2) was significantly related to psychopathic personality traits. Yet carrying two DRD4 risk alleles (as compared to zero) increased participants' mean psychopathy scores by only two points (from 55.97 to 57.99, on a scale of 23-115). [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] and [bib_ref] DRD2 and ANKK1 genotype in alcohol-dependent patients with psychopathic traits: Association and..., Ponce [/bib_ref] found DRD2 gene polymorphisms to be associated with higher scores on the International Personality Disorder Examination (IPDE). [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] reported these genetic markers (when combined) may be responsible for 11.4% of the variance in psychopathy scores.
## Other targets
Several other genetic polymorphisms have been analyzed for associations with psychopathy-related tendencies. These included SNAP25 t-snare protein gene [bib_ref] Synaptosomal-associated protein 25 gene polymorphisms and antisocial personality disorder: Association with temperament..., Basoglu [/bib_ref] [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] , OXT, the gene responsible for oxytocin production [bib_ref] Methylation of the oxytocin receptor gene and oxytocin blood levels in the..., Dadds [/bib_ref] , and the CNR1 and FAAH cannabinoid receptor gene polymorphisms [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref]. [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] found that variants of FAAH and CNR1 were related to an increase in PCL-R scores for their subjects, but found no impact of SNAP25 genes. In contrast, [bib_ref] Synaptosomal-associated protein 25 gene polymorphisms and antisocial personality disorder: Association with temperament..., Basoglu [/bib_ref] reported that a variant of the SNAP25 gene was much more common in antisocial personality subjects, and that these antisocial subjects had significantly higher novelty-seeking scores. [bib_ref] Methylation of the oxytocin receptor gene and oxytocin blood levels in the..., Dadds [/bib_ref] observed maturation-related differences in genetic activity, with the low-CU group showing lower methylation of the OXT gene in older children, while the opposite trend was observed in the high-CU group. In contrast, Rautiainen et al.'s (2016) genome-wide association study found none of these associations in their subjects, instead reporting an observed association between APD diagnosis (based on SCID-II interview) and Human Leukocyte Antigen (HLA) system genes, which impact the immune system and express in the brain.
## Reconsidering gene-environment interaction
Overall, it is well established that experiences and environmental influences can moderate the effects of individual polymorphisms on behavior and impact biological pathways that intersect with genetic influences, ultimately affecting gene expression [bib_ref] Gene-environment interaction, Manuck [/bib_ref]. The latter has been repeatedly shown to occur in maltreated children [bib_ref] Gene-environment interaction, Manuck [/bib_ref] [bib_ref] Social and physical environments early in development predict DNA methylation of inflammatory..., Mcdade [/bib_ref] , with children who experienced early stress showing different gene methylation, and thus expression, in adolescence than those whose environments were relatively low in stress [bib_ref] Social and physical environments early in development predict DNA methylation of inflammatory..., Mcdade [/bib_ref] [bib_ref] Epigenetic vestiges of early developmental adversity: Childhood stress exposure and DNA methylation..., Essex [/bib_ref]. Despite this, fewer than 50% of genetic studies included any environmental covariates, and none accounted for epigenetic change. Epigenetic mechanisms are rarely considered in relation to psychopathic trait development but may provide important insights regarding its etiology [bib_ref] The warrior gene: Epigenetic considerations, Gillett [/bib_ref].
While the aforementioned findings regarding genetic factors in psychopathy are variable, their interpretation is further complicated by gene-environment interaction research in antisocial personality traits [bib_ref] The effects of child maltreatment on early signs of antisocial behavior: Genetic..., Cicchetti [/bib_ref] [bib_ref] Callous-unemotional traits and antisocial behavior: Genetic, environmental, and early parenting characteristics, Larsson [/bib_ref]. Particularly, maltreatment and childhood adversity have been linked to increases in CU or APD traits [bib_ref] Methylation at SLC6A4 is linked to family history of child abuse: An..., Beach [/bib_ref] [bib_ref] Role of genotype in the cycle of violence in maltreated children, Caspi [/bib_ref] [bib_ref] Adverse childhood events as risk factors for negative mental health outcomes, Chapman [/bib_ref] [bib_ref] The effects of child maltreatment on early signs of antisocial behavior: Genetic..., Cicchetti [/bib_ref] [bib_ref] Callous-unemotional traits and antisocial behavior: Genetic, environmental, and early parenting characteristics, Larsson [/bib_ref]. Similarly, [bib_ref] Lower monoamine oxidase-a total distribution volume in impulsive and violent male offenders..., Kolla [/bib_ref] reported a higher prevalence of childhood physical abuse in offenders exhibiting psychopathic traits, and that these early experiences were associated with increases in reactive aggression. Finally, several investigations [bib_ref] Association in alcoholic patients between psychopathic traits and the additive effect of..., Hoenicka [/bib_ref] [bib_ref] DRD2 and ANKK1 genotype in alcohol-dependent patients with psychopathic traits: Association and..., Ponce [/bib_ref] have focused on psychopathy in those with substance use histories, and these have offered further support for the impact of childhood adversity on later substance use [bib_ref] Adverse childhood experiences, alcoholic parents, and later risk of alcoholism and depression, Anda [/bib_ref] [bib_ref] Childhood abuse, neglect, and household dysfunction and the risk of illicit drug..., Dube [/bib_ref].
Other behavioral and environmental factors may influence gene expression in ways that genetic research has yet to predict. For instance, several of the same genetic polymorphisms have been investigated in both psychopathy and ADHD research. Indeed, subjects with ADHD were the focus of [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref]. However, childhood ADHD is associated with significant problems in social, school, and emotional functioning [bib_ref] Preschool children with attention-deficit/hyperactivity disorder: Impairments in behavioral, social, and school functioning, Dupaul [/bib_ref]. These impairments have been linked with problematic family functioning, including greater family stress, higher rates of parental psychopathology, and conflicted parent-child relationships, which may produce a developmental cascade unaccounted for by currently available research.
## The role of neurotransmitters
Along with genetic polymorphism examinations, some researchers have sought to identify neurotransmitter involvement in psychopathy [bib_ref] Impulsive aggression in borderline personality disorder, Glenn [/bib_ref] [bib_ref] Two dopamine receptor genes (DRD2 and DRD4) predict psychopathic personality traits in..., Wu [/bib_ref]. This focus is not dissimilar to the interest in neurotransmitter activity underlying other mental disorders, based on the neurochemical imbalance hypothesis, which suggests that an imbalance in the brain chemistry of affected individuals leads to a variety of mental health problems [bib_ref] The biomedical model of mental disorder: A critical analysis of its validity,..., Deacon [/bib_ref] [bib_ref] The "chemical imbalance" explanation for depression: Origins, lay endorsement, and clinical implications, France [/bib_ref].
## Norepinephrine
Often known as noradrenaline, norepinephrine functions as an activation neurotransmitter in the threat-response system. It has been long hypothesized to interact with dopamine in a system of neurochemicals linked to reward-oriented behavior and response to stress, ultimately leading to undesirable or dysfunctional behavior such as aggression or absent empathy [bib_ref] Norepinephrine-dopamine interactions and behavior, Antelman [/bib_ref] [bib_ref] Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice..., Cases [/bib_ref] [bib_ref] Impaired maternal behavior in mice lacking norepinephrine and epinephrine, Thomas [/bib_ref]. Of the studies included in this review, the only one to investigate norepinephrine [bib_ref] Neuroendocrine correlates of antisocial personality disorder in abstinent heroin-dependent subjects, Gerra [/bib_ref] did not find a significant relationship with psychopathy.
## Serotonin
Researchers have long speculated about an association between core psychopathic traits and nonoptimal stability and efficiency
## 6
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in serotonin functioning, whereby healthy levels of baseline arousal dampen psychophysiological responses to threat, punishment, and social stimuli [bib_ref] Systematic review, structural analysis, and new theoretical perspectives on the role of..., Yildirim [/bib_ref]. These hypotheses have been supported by the finding that serotonin inhibits aggressive behavior [bib_ref] The effect of increased serotonergic neurotransmission on aggression: A critical meta-analytical review..., Carrillo [/bib_ref] [bib_ref] Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice..., Cases [/bib_ref] [bib_ref] 5-HT 1A and 5-HT 1B receptor agonists and aggression: A pharmacological challenge..., De Boer [/bib_ref]. Serotonin was, therefore, the focus of a significant number of recent studies on antisocial and psychopathic samples (da [bib_ref] Serotonin 1B receptor binding is associated with trait anger and level of..., Da Cunha-Bang [/bib_ref] [bib_ref] The relationship between serotonergic function and the psychopathy checklist: Screening version, Dolan [/bib_ref] [bib_ref] Serotonin (5-HT) augmentation reduces provoked aggression associated with primary psychopathy traits, Fanning [/bib_ref] [bib_ref] Neuroendocrine correlates of antisocial personality disorder in abstinent heroin-dependent subjects, Gerra [/bib_ref] [bib_ref] A meta-analysis of serotonin metabolite 5-HIAA and antisocial behavior, Moore [/bib_ref] [bib_ref] An exploration of the serotonin system in antisocial boys with high levels..., Moul [/bib_ref] [bib_ref] Increased serotonin 2A receptor availability in the orbitofrontal cortex of physically aggressive..., Rosell [/bib_ref] [bib_ref] New evidence for an association between the CSF HVA: 5-HIAA ratio and..., Soderstrom [/bib_ref] [bib_ref] Association of aggressive behavior with altered serotonergic function in patients who are..., Stanley [/bib_ref]. In the eight reviewed studies, lower serotonin levels were linked more often to increased impulsivity traits (da [bib_ref] Serotonin 1B receptor binding is associated with trait anger and level of..., Da Cunha-Bang [/bib_ref] [bib_ref] The relationship between serotonergic function and the psychopathy checklist: Screening version, Dolan [/bib_ref] [bib_ref] A meta-analysis of serotonin metabolite 5-HIAA and antisocial behavior, Moore [/bib_ref] than to callous-unemotional traits [bib_ref] An exploration of the serotonin system in antisocial boys with high levels..., Moul [/bib_ref] or aggression [bib_ref] Association of aggressive behavior with altered serotonergic function in patients who are..., Stanley [/bib_ref]. A deficiency in serotonin production may, therefore, not actually underlie aggressive, antisocial tendencies but rather impulse control problems that may lead to antisocial behaviors. Notably, [bib_ref] A meta-analysis of serotonin metabolite 5-HIAA and antisocial behavior, Moore [/bib_ref] reported that this effect was moderated by age, thus supporting the notion that differences in serotonin production are not lifecourse stable.
## Dopamine
Due to its association with hyperactivity, aggressive, and rewardmotivated behavior, dopamine has been investigated in three of the reviewed studies [bib_ref] Mesolimbic dopamine reward system hypersensitivity in individuals with psychopathic traits, Buckholtz [/bib_ref] [bib_ref] Neuroendocrine correlates of antisocial personality disorder in abstinent heroin-dependent subjects, Gerra [/bib_ref] [bib_ref] New evidence for an association between the CSF HVA: 5-HIAA ratio and..., Soderstrom [/bib_ref]. It is often assumed that dopaminergic genes cause an overabundance of the neurotransmitter, which produce some of the behavioral and emotional manifestations common in psychopathy. However, results of these studies have contradicted this idea. For instance, [bib_ref] Neuroendocrine correlates of antisocial personality disorder in abstinent heroin-dependent subjects, Gerra [/bib_ref] found that subjects with antisocial personalities showed reduced dopaminergic receptor sensitivity, suggesting that their subjects's brains were less responsive to dopamine and thus required more of it. In contrast, [bib_ref] Mesolimbic dopamine reward system hypersensitivity in individuals with psychopathic traits, Buckholtz [/bib_ref] reported dopamine system hyperactivity in their subjects, which they associated with antisocial-related impulsivity. [bib_ref] New evidence for an association between the CSF HVA: 5-HIAA ratio and..., Soderstrom [/bib_ref] found that aggression was related to a high dopamine turnover together with serotonergic dysregulation. Since not all psychopathic individuals are both aggressive and impulsive (i.e., some may only possess one of the two traits), these latter findings suggest that researchers may be observing correlates of aggression and impulsivity rather than psychopathy.
## Neurological differences
Observations of the emotional and empathic deficits in psychopathic offenders have led to hypotheses of functional and/or structural neurological abnormalities in antisocial individuals (Shamay-Tsoory, Harari, Aharon-Peretz, & Levkovitz, 2010; [bib_ref] Localization of deformations within the amygdala in individuals with psychopathy, Yang [/bib_ref]. Specifically, it has been suggested that, as a result of inherited genetic differences affecting neurotransmission and neurodevelopment, psychopathic individuals have decreased activation and volume in key areas of the brain, including the orbitofrontal cortex [bib_ref] Risky decisions and response reversal: Is there evidence of orbitofrontal cortex dysfunction..., Mitchell [/bib_ref] and amygdalae, as well as broader gray matter volume reductions [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref].
## Whole-brain studies
Multiple whole-brain volume [bib_ref] Volumetric structural brain abnormalities in men with schizophrenia or antisocial personality disorder, Barkataki [/bib_ref] [bib_ref] Corpus callosum abnormalities in psychopathic antisocial individuals, Raine [/bib_ref] , blood flow [bib_ref] Reduced frontotemporal perfusion in psychopathic personality, Soderstrom [/bib_ref] , Of the studies investigating whole-brain volume, one observed increased volume [bib_ref] Corpus callosum abnormalities in psychopathic antisocial individuals, Raine [/bib_ref] , while the other observed decreased volume [bib_ref] Volumetric structural brain abnormalities in men with schizophrenia or antisocial personality disorder, Barkataki [/bib_ref]. Oddly, both studies included participants with schizophrenia symptoms. [bib_ref] Volumetric structural brain abnormalities in men with schizophrenia or antisocial personality disorder, Barkataki [/bib_ref] , whose sample comprised 50% schizophrenia-affected participants, reported significant differences in intelligence between them and antisocial participants (who did not exhibit similar psychotic symptoms), along with reduced brain volume in comparison to nonviolent individuals and healthy controls. In contrast, [bib_ref] Corpus callosum abnormalities in psychopathic antisocial individuals, Raine [/bib_ref] reported that though the difference between participants and controls in psychotic symptoms was statistically significant (i.e., 40% met schizophrenia spectrum diagnosis criteria), they believed these symptoms did not significantly affect their participants' outcomes on the measures of interest, reporting increased volume as compared to controls. Yet such a conclusionthat the presence of psychosis would not be meaningful for a brain volume analysis of antisocial participantsis at odds with strong evidence for brain volume and function differences in psychotic individuals [bib_ref] Multimodal meta-analysis of structural and functional brain changes in first episode psychosis..., Radua [/bib_ref]. Further, the presence of psychotic symptoms is not typical or expected for antisocial or psychopathic individuals. Regionally, [bib_ref] Psychopathy and the posterior hippocampus, Laakso [/bib_ref] observed a positive correlation between the hippocampal volumes and age of the subjects, while also noting negative correlations between regional volume reductions and PCL-R Factor 1 scores. In analyzing blood circulation in the brain, [bib_ref] Reduced frontotemporal perfusion in psychopathic personality, Soderstrom [/bib_ref] reported no significant correlation between bloodflow and total PCL-R scores, though strong negative correlations were identified between the temporal blood flow and Factor 1 (emotional/affective) PCL-R scores, with a Spearman's rho of .34 for the right, and .49 for the left side. The highest-scoring subjects exhibited significantly lower bloodflow in the head of the caudate nucleus, the hippocampus, the left thalamus, the amygdala, and the medial and lateral frontal areas [bib_ref] Reduced frontotemporal perfusion in psychopathic personality, Soderstrom [/bib_ref].
Several studies have reported different activation patterns across a variety of tasks in the brains of individuals with psychopathic traits compared to those of controls. Specifically, reduced activation in areas influencing autonomic regulation (i.e., the rostral anterior cingulate cortex and right ventral striatum) in response to perceived personal distress or pain, and in the affective regulation areas (i.e., the amygdala and insula) in response to others' fear, distress and pain, has been reported in psychopathic individuals [bib_ref] Empathic responsiveness in amygdala and anterior cingulate cortex in youths with psychopathic..., Marsh [/bib_ref]. Similarly, reduced activation was found in areas responsible for reinforcement learning (i.e., the dorsal striatal circuits; [bib_ref] Empathic responsiveness in amygdala and anterior cingulate cortex in youths with psychopathic..., Marsh [/bib_ref] [bib_ref] Neural correlates of social cooperation and non-cooperation as a function of psychopathy, Rilling [/bib_ref].
Like , [bib_ref] Empathic responsiveness in amygdala and anterior cingulate cortex in youths with psychopathic..., Marsh [/bib_ref] subjects showed significantly less activation in the left amygdala, left middle and right anterior insula, anterior cingulate, right secondary somatosensory cortex, and the right ventromedial orbitofrontal cortex in the second half of the acquisition phase of the stimulus learning task. In contrast, [bib_ref] Temporal lobe abnormalities in semantic processing by criminal psychopaths as revealed by..., Kiehl [/bib_ref] reported that activation (in response to stimulus words) was overall similar between subjects and controls, except in the right anterior superior temporal gyrus for psychopathic individuals' responses between abstract and baseline phases [bib_ref] Temporal lobe abnormalities in semantic processing by criminal psychopaths as revealed by..., Kiehl [/bib_ref]. Further, subjects also exhibited reduced activation in these regions in response to concrete stimuli.
In a prisoners' dilemma game, male (but not female) subjects higher in psychopathy showed reduced activation in the right amygdala after a partner defected, and reduced activation within the rostral anterior cingulate cortex and dorsolateral prefrontal Personality Neuroscience cortex when choosing to defect [bib_ref] Neural correlates of social cooperation and non-cooperation as a function of psychopathy, Rilling [/bib_ref]. In response to the International Standardized Affective Picture System positive/ negative emotion task, subjects with higher psychopathy scores showed increased right-sided activation in prefrontal regions, anterior cingulate, and the amygdala for negative emotions, and reduced activation in the right subgenual cingulate and right medial temporal gyrus, left lobulus paracentralis, left dorsal cingulate, and left parahippocampal gyrus [bib_ref] Abnormalities in emotion processing within cortical and subcortical regions in criminal psychopaths:..., Müller [/bib_ref]. In contrast, positive emotions induced increased activation in the left gyrus frontalis, and right medial frontal and right medial temporal gyrus, leading to the conclusion that psychopathic individuals' emotional responsivity differed from that of other subjects [bib_ref] Abnormalities in emotion processing within cortical and subcortical regions in criminal psychopaths:..., Müller [/bib_ref].
## Gray matter
Multiple research papers [bib_ref] Psychopathy as a disorder of the moral brain: Fronto-temporo-limbic grey matter reductions..., De Oliveira-Souza [/bib_ref] [bib_ref] Aberrant paralimbic gray matter in criminal psychopathy, Ermer [/bib_ref] [bib_ref] Reduced prefrontal gray matter volume and reduced autonomic activity in antisocial personality..., Raine [/bib_ref] [bib_ref] Morphological alterations in the prefrontal cortex and the amygdala in unsuccessful psychopaths, Yang [/bib_ref] have reported observations of reduced gray matter volume in the orbitofrontal cortex of psychopathic individuals. [bib_ref] Morphological alterations in the prefrontal cortex and the amygdala in unsuccessful psychopaths, Yang [/bib_ref] , in comparing "successful" and "unsuccessful" psychopaths, reported that these (and other observed differences) applied only to the latter due to arrest histories. In addition, de [bib_ref] Psychopathy as a disorder of the moral brain: Fronto-temporo-limbic grey matter reductions..., De Oliveira-Souza [/bib_ref] observed gray matter volume reductions in the mid-anterior insula, and left anterior temporal cortex, while [bib_ref] Aberrant paralimbic gray matter in criminal psychopathy, Ermer [/bib_ref] noted gray matter volume reductions in the parahippocampal cortex, though these were not statistically significant in whole-brain analyses. [bib_ref] Aberrant paralimbic gray matter in criminal psychopathy, Ermer [/bib_ref] speculated that differences in gray matter in psychopathy are subtle and widespread, leading to minimal differentiation when whole-brain analyses are used. [bib_ref] Reduced prefrontal gray matter volume and reduced autonomic activity in antisocial personality..., Raine [/bib_ref] noted that observed reductions amounted to 11% compared to controls. These studies suggest that higher PCL-R scores may be associated with subtle reductions in gray matter volume across several paralimbic and limbic areas, implicated in emotion processing, theory-of-mind-related tasks, regulation, and behavioral control. However, distinction between individuals who have been arrested is important in interpreting these results given that the majority of psychopathy studies used forensic and institutional samples [bib_ref] Deficient fear conditioning in psychopathy: A functional magnetic resonance imaging study, Birbaumer [/bib_ref] [bib_ref] Psychopathy as a disorder of the moral brain: Fronto-temporo-limbic grey matter reductions..., De Oliveira-Souza [/bib_ref] [bib_ref] Aberrant paralimbic gray matter in criminal psychopathy, Ermer [/bib_ref] [bib_ref] Temporal lobe abnormalities in semantic processing by criminal psychopaths as revealed by..., Kiehl [/bib_ref] [bib_ref] Psychopathy and the posterior hippocampus, Laakso [/bib_ref] [bib_ref] Empathic responsiveness in amygdala and anterior cingulate cortex in youths with psychopathic..., Marsh [/bib_ref] [bib_ref] Abnormalities in emotion processing within cortical and subcortical regions in criminal psychopaths:..., Müller [/bib_ref] [bib_ref] Reduced frontotemporal perfusion in psychopathic personality, Soderstrom [/bib_ref] that is, these differences may not be evident in individuals with higher PCL-R scores but who avoid incarceration.
## The amygdala
Due to its association with emotional functioning, the amygdala has been a popular focus in neurobiological studies [bib_ref] Cortex and amygdala morphology in psychopathy, Boccardi [/bib_ref] [bib_ref] Amygdala reactivity and negative emotionality: Divergent correlates of antisocial personality and psychopathy..., Hyde [/bib_ref] [bib_ref] Amygdala hypoactivity to fearful faces in boys with conduct problems and callous-unemotional..., Jones [/bib_ref] [bib_ref] Reduced amygdala-orbitofrontal connectivity during moral judgments in youths with disruptive behavior disorders..., Marsh [/bib_ref] [bib_ref] Lower amygdala volume in men is associated with childhood aggression, early psychopathic..., Pardini [/bib_ref] [bib_ref] Reduced amygdala response in youths with disruptive behavior disorders and psychopathic traits:..., White [/bib_ref] [bib_ref] Localization of deformations within the amygdala in individuals with psychopathy, Yang [/bib_ref]. In studies that evaluated amygdala volume, one reported volume reductions [bib_ref] Localization of deformations within the amygdala in individuals with psychopathy, Yang [/bib_ref] , one reported increases [bib_ref] Cortex and amygdala morphology in psychopathy, Boccardi [/bib_ref] , and yet another reported no difference .
In observing amygdala activation patterns, right amygdala activation has been noted to increase in response to fearful faces [bib_ref] Amygdala hypoactivity to fearful faces in boys with conduct problems and callous-unemotional..., Jones [/bib_ref] , but decrease while comparing words related to legal and illegal acts [bib_ref] Reduced amygdala-orbitofrontal connectivity during moral judgments in youths with disruptive behavior disorders..., Marsh [/bib_ref]. Yet [bib_ref] Reduced amygdala response in youths with disruptive behavior disorders and psychopathic traits:..., White [/bib_ref] reported that during low attentional load trials subjects' amygdalae activation increased less than that of controls, as compared to high attentional load activation. Intrestingly, [bib_ref] Amygdala reactivity and negative emotionality: Divergent correlates of antisocial personality and psychopathy..., Hyde [/bib_ref] reported that when considered separately, APD and psychopathy traits did not have a significant relation to amygdala activation. Only when both were considered together did an association emerge. [bib_ref] Amygdala reactivity and negative emotionality: Divergent correlates of antisocial personality and psychopathy..., Hyde [/bib_ref] attributed this to suppression effects, whereby APD and psychopathy measures served as a "cooperative suppressor" for each other [bib_ref] Two replicable suppressor situations in personality research, Paulhus [/bib_ref] , amplifying the effects of the other on amygdala activation. Thus, [bib_ref] Amygdala reactivity and negative emotionality: Divergent correlates of antisocial personality and psychopathy..., Hyde [/bib_ref] argued that one of the two constructs (antisocial personality or psychopathy) was unrelated to amygdala activation, yet raised the impact of the other on the predictive ability of the model as a whole [bib_ref] Two replicable suppressor situations in personality research, Paulhus [/bib_ref].
## Integrating environmental influences into brain research
Environmental covariates were rarely considered in brain research. Only 4 of the aforementioned 20 studies included such variables. Yet, developmental research has consistently shown that neglected, maltreated, or stress-exposed children exhibit many of the same brain abnormalities observed in in these studies. For instance, smaller left amygdala volumes and significant whole-brain gray matter reductions have been reported in post-institutionalized children [bib_ref] Amygdala, hippocampal and corpus callosum size following severe early institutional deprivation: The..., Mehta [/bib_ref] , as well as localized hippocampal, insular, orbitofrontal cortex, anterior cingulate gyrus, and caudate volume reductions [bib_ref] Limbic scars: Long-term consequences of childhood maltreatment revealed by functional and structural..., Dannlowski [/bib_ref]. [bib_ref] Childhood experience and the expression of genetic potential: What childhood neglect tells..., Perry [/bib_ref] reported abnormal, atypical frontal-occipital circumference development of children who were chronically neglected. Though multiple studies have found increased amygdala responsivity to acute stress in people who have experienced significant early-life environmental stress or adversity [bib_ref] Developmental change in amygdala reactivity during adolescence: Effects of family history of..., Swartz [/bib_ref] [bib_ref] FKBP5 and emotional neglect interact to predict individual differences in amygdala reactivity, White [/bib_ref] , others have reported decreased amygdala activation in individuals resilient to post-traumatic stress disorder [bib_ref] Corticolimbic blood flow during nontraumatic emotional processing in posttraumatic stress disorder, Phan [/bib_ref] , and reciprocal and opposing relations between activation in the amygdala and medial prefrontal cortex [bib_ref] Regional cerebral blood flow in the amygdala and medial prefrontalcortex during traumatic..., Shin [/bib_ref].
In a first-of-its-kind recent study, [bib_ref] Primary and secondary variants of psychopathy in a volunteer sample are associated..., Sethi [/bib_ref] distinguished among psychopathic individuals with high anxiety and maltreatment (who may be classified with "secondary" psychopathy), and those without adversity or anxiety (i.e., with "primary" psychopathy). Despite similar interpersonal/affective facet scores among the two groups, the researchers indeed found differences among them in both fear-related brain activation patterns, and in amygdala activation (which was reportedly typical in those with maltreatment histories). This observation led the [bib_ref] Primary and secondary variants of psychopathy in a volunteer sample are associated..., Sethi [/bib_ref] to speculate that abnormalities in fear conditioning may play a role in the development of Factor 1 traits in the presence of anxiety, possibly in response to environmental trauma.
## Psychophysiological manifestations
Several psychophysiological differences have been noted in biological research. [bib_ref] Systematic review, structural analysis, and new theoretical perspectives on the role of..., Yildirim [/bib_ref] proposed that these result from neurological and neurotransmitter activity differences that produce typical baseline arousal levels while dampening arousal during stressful, frightening, or adverse social experiences. Indeed, [bib_ref] Reduced prefrontal gray matter volume and reduced autonomic activity in antisocial personality..., Raine [/bib_ref] reported that during a social stressor task, antisocial individuals exhibited reduced autonomic activity, skin conductance, and heart rate. In a meta-analysis of 95 studies, [bib_ref] Psychophysiology of aggression, psychopathy, and conduct problems: A meta-analysis, Lorber [/bib_ref] found that low resting electrodermal activity (EDA) and low task EDA were associated with psychopathy, and that psychopathy was negatively associated with EDA reactivity. However, Lorber's (2004) analysis did not identify a significant effect for 8 A Frazier et al.
resting heart-rate or heart-rate reactivity when aggression was considered separately from the psychopathy construct. In studying antisocial behavior,found that across 114 studies, antisocial behavior was related to low resting heart rate, with no moderating impact of age, sex, study design. However,metaanalysis demonstrated that across measurements of antisociality (e.g., offending behavior, violence, aggression, psychopathy) studies differed significantly in their findings. Similarly, in a sample of Asian "primary" and "secondary" psychopathic women, the latter exhibited much higher heart rate variability across rest and stress tasks, despite significant difference in interpersonal/affective facet scores in both groups [bib_ref] Female primary and secondary psychopathic variants show distinct endocrine and psychophysiological profiles, Goulter [/bib_ref]. In a sample of juveniles, a lower blink rate was observed among those labeled with "primary" (as compared to "secondary") psychopathy [bib_ref] Affective startle potentiation differentiates primary and secondary variants of juvenile psychopathy, Kimonis [/bib_ref]. [bib_ref] Psychophysiology of aggression, psychopathy, and conduct problems: A meta-analysis, Lorber [/bib_ref] suggested that contradictory findings may be attributable to heterogeneity in the behavioral construct (e.g., "successful" vs. "unsuccessful"; [bib_ref] Morphological alterations in the prefrontal cortex and the amygdala in unsuccessful psychopaths, Yang [/bib_ref]. Indeed, some have suggested that the construct includes so many diverse presentations that a multitude of etiological pathways should be considered [bib_ref] Two approaches to parsing the heterogeneity of psychopathy, Brinkley [/bib_ref]. A further consideration of this multiple-pathway hypothesis can benefit from integration of biological findings with developmental literature.
## Physiology and stress
Related to the documented neurological and neurochemical changes in stress-exposed children, alterations in physiological manifestation have been documented as well. The physiological consequences of these neurological changes are similarly diverse, with individuals' affected traumatic stress symptoms exhibiting increased ANS activation during acute stress, while others exhibit reduced activation along with fewer PTSD symptoms [bib_ref] Neurobiological sequelae of childhood trauma: PTSD in children, Perry [/bib_ref] [bib_ref] The neuroarcheology of child maltreatment: Evidence for altered neurodevelopment following traumatic abuse, Perry [/bib_ref] [bib_ref] Heart period and variability findings in preschool children with posttraumatic stress symptoms, Scheeringa [/bib_ref]. These findings have prompted speculation that adaptive dissociative states may stabilize into traits in certain instances [bib_ref] Childhood trauma, the neurobiology of adaptation, and use dependent development of the..., Perry [/bib_ref].
## Making sense of neurobiological findings
While overarching conclusions on the neuobiological mechanisms underlying psychopathic traits cannot be made due to contradictory findings, a theoretical trend emerges from the explored evidence. Suppressed activation has been noted across genetic, neurological, and psychophysiological findings. As for genetic evidence, the low-expression MAO-A variant is understood as having an inhibitory effect on the enzyme encoded by it [bib_ref] Role of genotype in the cycle of violence in maltreated children, Caspi [/bib_ref] [bib_ref] The effects of child maltreatment on early signs of antisocial behavior: Genetic..., Cicchetti [/bib_ref]. Although the exact nature of its influence is unclear [bib_ref] Serotonin transporter gene associations with psychopathic traits in youth vary as a..., Sadeh [/bib_ref] [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref] , the low-activity serotonin transporter (5-HTT) variant has also been linked with antisociality [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref]. As for neurological studies, many have reported a comparative suppression of neural activity and/or volume in designated brain structures like the orbitofrontal cortex, frontal lobes, and amygdalae [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref] [bib_ref] Risky decisions and response reversal: Is there evidence of orbitofrontal cortex dysfunction..., Mitchell [/bib_ref] [bib_ref] Reduced right hemisphere activation in severely abused violent offenders during a working..., Raine [/bib_ref]. Moreover, in considering psychophysiological evidence, a pattern of repressed activity and/or physical responses is also pertinent, as evidenced in studies revealing depressed skin conductance and heart rate in antisocial participants [bib_ref] Psychophysiology of aggression, psychopathy, and conduct problems: A meta-analysis, Lorber [/bib_ref] [bib_ref] Reduced prefrontal gray matter volume and reduced autonomic activity in antisocial personality..., Raine [/bib_ref].
Despite the noted trend, many findings challenge the proposed pattern of suppressed activation. For this reason, these observations allow for little in the way of definitive conclusions. For instance, a link between a high-activity COMT variant and higher "emotional dysfunction" has been found [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref]. A higher-activity 5-HTT variant has also been shown to exert greater influence on affective-interpersonal traits when compared to its lower-activity counterpart [bib_ref] Analysis of monoaminergic genes, childhood abuse, and dimensions of psychopathy, Sadeh [/bib_ref]. In addition, higher activation of certain brain structures (see [bib_ref] Abnormalities in emotion processing within cortical and subcortical regions in criminal psychopaths:..., Müller [/bib_ref] has been associated with emotional reactions to both negative and positive stimuli in psychopathic subjects.
When it comes to neurotransmitter evidence, applying the suppressed activation theory becomes an even greater challenge. If the low-expression MAO-A variant encodes a lower-activity enzyme that reduces the breakdown of monoamine neurotransmitters, it is reasonable to theorize that this may result in increased synaptic presence of the same neurotransmitters [bib_ref] Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice..., Cases [/bib_ref] [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref]. However, while increased presence (or decreased degradation) of neurotransmitters appears to contradict the overarching pattern of suppression, the absolute or partial inhibitory effects associated with some of these neurotransmitters (e.g., absent empathy, or decreased psychophysiological responses; [bib_ref] Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice..., Cases [/bib_ref] [bib_ref] Impaired maternal behavior in mice lacking norepinephrine and epinephrine, Thomas [/bib_ref] [bib_ref] Systematic review, structural analysis, and new theoretical perspectives on the role of..., Yildirim [/bib_ref] are in line with a suppression pattern.
One of the greatest challenges in drawing any trend-like conclusions concerning neurotransmitters is the fact that there appears to be a higher degree of inconsistency in the examined literature than that observed in genetic and brain structure research. For example, studies into the relationship between serotonin and aggression have produced only mixed results, with some offering support for a negative correlation (as cited by [bib_ref] Natural born killers: The genetic origins of extreme violence, Ferguson [/bib_ref] , while others point in the direction of a positive one [bib_ref] The effect of increased serotonergic neurotransmission on aggression: A critical meta-analytical review..., Carrillo [/bib_ref] [bib_ref] Serotonin transporter availability in impulsive aggressive personality disordered patients: A PET study..., Van De Giessen [/bib_ref]. It has been suggested that aggression type (reactive vs. proactive) is likely responsible for this inconsistency [bib_ref] The emergence of psychopathy: Implications for the neuropsychological approach to developmental disorders, Blair [/bib_ref] [bib_ref] The effect of increased serotonergic neurotransmission on aggression: A critical meta-analytical review..., Carrillo [/bib_ref] [bib_ref] Serotonin transporter availability in impulsive aggressive personality disordered patients: A PET study..., Van De Giessen [/bib_ref]. This proposed distinction underscores the intricacy of characteristics underlying psychopathy and its many risk factors. Similarly, the distinction between "primary" and "secondary" psychopathy, which has thus been little investigated in this context [bib_ref] Female primary and secondary psychopathic variants show distinct endocrine and psychophysiological profiles, Goulter [/bib_ref] [bib_ref] Primary and secondary variants of psychopathy in a volunteer sample are associated..., Sethi [/bib_ref] , may explain contradictory findings.
In addition to specific distinctions, determining the role individual neurotransmitters play in psychopathy is convoluted by the notion that some appear to have a differing, synergistic-like effect when interacting with one another. This is evident in the reported interactive effect of dopamine and epinephrine in producing both increased aggression and reduced empathy [bib_ref] Norepinephrine-dopamine interactions and behavior, Antelman [/bib_ref] [bib_ref] Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice..., Cases [/bib_ref] [bib_ref] Impaired maternal behavior in mice lacking norepinephrine and epinephrine, Thomas [/bib_ref] ; these qualities were not observed when epinephrine was examined in isolation [bib_ref] Neuroendocrine correlates of antisocial personality disorder in abstinent heroin-dependent subjects, Gerra [/bib_ref]. Furthermore, an interaction between cerebrospinal fluid dopaminergic and serotonergic metabolite levels has been speculated to underlie aggression [bib_ref] New evidence for an association between the CSF HVA: 5-HIAA ratio and..., Soderstrom [/bib_ref] , but this interaction has been rarely studied, while studies frequently examined serotonin alone (see [bib_ref] The effect of increased serotonergic neurotransmission on aggression: A critical meta-analytical review..., Carrillo [/bib_ref]. Because many studies have such a narrow scope, bridging the gap between their results becomes a difficult task. Moving beyond general observations of suppression, in a direction that leads to a fuller understanding of the interplay between all of the explored evidence, would presently be unfeasible. The inconsistency in findings appears to be rooted in a number of factors including variability in conceptualizations and measurements used across studies (Kim- [bib_ref] MAOA, maltreatment, and gene-environment interaction predicting children's mental health: New evidence and..., Kim-Cohen [/bib_ref] [bib_ref] Psychophysiology of aggression, psychopathy, and conduct problems: A meta-analysis, Lorber [/bib_ref] ; absent consideration of environmental experiences that interact with one's genotype, and their epigenetic outcomes [bib_ref] Psychopathy trait scores in adolescents with childhood ADHD: The contribution of genotypes..., Fowler [/bib_ref] ; and the use of broad categories and classifications for traits and behaviors that may, in fact, have different underlying neurobiological characteristics (e.g., measuring aggression without accounting for aggression types, as discussed earlier; [bib_ref] The effect of increased serotonergic neurotransmission on aggression: A critical meta-analytical review..., Carrillo [/bib_ref]. Similar arguments may be made based on emergent research into primary and secondary variants of psychopathy [bib_ref] Female primary and secondary psychopathic variants show distinct endocrine and psychophysiological profiles, Goulter [/bib_ref] [bib_ref] Affective startle potentiation differentiates primary and secondary variants of juvenile psychopathy, Kimonis [/bib_ref] [bib_ref] Primary and secondary variants of psychopathy in a volunteer sample are associated..., Sethi [/bib_ref]. It is probable that other distinctions and interactions have yet to be uncovered; and until they are, understanding the relationship between genes, neurotransmission, brain activity, and psychophysiology in psychopathy remains obscured for the most part.
## Neurobiology and development: directions for interdisciplinary collaboration
Considering the potential for early-life epigenetic change and early evolution of neurobiological differences produced by stress and adversity, it is critical that longitudinal perspectives be taken in psychopathy research utilizing neurobiological measures. In fact, cross-sectional designs using 7-year-olds may offer little added benefit to using adult participants, considering some changes are likely to occur in the first 3 years of life. In contrast, given reliance on incarcerated samples, much of the extant research excludes individuals who have entered and passed midlife, hindering predictions about stability of psychopathy traits over time. Furthermore, any observations made during adulthood are best interpreted as snapshots in time, which cannot inform our understanding of etiology nor prognosis, given later-in-life biological and behavioral changes (e.g., [bib_ref] Structural coherence and temporal stability of psychopathic personality features during emerging adulthood, Hawes [/bib_ref] [bib_ref] Understanding the downward extension of psychopathy to youth: Implications for risk assessment..., Vitacco [/bib_ref]. For instance, psychopathy reportedly increases in adolescence and diminishes as individuals reach young adulthood [bib_ref] Child and adolescent psychopathy: Like a painting by Monet, Salekin [/bib_ref]. Similarly, psychopathy scores (and offending behaviors) are generally significantly reduced as previously labeled ex-inmates enter into their 60s [bib_ref] Homicide, psychopathy, and aging -A nationwide register-based case-comparison study of homicide offenders..., Putkonen [/bib_ref] , suggesting an instability in psychopathy over the life course.
Moreover, the current understanding of genetic and neurobiological psychopathic mechanisms is based primarily on observations of incarcerated men of European descent. This limitation is particularly problematic to genetic research, since different ethnic groups vary in genetic manifestation of various conditions [bib_ref] The importance of race and ethnic background in biomedical research and clinical..., Burchard [/bib_ref] , including psychopathy [bib_ref] Neither antisocial personality disorder nor antisocial alcoholism is associated with the MAO-A..., Lu [/bib_ref] and its socio-cultural interpretation [bib_ref] Assessing measurement invariance of PCL-R assessments from file reviews of North American..., Mokros [/bib_ref]. Multicultural investigations can be uniquely informative, since non-invariance in the psychopathy construct between samples from similar cultural backgrounds (i.e., North Americans and Germans) has been noted [bib_ref] Assessing measurement invariance of PCL-R assessments from file reviews of North American..., Mokros [/bib_ref]. Research using community samples, women, and people of non-European heritage is necessary before any sweeping etiological conclusions can be drawn.
Also critical to consider are the ways constructs are measured across current research. Psychopathy is measured in a wide variety of ways, with some measures relying heavily on aggression and criminal justice involvement. Moreover, reliance on criminal history in the measurement of psychopathy is particularly problematic given findings that some physiological differences in individuals with arrest histories did not appear in those without such histories. Similarly, adversity-exposure measurement is often problematic in that different instruments capture different adverse events and all exclude adversity that occurs during the first 3 years of life. Furthermore, many measures do not assess the most invisible form of maltreatment: deprivation and neglect [bib_ref] Childhood experience and the expression of genetic potential: What childhood neglect tells..., Perry [/bib_ref]. Though an inherently challenging endeavor, researchers should go beyond self-report questionnaires onto developing and utilizing measures that assess early-life experience holistically.
# Limitations
In the current review, authors sought to summarize and evaluate the state of knowledge on the etiology of psychopathic traits, by focusing attention on research into genetic, neuroanatomical, and neurotransmission activity correlates of psychopathic and antisocial traits. Inevitably, such a pursuit is fraught with challenges related to the differing conceptualizations and measurements of the psychopathy construct. While a broad view of psychopathic personality is useful for a first effort, it should likely be preceded with more narrow reviews and meta-analytic efforts. Yet it is uncertain that these, too, would yield a clearer answer for the question at hand. For instance, a review of a subset of studies using PCL instruments (46% of the studies included in this review) did not produce increased consistency of findings regarding genetic or neurobiological mechanisms. That is, utilizingdefinition of psychopathy (to the exclusion of others) did not appear to lead to a better overall understanding of its etiology. It is possible that additional research exists that has not been included in this review (e.g., because it is yet to be published, or because it was unavailable at the time), and which may add support for some of the more pronounced findings. Furthermore, no statistical analysis was performed for the purpose of this review, and thus, it is possible that some findings, while inconsistent across studies, are nonetheless meaningful in the samples in which they were observed, or across several of these samples. Meta-analytic strategies to evaluate subsets of these findings (e.g., regarding genetic influences) can shed further light on these questions.
# Implications and conclusions
While it is important to consider the current status of knowledge and ways to advance research into psychopathic traits, it is equally important that an integrated perspective inform current practices in management and treatment of psychopathic individuals. Specifically, clinicians and legal professionals should consider how yet-unsubstantiated theories about its etiology have affected both prognosis and treatment of psychopathy. It is likely that, in the absence of a conceptualization of psychopathy as a biological malady, clinicians would perceive both psychopathy treatment and its desired outcomes differently. This is particularly important since the belief in its innate, immutable nature has affected the use of the psychopathy construct in high-stakes legal situations (e.g., death penalty sentencing hearings; [bib_ref] Juror perceptions of the interpersonal-affective traits of psychopathy predict sentence severity in..., Cox [/bib_ref] , with overwhelmingly negative outcomes.
Prognosis. Based on anecdotal evidence from case studies such as, along with hereditary research, psychopathy has largely been perceived as immutable and untreatable [bib_ref] Psychopathy, treatment change, and recidivism in high-risk, high-need sexual offenders, Looman [/bib_ref] [bib_ref] Psychopathy, treatment involvement, and subsequent violence among civil psychiatric patients, Skeem [/bib_ref]. Yet research demonstrates that, even without treatment, psychopathic traits and temperamental qualities are not stable over time [bib_ref] Ten-year rank-order stability of personality traits and disorders in a clinical sample, Hopwood [/bib_ref] [bib_ref] Maturity and change in personality: Developmental trends of temperament and character in..., Josefsson [/bib_ref] [bib_ref] Patterns and sources of continuity and change of energetic and temporal aspects..., Kandler [/bib_ref] [bib_ref] A test of the vulnerability model: Temperament and temperament change as predictors..., Laceulle [/bib_ref]. Even in actuarial recidivism risk assessment, considerations for the passage of time and aging have been recommended [bib_ref] Adjusting actuarial violence risk assessments based on aging or the passage of..., Harris [/bib_ref]. Given associations between psychopathy and early-life stress, it is notable that the idea 10 A Frazier et al.
of change has been even more pronounced across traumatic stress literature. [bib_ref] Childhood experience and the expression of genetic potential: What childhood neglect tells..., Perry [/bib_ref] found that in following chronically neglected children over time, recovery of brain structure and function over a year period was inversely proportional to age of removal from the neglectful environment, noting that even the oldest children made significant gains over time. Is it then possible that psychopathy is not as immutable as many believe? To assess this, it is worthwhile to consider common treatment strategies. Treatment. Many of the treatment paradigms targeting psychopathy are based in correctional settings, with the expressed goal of increasing remorse and reducing criminal or violent behavior [bib_ref] Treatment of psychopathy, Harris [/bib_ref] [bib_ref] Psychopathy, treatment change, and recidivism in high-risk, high-need sexual offenders, Looman [/bib_ref]. Yet few clinicians would recommend a similar treatment for trauma-affected populations; aggressive or violent behavior in this context is typically seen as one of many traumatic stress symptoms to be addressed in treatment, and self-reproachfulness is often explicitly discouraged [bib_ref] Complex trauma and aggression in secure juvenile justice settings, Ford [/bib_ref]. Further, trauma treatment commonly involves characteristics that are diametrically opposed to those seen in offender treatment. If clinicians treating individuals with psychopathic traits were to borrow from the complex trauma literature (e.g., [bib_ref] Examining child maltreatment through a neurodevelopmental lens: Clinical applications of the neurosequential..., Perry [/bib_ref] , they would recognize the possibility that many attempts at psychopathy treatment have not worked because of their failure to include the necessary elements for neurodevelopmental change: willing participation in consistent, patterned interaction with a caring, trusted person.
# Conclusion
For several decades, research has focused intensely on identifying the biological underpinnings of psychopathy, under the assumption that this construct represented a unification between life-course stable, immutable traits. Yet despite the passage of much time, a significant investment, and substantial technological advances in measurements of biological differences, few consistent conclusions can be made from this research. Indeed, some differences in neurological structure and activation patterns have been observed, but these leave the research community no closer to identifying an etiology for the construct. Practitioners, nonetheless, have eagerly adopted a biologically deterministic perspective on psychopathy, utilizing research measures to determine the fates of individuals accused of crimes (including children; for an example see [bib_ref] Diagnostic labeling in juvenile justice settings: Do psychopathy and conduct disorder findings..., Rockett [/bib_ref] , and to make decisions regarding treatment procedures and outcomes [bib_ref] A survey of psychological test use patterns among forensic psychologists, Archer [/bib_ref] [bib_ref] Nipping psychopathy in the bud: An examination of the convergent, predictive, and..., Odgers [/bib_ref] [bib_ref] Psychopathy and therapeutic pessimism: Clinical lore or clinical reality?, Salekin [/bib_ref]. This, in turn, could lead more individuals to experience the kind of treatment that may only serve to perpetuate the lack of remorse, empathy, and human connection presumed to underlie psychopathy. Ultimately, even the staunchest supporters of the biological hypothesis can agree that environmental influences cannot be excluded from a complete understanding of psychopathy. It is critical that in both research and practice, the influence of environment be evaluated alongside other factors affecting the development of psychopathic traits. Hence, future research should provide potential to evaluate neurodevelopmental change (whether through epigenetic mechanisms or neural plasticity) and predictors thereof, since these will likely serve to inform both prevention and intervention efforts targeting psychopathic individuals.
[table] Personality Neuroscience Table 1: Characteristics of the reviewed studies [/table]
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Cost and operational impact of promoting upfront GeneXpert MTB/RIF test referrals for presumptive pediatric tuberculosis patients in India
BackgroundOutreach and promotion programs are essential to ensuring uptake of new public health interventions and guidelines. We assessed the costs and operation dynamics of outreach and promotion efforts for up front Xpert MTB/RIF (Xpert) testing for pediatric presumptive tuberculosis (TB) patients in four major Indian cities.MethodsXpert test costs were assessed as weighted average per-test costs based on the daily workload dynamics matched by test volume specific Xpert unit cost at each study site. Costs of outreach programs to recruit health providers to refer pediatric patients for Xpert testing were assessed as cost per referral for each quarter based on total program costs and referral data. All costs were assessed in the health service provider's perspective and expressed in 2015 USD.ResultsWeighted average per-test costs ranged from $14.71 to $17.81 at the four laboratories assessed. Differences between laboratories were associated with unused testing capacity and/or frequencies of overtime work to cope with increasing demand and same-day testing requirements. Outreach activities generated between 825 and 2,065 Xpert testing referrals on average each quarter across the four study sites, translating into $0.63 to $2.55 per patient referred. Overall outreach costs per referral decreased with time, stabilizing at an average cost of $1.10, and demonstrated a clear association with increased referrals.ConclusionsXpert test and outreach program costs within and across study sites were mainly driven by the dynamics of Xpert testing demand resulting from the combined outreach activities. However, these increases in demand required considerable overtime work resulting in additional costs and operational challenges at the study laboratories. Therefore, careful laboratory operational adjustment should be evaluated at target areas in parallel to the anticipated demand from the Xpert referral outreach program scale-up in other Indian regions.
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# Introduction
In 2016, India accounted for more than one quarter of world's reported TB cases and deaths despite a dramatic 37% improvement in case notification rate between 2013 and 2016. Globally, children (aged <15 years) accounted for 6.9% of the notified new TB cases in 2016. Given that prompt diagnosis and linkage to care are integral components of TB control, the Indian government has made it a priority to expand rapid molecular diagnostic capacity for TB using Xpert MTB/RIF (referred to as Xpert from here on; Cepheid, Sunnyvale) test as part of India's plan to eliminate TB by 2025. However, scale-up of Xpert testing capacity and coverage alone does not ensure that all patients who could benefit from Xpert are referred for testing. Education and outreach activities to make health providers aware of the need and local testing availabilities are thus necessary to ensure uptake of Xpert testing.
Between April 2014 and June 2016, the Foundation for Innovative New Diagnostics (FIND), in collaboration with the Revised National Tuberculosis Control Program (RNTCP), conducted a large-scale project to catalyze the adoption of upfront Xpert testing in presumptive pediatric TB cases as a routine clinical practice utilizing multiple outreach interventions-phone calls, one-on-one provider sensitization meetings, and Continuing Medical Education (CME) workshops for public and private sector health providers. This project was undertaken in four major cities of India: New Delhi, Chennai, Hyderabad and Kolkata with a total of 3,670 health providers educated and informed of the upfront Xpert testing strategy for presumptive pediatric TB patients. The phone calls and one-on-one meetings with health providers included descriptions of project deliverables and mandates as well as informing the health providers of the availability of testing facilities and relevant patient referral procedures. CME workshops further included presentations by key speakers providing the details of the World Health Organization (WHO) guidelines on the use of Xpert for treatment of TB patients and promoting referrals of presumptive pediatric TB patients for Xpert testing.
These demand-generating activities were an integral component of the program and helped to increase access to testing and optimize efficiency of testing. During the study period, the program provided 46,879 GeneXpert tests to 42,238 children identifying 3,340 TB cases. More than 92% of the patients received test results within 24 hours of sample receipt at the lab. This program is now fully managed by the RNTCP and has been expanded to an additional six cities as of 2017 -Surat, Visakhapatnam, Guwahati, Bangalore, Nagpur and Indore-with plans to further scale-up to other cities in India.
Given the positive impact of the FIND/RNTCP pediatric Xpert implementation project and the RNTCP's commitment to expand the combined outreach program alongside of increasing Xpert testing coverage, it is important to evaluate the cost implications of the combined outreach efforts and their impact on laboratory operations and workloads. We used the FIND/ RNTCP pediatric Xpert implementation project data to provide empiric evidence on costs of outreach program operations, and their impact on cost and operation of the Xpert testing at four project laboratories.
# Methods
The objective of our work was to assess the economic costs of the FIND/RNTC outreach program in promoting upfront Xpert testing for pediatric presumptive TB patients and respective laboratory testing costs at the four study locations from the health systems perspective. As such, we 1) evaluated costs of the three outreach programs, 2) evaluated the cost dynamics of Xpert tests associated with changes in the workload observed at four laboratories during the study period; and 3) explored how outreach efforts influenced the demand for Xpert referrals for pediatric TB diagnosis.
## Find/rntcp pediatric xpert implementation project
Data were collected from a USAID-funded implementation project of upfront GeneXpert MTB/RIF testing of presumptive pediatric TB patients. The project enrolled pediatric (age 0-14 years) presumptive TB and Drug-Resistant (DR) TB patients in the catchment areas of the four RNTCP reference level laboratories in New Delhi, Chennai, Hyderabad and Kolkata. Patients provided various types of specimen (e.g. induced sputum, gastric lavage/aspirate, BAL, CSF, lymph node aspirates, etc.) for Xpert testing. Test results were promptly communicated to the health providers by e-mail and short messaging service (SMS).
## Evaluation of xpert per-test unit cost
For New Delhi, Hyderabad and Kolkata laboratories, we first calculated a reference table of Xpert per-test costs relevant to sample batch sizes ranging between 1 and 16 tests using bottom-up activity-based costing (ABC) framework. For Chennai, overhead costs were difficult to obtain; thus, we used the New Delhi lab costs based on its similarity in size and capacity. Data on all resource inputs required for Xpert testing was obtained from Time and Motion (TAM) studies [bib_ref] A comparison of four approaches for measuring clinician time use, Bratt [/bib_ref] conducted as part of previous FIND cost and cost-effectiveness studies of Xpert in India, Vietnam, and Malawi. The TAM data was collected based on repeated direct observations of sample batch sizes between 1 to 16 tests that typically covers a range of daily workload capacity using a single GeneXpert IV (GX4) machine. We opted to use the existing TAM data instead of conducting a new TAM as Xpert testing procedure is simple and highly standardized such that operator variability would not be a significant factor influencing the unit cost of an Xpert test. A resource-use and overhead cost questionnaire was utilized to reflect site specific costs, resource use and unit prices. Resource inputs were categorized as overhead, building, equipment, staff, laboratory reagents and chemicals, and consumables. Unit price of commodities (e.g. laboratory reagents, chemical, consumables), staff salaries, and overhead cost data were obtained from each laboratory's relevant accounting and inventory management records for the 2015 fiscal year. As government operated laboratories do not pay for building and utility related costs, these data were supplemented from a similar private non-profit laboratory in India offering Xpert test. Use and costs of capital assets were based on annualized costs using a 3% discount rate [bib_ref] The real and the nominal? Making inflationary adjustments to cost and other..., Kumaranayake [/bib_ref] and assumed expected life-years as specified in FIND's internal documents. All costs were evaluated and expressed in 2015 United States Dollars (USD) where cost data items in local currencies were converted to the USD according to the average UN operational exchange rate in 2015.
## Costs associated with unused genexpert equipment capacity
As underused Xpert testing capacity is an important opportunity cost associated with the implementation of Xpert testing, we sought to incorporate the costs of unused Xpert testing capacity as part of the per-test unit costs. In the case of Xpert testing, the type of GeneXpert testing platform likely determines the maximum daily testing capacity in a fixed laboratory operating time. The four study sites used GX4 units, which were equipped with four testing modules (each slot as able to hold one Xpert cartridge). Given that each Xpert test takes around 2 hours to complete, a single GX4 machine would have a daily maximum capacity to process 16 tests in a laboratory that normally operates between 8 and 10 hours each day. Any number of tests performed less than 16 in each operational day would result in incorporation of costs associated with unused GX4 module(s). For example, if a laboratory performed 10 Xpert tests in a given day, we added costs equivalent to 6 individual GX4 module capacities as part of the total testing costs for that day.
## Workload analysis and weighted per-test cost
Using the daily workload data at each laboratory, we assigned a reference per-test cost to each operational day and then calculated the weighted average per-test cost of an Xpert test unique for each study site. Since some laboratories had multiple GX4 units and had workload requiring overtime operations, we assumed sample batching would be cost-optimized for GX4 utilization (i.e. a combination that would have lowest cost associated with unused GX4 capacity). For example, if 28 tests were run on a given day, we determined three sample batching of one 16-test, one 10-test batch, and one 2-test batch would be cost-optimized. This process was repeated for each laboratory using laboratory specific reference per-test cost and averaged over the length of the experiment to obtain the average weighted per-test Xpert cost. A sample calculation can be viewed in S1 Calculation.
To ensure 24-hour turn-around of Xpert test results, some laboratories were equipped with more than one GX4 machines and operated overtime when workloads were beyond their normal testing capacity. To assess the burden of overtime work, we calculated the proportion of days with workloads exceeding each laboratory's normal testing capacity (determined based on the number of GX4 machines available and a standard 8 hour work day). Costs associated with overtime work were then assessed based on the total estimated hours of overtime multiplied by hourly staff wages and laboratory overhead costs. We assumed each incremental batch of 4 tests (1 to 4 tests) performed beyond normal testing capacity took at least two hours of operational time to run.
## Evaluation of outreach spending
Any health provider in the four cities with presumptive pediatric TB patients was eligible to refer patients for free pert testing. To promote referrals for Xpert testing during the study, our trained project staff placed phone calls, held one-on-one meetings and conducted CME workshops in the four cities.
Health providers catering to the healthcare needs of the pediatric population were mapped and were systematically approached first by telephone. Telephone contact was followed-up, where feasible, by a one-on-one meeting between the health provider and a member of the project team. These efforts were complemented by periodic comprehensive CME events. During telephone calls and one-on-one meetings, representatives of the project explained the Xpert test and how to refer presumptive pediatric TB patients for a free upfront Xpert test for bacteriologic diagnosis. CME events also covered topics relevant to the diagnosis and treatment of pediatric TB.
Cost data on each of the outreach efforts included staff wages, estimated staff and activity time associated with planning and carrying out each of the outreach activities, expenditure data on consumable, per-diem, phone call charges, travel related costs (for CMEs and one-onone visits), and time required for management and supervision. Regarding transportation costs, staff often visited multiple providers or locations per trip and used public transportation. However, detailed records were not kept, so our costing assumes one CME or meeting per trip and a standard distance of 10km travelled using a taxi.
Data on the telephone calls and one-on-one meetings (e.g. number calls or visits made, duration, expenses) were collected for a representative monthly sample of calls and visits made during the study period. Data on CME were obtained through retrospective review of all CME meeting and relevant FIND financial records.
We estimated the unit costs of each outreach activity using a top-down cost analysis method where the total expenditure on consumables, staff time, and transportation for a given recruitment activity was divided by the frequency of each activity conducted during the study period to compute the average unit costs. Overhead costs were calculated as 15% of operational costs as per FIND's overhead pricing structure. To express uncertainties in our cost estimates, we constructed high and low-cost estimates based on varied levels of staff and transportation costs (+/-20%) and percent allotment for overhead (ranges: 10 to 20%) respectively.
Data entry and analyses were performed using Excel 2016 (Microsoft Corporation) and R (version 3.5.0) for workload analyses.
# Funding
The FIND/RNTCP pediatric GeneXpert implementation project was funded by United States Agency for International Development (USAID) under the Challenge TB project. FIND was responsible for the implementation, training, coordination, monitoring, data analysis and writing of the report in close coordination with the Central TB Division, Ministry of Health and Family Welfare, Government of India.
## Ethics
Xpert testing for pediatric presumptive TB cases is an approved intervention under the RNTCP. The current project was undertaken by FIND, after approval from and in collaboration with RNTCP. As such, the results presented here are our experience-sharing of implementing approved interventions in a programmatic setting within the existing accredited RNTCP TB diagnostic lab network. Since the observations described here are a part of implementation of approved interventions under the RNTCP and a part of standard TB care in India, separate ethical clearance was not required. The only patient data used for this work was the date of GeneXpert testing and this data was fully anonymized before being shared for analysis.
# Results
## Xpert cost and laboratory operations
Reference per-test costs ranged from $13.74 to $32.14 in New Delhi with the lowest cost associated with a batch size of 16. In Kolkata, unit costs ranged from $13.55 to $30.23 and from $13.74 to $30.85 in Hyderabad (S1 . We were not able to evaluate overhead costs in Chennai due to the complexity of its operation (a mix of routine clinical and research tasks) making it difficult to disintegrate overhead cost for routine Xpert testing only. The major cost drivers were equipment costs and reagents (mainly the cost of the GeneXpert machine-decreasing with increasing batch size-and the Xpert cartridge) while overhead and direct staff hands-on cost had minimal impact. The weighted average per-test cost for each laboratory during this experiment ranged from $14.71 to $17.81, depending on the demand of Xpert test observed in this experiment [fig_ref] Table 1: Total per-test unit costs during the experiment, weighted to account for actual... [/fig_ref] and [fig_ref] Fig 2: Quarterly workload data by city [/fig_ref].
Overall, the demand for Xpert testing increased during the study period, particularly following the CME efforts. In New Delhi, it resulted in testing demand consistently exceeding the laboratory capacity [fig_ref] Fig 2: Quarterly workload data by city [/fig_ref]. Consequently, overtime work was most significant at the New Delhi laboratory, primarily at the later stages of the experiment, with 25.4% (179 days, inclusive of Sunday operations) of the 821 days in the study period recording overtime work,
## Fig 1. reference xpert per test costs for different testing workloads (batch size) categorized by type of resource inputs (new delhi tuberculosis centre).
Overhead includes electricity, gas, water, cleaning services etc. as well as staff involved in running the laboratories who were not employed by the FIND/RNTCP pediatric Xpert implementation project. Building space includes rent for rooms used for Xpert testing. Equipment includes the Xpert machine and other lab equipment. Staff includes wages for lab and managerial staff. Regents and chemicals consists of Xpert cartridge costs. Consumables include lab gloves and other personal protective equipment.
https://doi.org/10.1371/journal.pone.0214675.g001 [fig_ref] Table 1: Total per-test unit costs during the experiment, weighted to account for actual... [/fig_ref]. In New Delhi, Kolkata and Chennai overtime generally occurred as extended operating hours during the workweek, Monday through Saturday. In Hyderabad, 44 of the 114 overtime days were Sundays when the lab was scheduled to be closed. The majority of the costs associated with overtime testing were associated with theXpert cartridge and other consumables. Total costs associated with overtime staff and building costs were less than $1,000 in each city.
## Outreach and recruitment
A complete cost summary of outreach activities is presented in [fig_ref] Table 2: Summary of outreach efforts and costs by city [/fig_ref]. Unit costs for each type of outreach activity was $0.16 (range $0.12 to $0.20) per phone call, $7.14 (range $5.92 to $9.23) per one-on-one meetings with health providers, and $7.88 (range $6.03 to $9.86) per attendee at a CME. A total of 33 CMEs were held across the four cities with an average attendance of 58 health providers during the 26-month study period, an average of 3.8 CMEs per quarter. The number of 1-on-1 meetings and phone calls was recorded for between 9 and 20 months in each city with quarterly averages ranging between 98.1 and 199.5 and 31.8 and 145.5 for meetings and phone calls respectively [fig_ref] Table 2: Summary of outreach efforts and costs by city [/fig_ref]. There was substantial heterogeneity in the cost of CMEs and respective unit cost per attendee as the effort expended to organize a CME was decoupled from the resulting attendance.
In general, the combined outreach cost per Xpert testing referral generated decreased with time stabilizing at an average cost of $1.10 [fig_ref] Fig 3: Trends in outreach spending and number of successful patient referral for Xpert... [/fig_ref]. This ratio increased slightly in the final quarter as outreach efforts increased ahead of the project transition [fig_ref] Fig 3: Trends in outreach spending and number of successful patient referral for Xpert... [/fig_ref]. However, cost per referral was highly heterogeneous across the study sites, due to heterogeneous levels of demand for Xpert testing across the cities [fig_ref] Table 2: Summary of outreach efforts and costs by city [/fig_ref]
# Discussion
The FIND/RNTCP pediatric Xpert project demonstrated the importance and feasibility of providing a upfront Xpert testing for pediatric presumptive TB patients in four major Indian cities. Through this program, 92.6% of patients received results within 24 hours of sample receipt at the laboratories providing rapid diagnosis and drug sensitivity testing. More than 42,200 children were tested yielding 3,340 cases, including 295 cases of rifampicin-resistant TB. Subsequently, in this paper, we provide important insights and empiric evidence in respect to the costs and cost dynamics associated with the efforts in promoting and improving the utility of the Xpert test in India for diagnosis pediatric TB, which are critical in formulating future policies and guidelines for Xpert testing referral efforts and coping with increased demand for Xpert testing.
Our study demonstrated a clear empiric relationship between the demand generating outreach and promotion activities and the laboratory operational adjustments needed to adequately supply of Xpert testing capacities to cope with the increasing demand for Xpert testing at all study locations. For outreach activities, we observed significant upfront costs associated with initial recruitment efforts, where cost stabilized around $1 per patient referred as the program increased its operational efficiency. As shown in [fig_ref] Fig 3: Trends in outreach spending and number of successful patient referral for Xpert... [/fig_ref] the momentum towards program efficiency, measured as the number of patient referrals, was gained after one full year since the program initiation and directly corresponded to increasing frequency and spending on the combined outreach activities. This implies the importance of sustained periodic programming to ensure prolonged impact on health providers' awareness and use of the Xpert test for the diagnosis of pediatric TB.
On the laboratory operational side, Xpert testing capacity (under-or overuse) was a major cost driver for average Xpert per-test cost. Laboratories with excess capacity had higher average per-test costs whereas those that met or exceeded their standard workday capacity had lower average per-test costs. However, during periods of high demand, some laboratories required a substantial amount of overtime to meet demand for quick turnaround testing. In New Delhi, a significant proportion (13.9%) of the total expenditure on testing came from testing performed in excess of the standard workday capacity, particularly at a later stage of the study when the GeneXpert referrals rapidly increased immediately following CMEs. Likewise, as the outreach efforts to promote pediatric (and potentially general TB patients Xpert testing are scaled to other cities in India, evidence on projecting demand for Xpert testing in the target area should carefully be studied and be reflected in laboratory capacity and operational adjustments.
Methodologically, our cost analysis work builds upon earlier TB diagnostic costing studies [bib_ref] Using Top-down and Bottomup Costing Approaches in LMICs: The Case for Using..., Cunnama [/bib_ref] [bib_ref] Rapid diagnosis of tuberculosis with the Xpert MTB/RIF assay in high burden..., Vassall [/bib_ref] and incorporates a laboratory's key operational characteristics to appraise the unit cost of Xpert test unique to each laboratory in a two-step approach-1) establish reference pertest costs based on bottom-up micro-costing methods using TAM data and 2) calculate weighted-average per-test costs based on each laboratory's observed daily workloads. As such, the difference between the reference per-test cost for a 16-batch daily workload ($13.74) and the weighted-average per-test costs ($14.71) in New Delhi laboratory, for example, reflects the magnitude and cost associated with inefficiencies in laboratory operations (largely as a cost associated with under or over utilization of GX4 and human resource capacities). Subsequently, this measure could be used as an important indicator that measures the real-world efficiencies gaps in laboratory operations allowing programs to adjust staffing and operating hours to maximize testing efficiency.
There are several limitations in our work that may limit the interpretation and generalizability of our study findings. First, actual TAM data and parts of costing data were not directly assessed from the study laboratories due to logistical challenges and data availability issues. However, as Xpert testing procedure minimizes direct hands-on efforts of a laboratory personnel with a fixed overall test run time and use of a standardized test kit, variations in inter-operator and laboratory variability in the overall procedure and direct staff times as well as use of key laboratory consumables are likely not the major cost drivers of the Xpert test. As such, we addressed this limitation by calculating weighted per-test cost at each laboratory that reflects differences in operational conditions due to demand and capacity.
Second, unlike Xpert test costs, the unit cost of outreach spending was assessed based solely on the top-down method due to lack of adequate TAM and costing data. As transportation cost records were not kept, we had to assume a standard travel distance for relevant activities. This prevented us from directly exploring and quantifying the drivers of costs associated with inefficiencies in outreach spending as we did with the Xpert costs. Furthermore, a control group of health providers who were not reached through outreach activities was not available. Thus, we cannot directly assess the efficacy of each type of outreach activity or their combined effect relative to no direct outreach activity. However, using cost per successful patient referral for Xpert as an indicator of program efficiency, we were able to 1) demonstrate gain in program's efficiency in generating successful Xpert referrals [fig_ref] Fig 3: Trends in outreach spending and number of successful patient referral for Xpert... [/fig_ref] and key cost driving components of the outreach efforts across the four study sites [fig_ref] Table 2: Summary of outreach efforts and costs by city [/fig_ref].
Additionally, the variability of operations and costs observed at the four study sites may not adequately represent potential heterogeneities in costs and operations associated with the scale-up of this program in other areas in India. However, we provide a data and analytic framework that can be adopted by the on-going program expansion (or similar types of public health program implementation and scale-up) led by the RNTCP in other regions of India. Likewise, our study estimates provide an important baseline for on-going and future program scale-up operations to as a point of comparison for the outreach program and Xpert testing costs.
Our study offers a novel method to incorporate operational factors in assessing the cost of laboratory diagnostics using TAM and dynamic workload data unique to each laboratory. Given India's plan for universal access to drug-susceptibility testing and expanded pediatric Xpert testing, our findings can serve as important evidence in efficiently balancing Xpert testing capacity and demand in areas where pediatric Xpert testing referrals may be expanded.
## Supporting information
## S1
[fig] Fig 2: Quarterly workload data by city. Black indicates quarterly workload and red indicates quarterly testing capacity based on standard operating hours. Values in parentheses are the average workload-weighted Xpert per-test unit costs. https://doi.org/10.1371/journal.pone.0214675.g002 [/fig]
[fig] Fig 3: Trends in outreach spending and number of successful patient referral for Xpert testing. Fig 3A represents the trend of outreach spending cost per patient (solid line) referred plotted against the total number of patients referred (dotted line). Fig 3B plots the combined total outreach spending (solid line) against the number of patients referred (dotted line) for each quarter of the study period. Blue arrows indicate quarters where at least one CME was conducted. https://doi.org/10.1371/journal.pone.0214675.g003 [/fig]
[table] Table 1: Total per-test unit costs during the experiment, weighted to account for actual different unit costs by workloads, and costs associated with overtime work and laboratory operations to meet same-day testing.https://doi.org/10.1371/journal.pone.0214675.t001 followed by 16.2% (114 days) in Hyderabad, 5.3% (37 days) in Kolkata and 4.1% (29 days) in Chennai [/table]
[table] Table 2: Summary of outreach efforts and costs by city. [/table]
[table] Table: Breakdown of Xpert per-test unit costs. (DOCX) S2 Table. Summary of overtime days at each study laboratory according to the days of the week. (DOCX) S3 Table. Number of one-on-one meetings and phone calls recorded during observation period by city. (DOCX) S4 Table. Summary of average per attendee cost for CMEs. (DOCX) S1 Fig. Example calculation of average workload-weighted Xpert per-test unit cost. (TIFF) S1 Calculation. Example calculation of average workload-weighted Xpert per-test unit cost. (DOCX) S1 File. Costing sheet for Xpert per-test unit cost. (XLSX) S2 File. Dataset of daily lab workloads. (CSV) Conceptualization: Sanjay Sarin, Sophie Huddart, Neeraj Raizada, Catharina Boehme, Claudia M. Denkinger, Hojoon Sohn. curation: Aakshi Kalra. analysis: Sophie Huddart, Hojoon Sohn. acquisition: Sanjay Sarin, Neeraj Raizada, Catharina Boehme, Claudia M. Denkinger. Investigation: Sanjay Sarin, Neeraj Raizada, Hojoon Sohn. Methodology: Sophie Huddart, Claudia M. Denkinger, Hojoon Sohn. administration: Sanjay Sarin, Neeraj Raizada, Debadutta Parija, Aakshi Kalra, Claudia M. Denkinger. Resources: Aakshi Kalra, Raghuram Rao, Virender Singh Salhotra, Sunil D. Khaparde. Software: Sophie Huddart. Supervision: Sanjay Sarin, Neeraj Raizada, Debadutta Parija, Hojoon Sohn. [/table]
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Late Post Traumatic Right-sided Diaphragmatic Hernia Presenting with Acute Intestinal Obstruction and Strangulation: A Rare Presentation
We report a case of 16 year old male, who was presented to Jawaharlal Nehru Medical College, AMU, Aligarh, India as an emergency case with complaints of severe abdominal pain, difficulty in breathing and bilious vomiting. He was diagnosed as a case of late post traumatic right side diaphragmatic hernia with acute intestinal obstruction. He was successfully treated surgically by laparotomy and the defect was closed with polypropylene suture. The patient was followed for 3 months after operation and there was no complication.
# Introduction
The early diagnosis of post-traumatic diaphragmatic lesions is often difficult, which explains the 30 to 50% of non-diagnosed cases. [bib_ref] Right-sided posttraumatic diaphragmatic rupture and delayed hepatic hernia, Seket [/bib_ref] Traumatic injuries of the diaphragm are uncommon, and it is difficult to establish the exact incidence, but by autopsy studies, the incidence of these injuries range between 5.2% and 17%. [bib_ref] Missed diaphragmatic injuries and their long-term sequelae, Reber [/bib_ref] Only 0.8% to 1.6% of the total lesions observed in these patients are due to blunt trauma. [bib_ref] Trauma to the diaphragm, Mansour [/bib_ref] Post-traumatic diaphragmatic hernia is a particular lesion in traumatology that may be neglected.Besides the clinical signs and the modality of presentation, the lesions appear absolutely nonspecific.Incorrect interpretation of the X-ray or only intermittent hernial symptoms are frequent reasons for incorrect diagnosis. [bib_ref] A review on delayed presentation of diaphragmatic rupture, Rashid [/bib_ref] Also the initial non-recognition of the possible manifestation of the diaphragmatic hernia following blunt or penetrating injuries is usually because the practitioner has not sought it. Thus, the diagnosis may be delayed for a few days to several months and only be made following a complication. Review of the historical clinical literature, including the series of Carter et al reveals that the majority (80-90%) of blunt diaphragmatic ruptures have occurred on the left side. They required greater force of impact, possibly because the liver provides protection or because of a weakness in the left diaphragm. [bib_ref] Traumatic diaphragmatic hernia, Carter [/bib_ref] Obstruction can occur in diaphragmatic hernias but strangulation is rare. 8
## Case report
A 16 yr old boy presented as an emergency case with complaints of vomiting, abdominal pain and difficulty in breathing. We learned from the patient's history that he had been run over the chest by a motor vehicle 11 years back and since then he has been having episodes of dyspnea. The colour of the vomitus was bilious. On examination there was decreased air entry on the right side of the chest and bowel sounds were present in the right hemithorax which were exaggerated. Radiographs of the chest [fig_ref] Figure 1: Radiograph of the patient showing right side diaphragmatic hernia with multiple air... [/fig_ref] were suggestive of bowel loops in the right hemithorax and radiographs of abdomen were suggestive of multiple air fluid levels and distension of the small bowel.
Emergency laparotomy was done by midline incision. Terminal ileum and transverse colon along with liver was present in the right hemithorax and there were pregangrenous changes in the terminal ileum [fig_ref] Figure 2: Operative photograph of the patient showing diaphragmatic rupture and pregangrenous condition of... [/fig_ref]. There was a defect in the right diaphragmatic cupola present posteriorly and ipsilateral lung was collapsed. Adhesions were present in the small bowel and the transverse colon [fig_ref] Figure 3: Mobilization and adhesiolysis of transverse colon, small bowel regaining its color after... [/fig_ref]. The bowel loops were free and slowly the color of the bowel became normal. The liver was mobilized and the diaphragmatic rent was closed using polypropylene sutures. There was no tension over the rent after the repair. Intercostal tube drainage was done on the right side. Conversion to thoracotomy was not done as the contents were easily mobilized by laparotomy. The patient was followed for 3 months post operation and there was no complication.
# Discussion
Traumatic Diaphragmatic Hernias usually result from severe external blunt injury or penetrating injuries such as a knife or bullet wound. The hernias may be recognized during the period of hospitalization immediately following the trauma, the immediate type of hernia is described by Carter et al. [bib_ref] Traumatic diaphragmatic hernia, Carter [/bib_ref] However, it is widely accepted that herniation may be delayed.
If the diaphragmatic injury is not recognized during the immediate post-traumatic period, the patient may: 1) recover and remain symptom free; 2) suffer from chronic abdominal and/or chest symptoms, or 3) present with an acute crisis, often with signs of intestinal obstruction or strangulation. [bib_ref] Delayed presentation of traumatic diaphragmatic hernia, Hegarty [/bib_ref] In a review of 276 patients with traumatic diaphragmatic hernia, Hood found only 13% on the right side. 10 This is probably due to the liver cushioning the diaphragm in blunt trauma and plugging the defect in the penetrating trauma.
# Conclusion
Recognition of a traumatic diaphragmatic hernia in the immediate post-traumatic period is difficult, due to associated injuries and to the fact that several radiological features suggestive of hernia may mimic those of chest injuries. A careful history, examination, and awareness of the possibility of the condition and its complications are essential if these patients are to be managed successfully.
[fig] Figure 1: Radiograph of the patient showing right side diaphragmatic hernia with multiple air fluid levels in small intestine suggestive of Acute Intestinal Obstruction. [/fig]
[fig] Figure 2: Operative photograph of the patient showing diaphragmatic rupture and pregangrenous condition of bowel loops. [/fig]
[fig] Figure 3: Mobilization and adhesiolysis of transverse colon, small bowel regaining its color after being taken out from thoracic cavity. [/fig]
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Pneumatocele in a Ugandan female with SARS‐CoV2 infection: A case report
This 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.AbstractPneumatocele may complicate the course of SARS-CoV2 infection. Our article exhibits the value of early radiological imaging for the timely diagnosis and management of COVID-19 and its complications. Conservative management is the mainstay of the treatment of pneumatoceles; however, prompt surgical intervention is imperative for complicated pneumatoceles.
# | introduction
Coronavirus disease 2019 (COVID-19) caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; previously known as 2019-nCoV), has spread to more than 220 countries around the world since its discovery in 2019. More than 246 million cases and more than 5 million deaths have been reported worldwide,whereas Uganda alone has reported 126,272 cases and 3217 deaths.COVID-19 presents with a clinical syndrome caused by an intense inflammatory reaction due to a cytokine storm. [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref] COVID-19 patients usually present with radiologic findings of ground-glass opacities (83%), ground-glass opacities with consolidation (58%), pleural thickening (52%), interlobular septal thickening (48%), or air bronchograms (46%). [bib_ref] Coronavirus disease 2019 (COVID-19) CT findings: a systematic review and metaanalysis, Bao [/bib_ref] [bib_ref] Potential links between COVID-19-associated pulmonary aspergillosis and bronchiectasis as detected by high..., Baratella [/bib_ref] Pneumomediastinum and pneumothorax are the commonly reported complications majorly resulting from mechanical or positive pressure ventilation, or directly related to COVID-19 pneumonitis. [bib_ref] Interstitial lung disease-related pneumomediastinum in COVID-19 patients, Blanc [/bib_ref] Pneumatocele, a thin-walled air-filled cystic lesion in the lung commonly develops, associated with acute pneumonia or after a severe infection like empyema. [bib_ref] Complicated pneumonias with empyema and/or pneumatocele in children, Kunyoshi [/bib_ref] [bib_ref] Pulmonary pneumatocele: pathology and pathogenesis, Quigley [/bib_ref] The development of pneumatocele secondary to COVID-19 pneumonitis appears rarer. COVID-19 pulmonary lesions can also present as pneumatocele in the form of emphysema (5.3%),cystic air spaces (37.5%), 10 or cystic changes (10%). [bib_ref] Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a..., Shi [/bib_ref] In this article, we present an unusual complication of SARS-CoV-2 infection, highlighting a pneumatocele secondary to COVID-19.
## | case presentation
We report a 67-year-old female, non-smoker, nonalcoholic patient, known hypertensive for about 10 years on medication: amlodipine and losartan, but no other prior history of lung disease and no history of vaccination against SARS-CoV-2. She had a week-long history of dry cough and high-grade fever with chills and rigors prior to admission. She was earlier diagnosed with COVID-19 by COVID rapid antigen test from a peripheral clinic and was being managed as an outpatient on zinc, vitamin D, ceftriaxone-sulbactam, dexamethasone, gentamicin, and ascorbic acid.
Two days prior to presenting to our hospital, she developed acute right-sided chest pain, which worsened with inspiration, and progressive shortness of breath. The diagnosis of COVID-19 was confirmed by reverse transcription-PCR for SARS-CoV-2.
On physical examination, she was significantly in severe respiratory distress, tachypneic at 44 breaths/minute, pulse oximetry of 57% at room air, followed by 91% on oxygen via non-rebreather mask at 10 liters/minute flow, temperature 37.5 0 C, blood pressure 124/64 mmHg, and heart rate 110 beats/minute. She also had stress hyperglycemia of 8.4 mmol/L secondary to the infection. Her respiratory examination revealed reduced air entry on the right lower lung zones with diffuse crepitations on the right middle and upper lung zones and lower left lung zones. Other systemic examinations were unremarkable.
## | laboratory
## Investigations and imaging
At admission, her C-reactive protein (CRP) was significantly elevated: 96.2 mg/L, total white blood cell count: 15.10 × 10 3 /µl, lymphocytes: 1.46 × 10 3 /µl, neutrophils: 12.65 × 10 3 /µl, hemoglobin: 13.32 g/dl, and total Platelet count: 444.5 × 10 3 /µl. Renal and liver function tests were normal.
High-resolution chest computed tomography (HRCCT) at admission revealed features of COVID-19 Reporting and Data System (CO-RADS)-5 with about 75% pulmonary involvement. Multifocal bilateral ground-glass opacities plus bilateral consolidations in the left lower lobe and right upper lobe, and a pneumatocele, a thin-walled round cystic space within the lung parenchyma in the right lower lobe measuring 8.54 × 5.27 cm .
## | management and follow-
## Up
The patient was managed conservatively on a 7-day course of dexamethasone intravenous (IV) 6 mg once daily, piperacillin-tazobactam (IV) 4.5 g thrice daily, zinc sulfate per oral 20 mg once daily, enoxaparin subcutaneous 60 mg once daily, vitamin c per oral 1000 mg once daily, paracetamol (IV) 1 g thrice daily, and oxygen therapy as per requirement initially via non-rebreather mask, which was successfully progressively weaned off to nasal prongs, and finally, she was completely taken off oxygen. She showed marked clinical improvement with a significant improvement in CRP and white blood count. She was eventually discharged without any sequelae in stable condition, on a 5-day course of amoxicillin/clavulanic acid, and 7 days of montelukast, vitamin C, and vitamin D. She also continued her antihypertensive drugs throughout her hospital stay and after discharge. She was tested COVID-19 negative at 2 weeks; the review was unremarkable at 6 weeks, and she had no complaints. The repeat high-resolution chest computed tomography (HRCCT) at 10 weeks revealed regressing pneumatocele size with fibrotic changes .
# | discussion
SARS-CoV-2 infection causes a cytokine storm leading to an intense inflammatory reaction. The mechanism of pneumatocele formation, subsidiary to this inflammatory reaction caused by SARS-CoV-2 infection, appears to be a combination of diffuse alveolar damage followed by necrosis of the airway walls, [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref] [bib_ref] Pulmonary pneumatocele: pathology and pathogenesis, Quigley [/bib_ref] and obstruction and dilation of bronchi secondary to an intraluminal check-valve formation, which later ruptures causing air-filled cysts. [bib_ref] Pulmonary pneumatocele: pathology and pathogenesis, Quigley [/bib_ref] [bib_ref] Subpleural emphysema complicating Staphylococcal and other pneumonias, Boisset [/bib_ref] Pneumatoceles often resolve spontaneously, and management is mostly conservative; however, in some
## F i g u r e 1 axial view of high resolution chest ct (hrcct)
showing multifocal bilateral ground-glass opacities and 8.54 × 5.27 cm pneumatocele in anterior and medial segments of right lower lobe instances, they can dissect through the pleural membrane and rupture to cause pneumothorax, pneumomediastinum, or pneumopericardium. [bib_ref] Air leaks, pneumatoceles, and air spaces in Covid-19 pneumonia, Jolobe [/bib_ref] [bib_ref] Pneumatocele in infants and children: report of 12 cases, Amitai [/bib_ref] They may as well undergo drastic changes in size, especially in patients on mechanical ventilation, compression of surrounding lung, and cardiorespiratory compromise or infected plus accumulation of pus. In our case, the patient was diagnosed with COVID-19 and was earlier being managed as an outpatient. However, later she deteriorated, and radiological imaging with high-resolution chest computed tomography (HRCCT) revealed multifocal bilateral ground-glass opacities and a large pneumatocele. She was managed conservatively and discharged on the 8th day with a marked improvement and an unremarkable follow-up.
Complicated pneumatocele warrants intervention; however, no firm algorithm has been found in the literature. Previously reported case reports and series have stressed successful image-guided percutaneous transcatheter aspiration and drainage of complicated pneumatoceles. [bib_ref] Decompression of pneumatocele in a neonate by percutaneous catheter placement, Kogutt [/bib_ref] [bib_ref] Percutaneous management of intrapulmonary air and fluid collections, Erasmus [/bib_ref] Failure of this technique should lead to surgical pneumonostomy 17 or resection. [bib_ref] Surgical resections of superinfected pneumatoceles in a COVID-19 patient, Castiglioni [/bib_ref] [bib_ref] Post COVID-19 large pneumatocele: clinical and pathological perspectives, Hamad [/bib_ref] On the contrary, in critically ill patients, surgical approach seems more reasonable as the first choice of intervention, since it may be life-saving. In this case, the patient substantively recovered on conservative management without any need for surgical intervention. Although the patient was asymptomatic and in good general condition at a 6-week follow-up, the repeat high-resolution chest computed tomography (HRCCT) at 10th week revealed regressing pneumatocele size with fibrotic changes.
# | conclusion
Pneumatocele may complicate the course of SARS-CoV2 infection. Early radiological imaging significantly improves the timely diagnosis and monitoring of COVID-19 and its complications.
## Acknowledgements
Special thanks to the staff at the Department of Medicine at Uganda Martyrs Hospital Lubaga that were involved in the management of this patient.
## Conflicts of interest
The authors declare no conflicts of interest.
# Author contribution
Sanjanaa Srikant conceptualized the study, wrote the first draft, and edited and approved the final manuscript. Dave Darshit conceptualized the study, involved in patient follow-up, wrote the first draft, and edited and approved the final manuscript. Dhara Dave reviewed the manuscript for important intellectual content and contributed to editing, supervision, and the final approval of the case report.
# Ethical approval
Institutional Review Board approval was not required to publish this article.
## Consent
Written informed consent was obtained from the patient for the publication of this case report and accompanying images.
[fig] F: I G U R E 2 Coronal view of high resolution Chest CT (HRCCT) showing multifocal bilateral ground-glass opacities and 8.54 × 5.27 cm pneumatocele in anterior and medial segments of the right lower lobe I G U R E 3 Axial view of high resolution chest CT (HRCCT) showing regressing pneumatocele and fibrotic changes DATA AVAILABILITY STATEMENT None. ORCID Sanjanaa Srikant https://orcid.org/0000-0003-3775-0123 Darshit Dave https://orcid.org/0000-0002-1762-5918 [/fig]
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A Simple Biomimetic Receptor Selectively Recognizing the GlcNAc2 Disaccharide in Water
Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.
## In memory of professor alberto vacca
Abstract: GlcNAc 2 is the core disaccharide fragment present in N-glycans exposed on the surface of enveloped viruses of high health concern, such as coronaviruses. Because N-glycans are directly involved in the docking of viruses to host cells, recognition of GlcNAc 2 by a biomimetic receptor may be a convenient alternative to the use of lectins to interfere with viral entry and infection. Herein, we describe a simple biomimetic receptor recognizing the methyl-b-glycoside of GlcNAc 2 in water with an unprecedented affinity of 160 mM, exceeding that of more structurally complex receptors reported in the literature. The tweezers-shaped acyclic structure exhibits marked selectivity among structurally related disaccharides, and complete discrimination between mono-and disaccharides. Molecular modelling calculations supported by NOE data provided a three-dimensional description of the binding mode, shedding light on the origin of the affinities and selectivities exhibited by the receptor.
Enveloped viruses are a broad class of highly glycosylated viroids of high health concern, including coronaviruses (SARS-CoV-2, SARS-CoV and MERS), retroviruses (HIV and hepatitis B), orthomyxoviruses (influenza A-C), flaviviruses (dengue, hepatitis C, yellow fever, Zika) and filoviruses (Ebola and Marburg fever), among others.Viral adhesion to host cells is often mediated by specific carbohydrate-protein interactions, which occur through the glycans exposed on the surface of the viral envelope.Biomimetic receptors for carbohydrates targeting these saccharides may inhibit viruscell interaction, thereby preventing viral entry and infection. In this context, among biologically relevant oligosaccharides, N,N'-diacetylchitobiose (GlcNAc 2 ) holds a pivotal role, because is part of the highly conserved GlcNAc 2 Man 3 core fragment of N-glycans present on the surface of enveloped viruses, constituting the disaccharidic unit Nlinked to membrane proteins through an asparagine residue, which often get exposed by mannoside deletions due to virus mutations.Unsurprisingly, GlcNAc-binding lectins, such as NICTABA from Nicotiana tabacum and Urtica dioica Agglutinin (UDA), which target GlcNAc 2 at the stem of Nglycosilation sites, possess a broad-spectrum activity against several families of enveloped viruses,Thus, effective molecular recognition of GlcNAc 2 in water by a simple and easily accessible biomimetic receptor can potentially represent a convenient alternative to natural lectins, because of advantages in terms of availability, molecular weight, purity, stability and immunogenicity.
Selective recognition of neutral glycans by biomimetic receptors in physiological medium represents a major challenge of current research,because water is a strong competitor for recognition of polar molecules such as carbohydrates.Nevertheless, in the last few years, significant steps forward have been made in the design of receptors for mono-and oligosaccharides, mainly developing appropriately sized macrocyclic architectures.The main drawback of the latter strategy is that macrocyclic architectures must be precisely tailored on specific saccharidic targets and often require lengthy multistep syntheses with relatively low overall yields, due to the critical macrocyclization step.
We have recently developed a new generation biomimetic receptor for monosaccharides in water, by assembling into a macrocyclic architecture a tridentate hydrogen binding motif (1,8-diaminocarbazole endowed with two hydrosolubilizing phosphonate groups) with anthracene moieties providing extended CH-p interactions with the saccharidic backbone.Interestingly, the corresponding adaptive tweezersshaped liposoluble receptor proved to effectively recognize biologically relevant xanthines in organic solvents.Following the idea that an acyclic adaptive structure may accommodate disaccharides more effectively than its macrocyclic counterpart, we tested receptor 1, the hydrosoluble version of the parent receptor 2,vs. a set of mono-and disaccharides in water, in the belief that effective recognition of disaccharides may be achieved by a simple, easily available structure. [bib_ref] Examples of adaptive architectures in carbohydrate recognition: a), Kim [/bib_ref] We report here that this is true indeed, and that 1 is not only a simple and easily accessible receptor for disaccharides, but also the most effective biomimetic receptor for GlcNAc 2 in the literature up to date.
Compound 1 was easily obtained by hydrolysis of the previously reported ester 2 (Scheme 1).1 is freely soluble in water under both, mild alkaline (pH 11) and physiological (pH 7.4) conditions, whereas precipitates at acidic pH, due to high degree of protonation of phosphonate groups. Receptor 1 shows sharp 1 H NMR signals at low concentration values (5 10 4 M), broadening at higher values at pH 7.4, but not at pH 11, suggesting concentration-dependent self-association, supported by chemical shift changes.
The binding properties of receptor 1 were qualitatively screened by 1 H NMR spectroscopy toward a set of monosaccharides, including glucose, rhamnose, fucose, xylose, sialic acid, a and b methyl glucosides, galactosides, mannosides, and N-acetylglucosamine [fig_ref] Scheme 1: Synthesis of receptor 1 and proton labels [/fig_ref] , together with a set of disaccharides, including sucrose (Suc), trehalose (Tre), cellobiose (CeB), maltose (Mal) and lactose (Lac) [fig_ref] Scheme 1: Synthesis of receptor 1 and proton labels [/fig_ref] by monitoring the shifts of the proton signals of the sugar upon addition of an equimolar amount of 1. Surprisingly, little (Dd 0.03 ppm) or no variations were observed for all the investigated monosaccharides and for Suc and Tre, whereas a marked upfield shift was observed for CeB, Mal and Lac, which were larger for the b than for the a anomer (Figures S3-S5), reasonably due to the shielding effect of the anthracene moieties in the binding cavity. A concomitant broadening of signals, larger for the CeB, indicated slow chemical exchange, most likely due to strong binding, suggesting a preference for all-equatorial disaccharides.
A quantitative investigation was then carried out by NMR spectroscopy, extending the study to the all-equatorial GlcNAc 2 . Because in N-glycans the GlcNAc 2 glycoside unit is present as the b anomer, methyl-b-glycosides of GlcNAc 2 (MebGlcNAc 2 ), cellobiose (MebCeB), maltose (MebMal) and lactose (MebLac) [fig_ref] Scheme 1: Synthesis of receptor 1 and proton labels [/fig_ref] were employed, to avoid interconversion equilibria between anomers. Dilution experiments of receptor 1 were preliminary carried out at pH 7.4, fitting a self-association model featuring two clusters, in which the dimer was the predominant species at low concentrations. The fit gave a dimerization constant of logb dim = 2.65 AE 0.07, most likely due to p stacking of the aromatic moieties, which was set invariant in the nonlinear regression analysis of the glycoside binding experiments. The cumulative associationcalculated from the measured binding constants and reported in [fig_ref] Table 1: Cumulative formation constants [/fig_ref]. Amazingly, results show that receptor 1 binds MebGlcNAc 2 with an affinity of 160 mM which is unprecedented in the literature for a synthetic receptor. Indeed, to the best of our knowledge, the highest affinity reported to date for MebGlcNAc 2 by a biomimetic receptor is that observed by Davis and co-workers with a bicyclic polyamidic receptor, showing a 3-fold lower affinity than 1 (BC 50 0 = 455 mM, K a = 2200 M À1 ).The affinity of 1 for MebGlcNAc 2 exceeds that of some lectin-like proteins, such as hevein from Hevea brasiliensis, which shows for GlcNAc 2 an affinity one order of magnitude smaller (BC 50 0 = 1.61 mM, K a = 620 AE 50).Moreover, receptor 1 exhibits a marked selectivity, showing an affinity for MebCeB nearly one order of magnitude smaller, and a 200fold drop of affinity for MebMal and MebLac. 1 H NMR titrations with MebCeB were also duplicated at pD 11 (Table S1, Supporting Information) and fitted to the association model obtained at pD 7.4. The closely comparable affinities confirmed that the degree of protonation of the phosphonate groups does not affect the binding ability of receptor 1. Most remarkably, recognition of monosaccharides appears completely depleted, as appreciated from the preliminary screening and from the titration of MebGlcNAc, in which no significant variation of chemical shifts was observed [fig_ref] Scheme 1: Synthesis of receptor 1 and proton labels [/fig_ref]. Somewhat counterintuitively, a flexible acyclic structure exhibits excellent affinities and selectivities, overriding those of more structurally complicated macrocyclic architectures.The binding affinities obtained by NMR were further confirmed by ITC in H 2 O at physiological pH. Data from two to three independent titrations run at different reactant concentrations were combined and simultaneously fitted to remove ambiguities in the definition of the binding model. The dimerization constant obtained by NMR dilution experi-ments at pD 7.4 was set invariant in the nonlinear regression analysis of ITC data. Cumulative binding constants, together with affinity values, were reported in [fig_ref] Table 1: Cumulative formation constants [/fig_ref] for a direct comparison with NMR results. The good agreement between the two independent techniques confirmed the observed affinities. Unfortunately, because of the strong self-association of receptor 1, ITC measurements did not provide reliable thermodynamic parameters.
To shed light on the origin of the affinities and selectivities exhibited by receptor 1, a three-dimensional description of the binding mode was attempted for the 1:1 complex of 1 with MebGlcNAc 2 , using molecular modelling calculations supported by NOE data from NMR spectroscopy. NOESY spectra carried out on an equimolar mixture of 1 and MebGlcNAc 2 showed unambiguous intermolecular NOE contacts between the H-2 and H'À6' protons, belonging to the two monosaccharide units, and the H-C protons of the anthracenes [fig_ref] Figure 2: Global minimum structure of the 1 MebGlcNAc 2 complex in two different... [/fig_ref]. In addition, NOE contacts were found between the methyl protons of the N-acetyl group of the methylglycosidic unit with the H-C protons of the anthracene and the H-A and H-B protons of the carbazole. In contrast, no NOE contacts could be found for the second N-acetyl group.
A conformational search carried out on the 1:1 complex returned a single family of minimum energy conformers within 10.0 kJ mol À1 from the global minimum. The minimum energy structure depicted in [fig_ref] Figure 2: Global minimum structure of the 1 MebGlcNAc 2 complex in two different... [/fig_ref] shows the disaccharide entirely located inside the binding cleft, within the two anthracene faces, with the H-2 and H'À6' protons pointing toward the H-C protons of the anthracenes, in agreement with the proximities inferred by NOE contacts [fig_ref] Figure 2: Global minimum structure of the 1 MebGlcNAc 2 complex in two different... [/fig_ref]. In addition, the N-acetyl group of the methylglycosidic unit faces the diaminocarbazole moiety, pointing the methyl protons toward the H-A and H-B protons, and to one of the H-C protons of the anthracene. From the above model, all O···H interatomic distances shorter than the sum of the van der Waals radii and compliant with hydrogen bonding criteria were calculated and four hydrogen bonding interactions were found between 1 and MebGlcNAc 2 [fig_ref] Figure 2: Global minimum structure of the 1 MebGlcNAc 2 complex in two different... [/fig_ref] : the carbazole NH and one of the aminic NH behave as donors toward OH-3 of the methylglycosidic unit, whereas the other aminic NH of the receptor acts as both, donor to the OH-3 and acceptor from the amidic NH of the N-acetyl group. In addition to CHp interactions with the anthracene units, the N-acetyl group contributes to stabilize the complex through CH-p interactions with the carbazole unit. Most likely, the substantial participation to binding of the N-acetyl group may account for the observed selectivity over other disaccharides.
In this context, it is worth mentioning that, interestingly, the binding mode of 1 with MebGlcNAc 2 is reminiscent of that between hevein and the corresponding trisaccharide chitotriose, as reported by the group of J. JimØnez-Barbero [fig_ref] Figure 3: Comparison of the global minimum structures of a [/fig_ref].A close similarity can be appreciated between the binding mode of chitotriose to Tyr30 of hevein and of chitobiose to the aminocarbazole of 1. Indeed, both receptors engage hydrogen-bonding with the saccharidic OH-3 and with the acetamidic NH, while in both cases the methyl group stabilizes the complex through CH-p interactions with the aromatic ring. Altogether, the present work shows that molecular recognition of a disaccharide can effectively and selectively be achieved with a well-designed acyclic host featuring an adaptive architecture. The tweezers-shaped receptor 1 fully discriminates disaccharides from monosaccharides, selectively recognizes all-equatorial from non all-equatorial disaccharides, and shows an unprecedented affinity for GlcNAc 2 , the core glycosidic fragment of viral N-glycans. The hydrogenbonding and CH-p interactions established by receptor 1 with the N-acetyl group most likely account for the selectivity observed for MebGlcNAc 2 over other all-equatorial disaccharides. Because of simple structure, easy synthetic availability, and accessible structure modifications, receptor 1 stands as a promising tool for saccharide recognition.
[fig] Scheme 1: Synthesis of receptor 1 and proton labels. [/fig]
[fig] Figure 1: Structure of the investigated a) monosaccharides and b) disaccharides and their abbreviations. [/fig]
[fig] Figure 2: Global minimum structure of the 1 MebGlcNAc 2 complex in two different projections. a) The strongest intermolecular NOEs found between 1 and MebGlcNAc 2 are indicated as solid lines with corresponding distances [] calculated from the lowest energy conformer. b) Intermolecular hydrogen-bonding interactions found in the calculated structure are indicated as dashed lines with corresponding oxygen/hydrogen and nitrogen/hydrogen distances []. [/fig]
[fig] Figure 3: Comparison of the global minimum structures of a) hevein·chitotriose complex and b) 1C MebGlcNAc 2 complex. Intermolecular hydrogen-bonding and CH-p interactions involving Tyr30 and the arylamine moiety of 1 are indicated as dashed and solid lines, respectively. Protein backbone was drawn as a ribbon for clarity. [/fig]
[table] Table 1: Cumulative formation constants (log b n )[a] and intrinsic median binding concentration (BC 50 0 , mM)[b] for receptor to glycoside (R:G) complexes of 1 with methyl glycosides, measured at 298 K from NMR data in D 2 O at pD 7.4 and from ITC data in H 2 O at pH 7.4. [c] MebGlcNAc 2 1:1 3.55 AE 0.04 0.16 AE 0.01 3.49 AE 0.07 0.12 AE 0Formation constants were obtained by nonlinear least-square regression analysis of NMR and ITC data.[b] Calculated from the log b values using the "BC50 Calculator" program.[15] [c] Receptor 1 dimerization constant (log b dim = 2.65 AE 0.07) was set invariant in the nonlinear regression analysis of NMR and ITC data. [/table]
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Imbalanced Nutrient Intake in Cancer Survivors from the Examination from the Nationwide Health Examination Center-Based Cohort
This study was conducted to examine the nutrient intake status of cancer survivors. A total of 5224 cancer survivors, 19,926 non-cancer individuals without comorbidities (non-cancer I), and 20,622 non-cancer individuals with comorbidities, matched by age, gender, and recruitment center location were included in the analysis. Generally, the proportion of total energy from carbohydrates was higher and the proportion from fat was lower in cancer survivors. The odds ratios (ORs) for total energy (OR = 0.92, 95% confidence interval (CI) = 0.86-0.99), proportion of total energy from fat (OR = 0.54, 95% CI = 0.35-0.83), and protein (OR = 0.85, 95% CI = 0.79-0.90) were significantly lower, and the OR for the proportion of total energy from carbohydrates was higher (OR = 1.21, 95% CI = 1.10-1.33) in the cancer survivors than in non-cancer I. Additionally, the cancer survivors' protein, vitamin B 1 , vitamin B 2 , niacin, and phosphorus intakes were lower, whereas their vitamin C intake was higher. When divided by cancer type, the ORs for the carbohydrate percentages were significantly higher in the colon and breast cancer survivors, whereas protein intake was lower in gastric, breast, and cervical cancer survivors. The nutrient intake patterns in Asian cancer survivors are poor, with higher carbohydrate and lower fat and protein intakes.However, the few studies in Western countries that have focused on the adherence of cancer survivors to dietary guidelines have shown poor adherence in cancer survivors[8,13]. Given the diverse dietary patterns[14]and different cancer patterns between countries, regions, and ethnicities[15], the nutritional status in cancer survivors needs to be investigated in Asian countries. However, these studies are lacking.Therefore, we conducted this study to evaluate the extent of adherence of cancer survivors to the dietary reference intakes for Koreans, compared to people without experience with cancer. Considering the emerging concept that cancer should be considered a chronic disease[16,17], we divided the people without experience with cancer into groups based on the presence or absence of comorbidities. Additionally, we evaluated the nutrient intake status by cancer type to identify whether dietary intake in cancer survivors was affected by cancer type.Materials and MethodsData Source and Study PopulationThis study used baseline survey data from a nationwide health examination center-based cohort (the Health Examinee (HEXA) cohort). The HEXA cohort is one part of the Korea Genome Epidemiology Study (KoGES), which is an ongoing population-based cohort study that began in 2001. In the HEXA cohort, the participants were recruited from health examination centers for regular check-ups in 14 Korean urban areas. The health examinees were aged 40-79 years, gave informed consent, and were asked to complete an interviewer-administered questionnaire that included sociodemographic factors, past medical history and family history, behavioral characteristics, and a validated food frequency questionnaire designed for Koreans. Additionally, the results of clinical tests and physical examinations from medical check-ups were collected. Details of the KoGES and HEXA cohort are described elsewhere[18,19]and on the Korea National Institute of Health website[20].Based on information from the past medical history collected in the baseline questionnaire, the cancer survivors were individuals who responded that they had been diagnosed with any type of cancer by a doctor, using the definition of DeSantis et al., who defined cancer survivors as any person who had been diagnosed with any type of cancer, including both patients under treatment and patients who had completely recovered[6]. A total of 5274 cancer survivors were identified. Participants without a self-reported cancer history were divided into two groups, as follows: non-cancer individuals without comorbidities (non-cancer I) and non-cancer individuals with comorbidities (non-cancer II). The collected comorbidities through the questionnaire included hypertension, diabetes, dyslipidemia, stroke, angina or myocardial infarction, gastrointestinal disease, intestinal polyps, fatty liver, chronic liver disease or liver cirrhosis, gallbladder, respiratory disease, thyroid disease, arthritis, osteoporosis, gout, glaucoma or cataracts and depression, and those who reported that they had been diagnosed with any type of above diseases by a doctor were considered non-cancer II group. This selection resulted in 78,199 subjects in the non-cancer I group and 89,884 subjects in the non-cancer II group. Through individual matching by age (±2 years), gender (male and female), and recruitment health examination location (39 centers) with a 1:4 ratio, 5272 cancer survivors, 21,085 people in non-cancer I, and 21,085 people in non-cancer II were included in the analysis.(Figure 1).
# Introduction
The number of cancer survivors has rapidly increased with the increases in both cancer incidence and survival rates over the last several decades [bib_ref] The global burden of cancer, Fitzmaurice [/bib_ref] , with 32.5 million 5-year cancer survivors worldwide. In Korea, the 5-year cancer relative survival rate has improved compared with the rate 15 years ago, from 41% to 68%. The prevalence of cancer survivors was 2453 per 100,000 individuals in 2012. Compared with Western countries, the proportion of cancer survivors of gastric and thyroid cancer was higher due to higher incidence and relatively lower 5-year mortality rate [bib_ref] Cancer statistics in Korea: Incidence, mortality, survival, and prevalence in 2012, Jung [/bib_ref]. Cancer survivors have an increased risk of not only secondary cancer but also other chronic diseases, including cardiovascular disease, endocrine diseases, and diabetes [bib_ref] Non-cancer adverse health conditions and perceived health and function among cancer survivors..., Gallicchio [/bib_ref] [bib_ref] Health status of long-term cancer survivors: Results from an Australian population-based sample, Eakin [/bib_ref]. These adverse outcomes following cancer may be caused by the cancer itself, the treatments [bib_ref] Cancer treatment and survivorship statistics, Desantis [/bib_ref] or common modifiable lifestyle behaviors, suggesting the importance of informed lifestyle choices for cancer survivors [bib_ref] Lifestyle interventions in cancer survivors: Designing programs that meet the needs of..., Stull [/bib_ref] [bib_ref] Cancer survivors' adherence to lifestyle behavior recommendations and associations with health-related quality..., Blanchard [/bib_ref] [bib_ref] Nutrition and physical activity guidelines for cancer survivors, Rock [/bib_ref]. Among the modifiable factors, nutrition or dietary habits play an important role not only in the development of cancer [bib_ref] Meat consumption and cancer risk: A case-control study in Uruguay. Asian Pac, Aune [/bib_ref] [bib_ref] A multicountry ecological study of cancer incidence rates in 2008 with respect..., Grant [/bib_ref] , but also in the etiology of chronic diseases, quality of life, or prognosis, including mortality from cardiovascular diseases in cancer survivors after cancer diagnosis [bib_ref] Nutrition and physical activity guidelines for cancer survivors, Rock [/bib_ref] [bib_ref] Association between post-cancer diagnosis dietary inflammatory potential and mortality among invasive breast..., Zheng [/bib_ref]. Thus, identifying nutritional status and patterns might be a priority for cancer survivors, to improve long-term health and survival by planning and conducting appropriate nutritional interventions in these populations.
## Nutrient intake
Nutrient intake was assessed using a validated semi-quantitative food frequency questionnaire consisting of 106 food items, frequencies of servings, and portion sizes. Daily nutrient intake values were estimated by the sum of the nutrients of each food item using the Food Korea [bib_ref] Validation and reproducibility of food frequency questionnaire for Korean genome epidemiologic study, Ahn [/bib_ref] [bib_ref] Development of a semi-quantitative food frequency questionnaire based on dietary data from..., Ahn [/bib_ref]. Among the estimated nutrient intakes, total energy (kilocalories), protein, fat, carbohydrates (% of total energy), vitamin A, vitamin B1, vitamin B2, niacin, folate, vitamin B6, vitamin C, calcium, phosphorus, iron, and zinc were investigated in detail using the recommended dietary intakes established for Koreans.
# Statistical analysis
Of the matched participants, 1106 individuals had incomplete dietary questionnaires, including missing values for nutrient intake (48 cancer survivors, 595 non-cancer I, and 463 non-cancer II). After excluding these individuals, nutrient intake was compared between 5224 cancer survivors, 19,926 non-cancer I individuals, and 20,622 non-cancer II individuals using two methods: the mean intake of each nutrient between the three groups and the proportion of subjects with an intake of each nutrient higher than the recommended value compared to non-cancer I (reference group).
The baseline characteristics of the cancer survivors, non-cancer I individuals, and non-cancer II individuals were compared using the Chi-square test, and the mean of each nutrient level was compared by analysis of variance (ANOVA) with a post hoc comparison (Tukey's test). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate whether the cancer survivors and non-cancer individuals with comorbidities had nutrient intakes that were better than the intakes of the non-cancer I individuals by conditional logistic regression, adjusted for age, marital status, education level, income level, job status, smoking status, drinking status, current physical activity, and body mass index, based on measured height and weight. Although age was used as one of the matching variables, we also adjusted for age in the conditional logistic regression analysis due to the significant differences in age between the three groups. Body mass index was categorized as <23, , and ≥25 kg/m 2 according to the report from the World Health Organization for Asia-Pacific regions. Additionally, the Wald test was applied for differences in nutrient intakes between the cancer survivors and non-cancer II individuals. A subgroup analysis for each cancer type with 300 or more cases was conducted for the mean nutrient comparison, and the ORs were compared with non-cancer I. Subgroups with less than 300 cases were classified as an "other cancer" group. Cancer survivors diagnosed with more than one cancer
## Nutrient intake
Nutrient intake was assessed using a validated semi-quantitative food frequency questionnaire consisting of 106 food items, frequencies of servings, and portion sizes. Daily nutrient intake values were estimated by the sum of the nutrients of each food item using the Food Composition Korea [bib_ref] Validation and reproducibility of food frequency questionnaire for Korean genome epidemiologic study, Ahn [/bib_ref] [bib_ref] Development of a semi-quantitative food frequency questionnaire based on dietary data from..., Ahn [/bib_ref]. Among the estimated nutrient intakes, total energy (kilocalories), protein, fat, carbohydrates (% of total energy), vitamin A, vitamin B 1 , vitamin B 2 , niacin, folate, vitamin B 6 , vitamin C, calcium, phosphorus, iron, and zinc were investigated in detail using the recommended dietary intakes established for Koreans.
# Statistical analysis
Of the matched participants, 1106 individuals had incomplete dietary questionnaires, including missing values for nutrient intake (48 cancer survivors, 595 non-cancer I, and 463 non-cancer II). After excluding these individuals, nutrient intake was compared between 5224 cancer survivors, 19,926 non-cancer I individuals, and 20,622 non-cancer II individuals using two methods: the mean intake of each nutrient between the three groups and the proportion of subjects with an intake of each nutrient higher than the recommended value compared to non-cancer I (reference group).
The baseline characteristics of the cancer survivors, non-cancer I individuals, and non-cancer II individuals were compared using the Chi-square test, and the mean of each nutrient level was compared by analysis of variance (ANOVA) with a post hoc comparison (Tukey's test). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate whether the cancer survivors and non-cancer individuals with comorbidities had nutrient intakes that were better than the intakes of the non-cancer I individuals by conditional logistic regression, adjusted for age, marital status, education level, income level, job status, smoking status, drinking status, current physical activity, and body mass index, based on measured height and weight. Although age was used as one of the matching variables, we also adjusted for age in the conditional logistic regression analysis due to the significant differences in age between the three groups. Body mass index was categorized as <23, , and ≥25 kg/m 2 according to the report from the World Health Organization for Asia-Pacific regions. Additionally, the Wald test was applied for differences in nutrient intakes between the cancer survivors and non-cancer II individuals. A subgroup analysis for each cancer type with 300 or more cases was conducted for the mean nutrient comparison, and the ORs were compared with non-cancer I. Subgroups with less than 300 cases were classified as an "other cancer" group. Cancer survivors diagnosed with more than one cancer type were classified as a "multiple cancer" group. The statistical software package SAS version 9.2 (SAS Institute, Cary, NC, USA) and R version 3.2.2 (R Foundation, Vienna, Austria) was used for all analyses.
# Results
The mean age of the 5224 cancer survivors was 55.8 years, and 74.9% were women. The top five cancer types (stomach, colon, breast, cervix, and thyroid) accounted for 66% of the cancer survivors, and the mean interval from diagnosis was 6.6 years. [fig_ref] Table 1: Baseline characteristics of cancer survivors, non-cancer individuals without comorbidities [/fig_ref] shows the baseline characteristics of study participants in three groups. shows the comparison of the means of each nutrient intake. The daily total energy, protein, proportion of total energy from protein, fat, proportion of total energy from fat intake, as well as vitamin B 1 , vitamin B 2 , niacin, phosphorus, and zinc intakes were lower in the cancer survivors than in the non-cancer I individuals. However, the proportion of total energy from carbohydrates, fiber and vitamin C intakes were higher compared to the non-cancer I individuals. When compared with the non-cancer II individuals, the cancer survivors' protein, proportion of total energy from protein, fat, proportion of total energy from fat, and vitamin B 1 intakes were lower, whereas the proportion of total energy from carbohydrates and the fiber, folate, vitamin C, vitamin E, iron, and potassium intakes were higher. [fig_ref] Table 3: Proportion and conditional logistic regression for higher dietary nutrient intake than recommended... [/fig_ref] shows the proportions and adjusted conditional ORs of the proportions of nutrient intakes that were higher than the recommended value for each nutrient. In all three groups, more than 80% of the subjects had a proportion of energy intake from carbohydrates that was higher than the recommended value and 1% or less of the subjects had a higher proportion of energy intake from fat than the recommended value. The cancer survivors consumed more energy from carbohydrates and less from fat.
The ORs for total energy, proportion of total energy from fat, and protein were significantly lower for the cancer survivors than they were for non-cancer I individuals (OR = 0.92, 95% CI = 0.86-0.99; OR = 0.54, 95% CI = 0.35-0.83; and OR = 0.85, 95% CI = 0.79-0.90, respectively), whereas the OR for the proportion of total energy from carbohydrates was higher (OR = 1.21, 95% CI = 1.10-1.33). Moreover, the cancer survivors' protein, vitamin B 1 , vitamin B 2 , niacin, and phosphorus intakes were lower than the non-cancer I individuals. Conversely, the proportion of cancer survivors with a vitamin C intake above the recommended value was higher (OR = 1.16, 95% CI = 1.08-1.23). Compared with the non-cancer II individuals, the cancer survivors' macronutrient intakes, including the proportions of total energy from fat intake and protein, were lower, whereas the proportion of total energy from carbohydrates and the vitamin A, folate, vitamin B 6 , vitamin C, and iron intakes were higher (p < 0.05, [fig_ref] Table 3: Proportion and conditional logistic regression for higher dietary nutrient intake than recommended... [/fig_ref].
When we divided the cancer survivors by cancer type, the thyroid cancer survivors did not show significant differences in their intakes of any nutrients. However, the proportion of total energy from fat intake was lower and the proportion of total energy from carbohydrate intake was higher in all other cancer types than they were in the non-cancer I individuals. Additionally, the gastric cancer survivors' protein, vitamin B 1 , vitamin B 2 , calcium, and phosphorus intakes were lower and the breast and cervical cancer survivors' total energy, protein, and vitamin B 1 intakes were lower [fig_ref] Table 4: Dietary intake in cancer survivors according to cancer type [/fig_ref]. [fig_ref] Table 5: Conditional logistic regression for higher dietary nutrient intakes than recommended in survivors... [/fig_ref] shows the adjusted ORs for intakes higher than the recommended levels for each nutrient in each type of cancer survivor compared with the non-cancer I individuals. The ORs of the proportion of total energy from carbohydrates were significantly higher in the colon and breast cancer survivors, suggesting that these individuals consumed more carbohydrates than the non-cancer population. Additionally, protein intakes were lower in the gastric, breast, and cervical cancer survivors. The ORs of vitamin B 1 intake were significantly lower in the gastric, colon, and breast cancer survivors, niacin intake ORs were lower in the breast cancer survivors, and phosphorus intake ORs were lower in the gastric, breast, and cervical cancer survivors. Conversely, for vitamin C intake, ORs were significantly higher in the gastric and colon cancer survivors.
[formula] Fat (% of total energy) -b -b -b -b -b -b -b [/formula]
# Discussion
This study is one of the few studies to compare nutrient intake in cancer survivors with matched, unaffected people, based on population study results. The results suggested that cancer survivors' nutrient intake statuses were poor, compared with non-cancer individuals without comorbidities, in terms of total energy, the proportion of energy from carbohydrates and fat, B vitamins, niacin, and phosphorus, but were better in regard to vitamin C intake. Even when compared with non-cancer individuals with comorbidities, the cancer survivors' proportions of energy from carbohydrates and fat and their protein intakes were poor, although their micronutrient intakes were better.
Although several previous studies have reported the poor dietary intake of adult cancer survivors, these studies considered specific types of foods, such as only fruit and vegetable consumption (5-A-Day) [bib_ref] Cancer survivors' adherence to lifestyle behavior recommendations and associations with health-related quality..., Blanchard [/bib_ref] [bib_ref] A population-based estimate of the prevalence of behavioral risk factors among adult..., Coups [/bib_ref] , specific guideline adherence [bib_ref] Adherence to the World Cancer Research Fund/American Institute for cancer research recommendations..., Inoue-Choi [/bib_ref] , or weight change as markers for nutrient intake [bib_ref] Role of nutritional status in predicting quality of life outcomes in cancer-A..., Lis [/bib_ref]. One recent study conducted in a Western population estimated all nutrient intake levels and suggested poor adherence to dietary guidelines in cancer survivors, which was similar to our results. However, the nutrients with poor intakes differed between previous Western studies and this study. Zhang et al. showed higher energy intakes, saturated fat, and sodium and lower vitamin D, vitamin E, potassium, fiber, and calcium intakes in cancer survivors [bib_ref] Demark-Wahnefried, W. Diet quality of cancer survivors and noncancer individuals: Results from..., Zhang [/bib_ref] , which was in contrast to our results in which lower calorie, protein, and fat intakes were found. These different patterns of dietary nutrient intakes in cancer survivors may be affected by both different eating patterns in cancer survivors and background nutrient intake patterns in the general Western and Asian populations. The dietary protein, carbohydrate, saturated fat, B vitamin, niacin, and folate intakes were higher than the recommended levels in people without a history of cancer in the study by Zhang et al. [bib_ref] Demark-Wahnefried, W. Diet quality of cancer survivors and noncancer individuals: Results from..., Zhang [/bib_ref] , whereas the intakes of these nutrients were lower (with the exception of the carbohydrate intake) in this study population, even in the non-cancer individuals.
In Asian countries where polished rice-based meals are common, the diet is higher in carbohydrates but low in animal fat [bib_ref] The cardiovascular continuum in Asia-A new paradigm for the metabolic syndrome, Cheung [/bib_ref]. This difference may affect the lower levels of nutrient intake from animal products, such as fat, protein, and B vitamins. The mean values of all nutrients were comparable with previous nationally representative studies in Korea [bib_ref] Nutritional intake of Korean population before and after adjusting for within-individual variations:..., Kim [/bib_ref] , suggesting that carbohydrate intake was higher and intake of nutrients from animal products was generally lower than recommended in Korea. Furthermore, several other studies conducted in Asian countries showed lower nutrient intake levels, especially for total energy, protein, and fat, in both the general population [bib_ref] Intake of fatty acids in general populations worldwide does not meet dietary..., Harika [/bib_ref] and cancer survivors [bib_ref] Effects of nutritional and psychological status of the patients with advanced stomach..., Tian [/bib_ref] [bib_ref] Nutritional status and quality of life of the gastric cancer patients in..., Tian [/bib_ref] [bib_ref] Association of nutritional status with quality of life in breast cancer survivors...., Mohammadi [/bib_ref].
When we focused on the comparison of nutrient intakes in cancer survivors and non-cancer individuals, fat, protein, vitamin A, B vitamins, niacin, and phosphorus, which are primarily ingested from animal food sources, showed significantly lower intakes in the cancer survivors. The proportion of total energy from fat and the protein intake were even lower than the intakes in non-cancer II individuals, who generally require dietary restrictions due to comorbidities. Cancer survivors are highly motivated to adopt healthy lifestyle behaviors [bib_ref] Lifestyle interventions in cancer survivors: Designing programs that meet the needs of..., Stull [/bib_ref] [bib_ref] Understanding the potential of teachable moments: The case of smoking cessation, Mcbride [/bib_ref] , and their interest in a healthy lifestyle has been shown to mostly focus on diet, especially a low-fat diet [bib_ref] Current health behaviors and readiness to pursue life-style changes among men and..., Demark-Wahnefried [/bib_ref]. The nutrition guidelines for cancer survivors in Western countries generally recommend diets high in vegetables, fruits, and whole grains and low in fat [bib_ref] Nutrition and physical activity guidelines for cancer survivors, Rock [/bib_ref]. However, for cancer survivors in Asian countries, such recommendations might make them increase their carbohydrate intake and reduce their intake of animal products, inducing far lower levels of protein and fat as proportions of the total caloric intake and relatively higher carbohydrate intakes. The higher vitamin C intake levels in survivors could suggest a higher intake of vegetables and fruits in cancer survivors, which follows the current guidelines well. Therefore, the adaptation of nutrient intake guidelines from Western countries without considering the differences in dietary intakes between Western and Asian countries in the general population may produce negative effects on nutrient intakes in cancer survivors.
To the best of our knowledge, few studies have investigated the differences in nutrient intakes between survivors by cancer type. Survivors of gastric, colon, breast, cervical, and thyroid cancer, which are the five major prevalent cancers in Korea [bib_ref] Cancer statistics in Korea: Incidence, mortality, survival, and prevalence in 2012, Jung [/bib_ref] , were compared with non-cancer I individuals.
The intake patterns of survivors of thyroid cancer, which is the most common cancer in Korea, were similar or better than the non-cancer I individuals. However, gastric, breast, and cervical cancer survivors showed worse nutrient intake qualities with lower protein intake. Additionally, gastric and breast cancer survivors showed poor nutrient intakes for several micronutrients and minerals. The nutrient intakes of gastric cancer patients generally deteriorate due to gastrectomy and chemotherapy [bib_ref] Importance of early nutritional screening in patients with gastric cancer, Gavazzi [/bib_ref] , which was supported by our results. However, a previous study in the United States proposed that breast cancer survivors have the best diet quality among the major types of cancer [bib_ref] Demark-Wahnefried, W. Diet quality of cancer survivors and noncancer individuals: Results from..., Zhang [/bib_ref] , indicating that a regional approach to nutrient intake was needed.
Although our study has strengths, several limitations should be considered. First, the cross-sectional design based on baseline measurements of a cohort prevented the assessment of changes or trends in dietary intake after cancer diagnosis in cancer survivors. Thus, we could not investigate the changes in nutrient intake before and after cancer diagnosis in cancer survivors. Second, the cancer survivors and non-cancer individuals with/without comorbidities were divided based on self-reports, which could cause misclassification. However, previous studies reported a reasonably high validity for self-reported cancer histories [bib_ref] Validation of self-reported cancer incidence at follow-up in a prospective cohort study, Cho [/bib_ref] , and we considered the risk for misclassification bias to be minimal. Additionally, because the cancer stage and treatment information was not accessed by the questionnaires, evaluating their effects on dietary intakes in cancer survivors was not possible. Third, the subjects were recruited from health examination centers. Thus, they might be healthier than general cancer survivors or people without cancer and more concerned about healthy lifestyle, limiting the ability to generalize the results. Fourth, although we matched cancer survivors with non-cancer groups by age, gender, and recruitment center to eliminate their effects on dietary intake, the age distribution was still significantly different. We included age as one of the covariates in the conditional logistic regression model to reduce its effect. Additionally, we confirmed that the results were not significantly different, with the exception of minor changes in point estimates based on both the conditional analysis and logistic regression analysis (data not shown). Fifth, although food frequency questionnaires have been widely applied to access dietary intake in epidemiological studies, the measurement errors including the portion size or lower correlation with other tools, especially in Asian countries, should be considered [bib_ref] Dietary assessment methods in epidemiologic studies, Shim [/bib_ref]. Despite these limitations, to the best of our knowledge, this study is the first study to evaluate nutrient intake in cancer survivors as a whole and by cancer type compared with the non-cancer population using a relatively large sample size and a validated food frequency questionnaire. To increase the comparability, cancer survivors and two non-cancer groups were matched by age, gender, and recruitment center, and numerous possible covariates were adjusted for in the analysis. Additionally, the nutrient intake was compared with non-cancer individuals with comorbidities to obtain a perspective of cancer as a chronic disease.
# Conclusions
A healthy diet is important for decreasing cancer-related adverse sequelae, comorbidities, and death and improving quality of life [bib_ref] Lifestyle interventions in cancer survivors: Designing programs that meet the needs of..., Stull [/bib_ref] [bib_ref] Role of nutritional status in predicting quality of life outcomes in cancer-A..., Lis [/bib_ref]. We observed poor nutrient intakes in the cancer survivors, especially a lower calorie intake, inappropriate proportions of total energy (higher proportion of carbohydrate and lower proportion of fat and protein), and lower micronutrients derived from animal products. This pattern was different from observations in Western countries, where the higher intakes of energy and fat are important issues. Therefore, the adaptation of nutrition guidelines in Western countries for cancer survivors in Asia needs to be re-considered, and guidelines for Asian cancer survivors should be established based on dietary intake patterns in the general population.
[fig] Figure 1: Flow chart of the study participants selection process. [/fig]
[table] Table 1: Baseline characteristics of cancer survivors, non-cancer individuals without comorbidities (non-cancer I), and non-cancer individuals with comorbidities (non-cancer II). [/table]
[table] Table 3: Proportion and conditional logistic regression for higher dietary nutrient intake than recommended in cancer survivors and non-cancer individuals with comorbidities (non-cancer II) compared with non-cancer individuals without comorbidities (non-cancer I). Analyses were adjusted for age, marital status, education level, income level, job status, smoking status, drinking status, current physical activity, and body mass index; b Differences between non-cancer individuals with comorbidities and cancer survivors in the Wald test. [/table]
[table] Table 4: Dietary intake in cancer survivors according to cancer type. [/table]
[table] Table 5: Conditional logistic regression for higher dietary nutrient intakes than recommended in survivors of each type of cancer compared with non-cancer individuals without comorbidities (non-cancer I). [/table]
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Safety and Efficacy of Indocyanine Green in Colorectal Cancer Surgery: A Systematic Review and Meta-Analysis of 11,047 Patients
Citation: Safiejko, K.; Tarkowski, R.; Kozlowski, T.P.; Koselak, M.; Jachimiuk, M.; Tarasik, A.; Pruc, M.; Smereka, J.; Szarpak, L. Safety and Efficacy of Indocyanine Green in Colorectal Cancer Surgery: A Systematic Review and Meta-Analysis of 11,047 Patients.
# Introduction
Indocyanine green (ICG) was approved for clinical use in 1959. It is an inexpensive, readily available, and simple-to-use compound characterized by low toxicity [bib_ref] Light-absorbing properties, stability, and spectral stabilization of indocyanine green, Landsman [/bib_ref]. In the recent decade, owing to the rapid development of medical devices in surgery, including laparoscopic and robotic systems, the compound has found a variety of applications in abdominal organ surgery. The ICG technique has become particularly popular among surgeons performing colorectal surgery. It allows intraoperative real-time assessment of the blood supply to the stumps of the large intestine after resection and to the intestine after anastomosis [bib_ref] Intraoperative use of fluorescence with indocyanine green reduces anastomotic leak rates in..., Arezzo [/bib_ref]. Good blood supply to the anastomosis is crucial for proper healing and preventing intestinal anastomotic leak [bib_ref] ICG fluorescence imaging in colorectal surgery: A snapshot from the ICRAL study..., Baiocchi [/bib_ref].
Anastomotic leak is one of the most serious colorectal and rectal surgery complications, significantly increasing postoperative morbidity and mortality. It also worsens the final oncological outcome and decreases the patients' quality of life [bib_ref] A collaborative review of the current concepts and challenges of anastomotic leaks..., Vallance [/bib_ref]. Currently available data show that the percentage of anastomotic leak in colorectal and rectal surgery ranges from 1% to 19%. Undoubtedly, the incidence of anastomotic leak is highly variable and depends on the anatomical location of the anastomosis, ranging 1-8% in ileocecal anastomoses and 5-19% in colorectal anastomoses [bib_ref] Systematic review of preoperative, intraoperative and postoperative risk factors for colorectal anastomotic..., Mcdermott [/bib_ref] [bib_ref] Hospital variation in anastomotic leakage after rectal cancer surgery in the Spanish..., Ortiz [/bib_ref].
It has been confirmed that the incidence of anastomotic leak in colorectal and rectal surgery depends on many factors. These are surgeon-dependent factors, such as the technique of anastomosis, blood supply to the anastomosis, lack of tension of the anastomosed bowel, timing of surgery, perioperative blood loss, restrictive perioperative fluid therapy, nutritional support, as well as surgeon-independent factors, such as age, male gender, comorbidities, malnutrition, obesity, stimulants, immunosuppression, preoperative chemotherapy and radiotherapy, advanced stage of cancer, and inflammatory bowel disease [bib_ref] Systematic review of preoperative, intraoperative and postoperative risk factors for colorectal anastomotic..., Mcdermott [/bib_ref] [bib_ref] Emerging trends in the etiology, prevention, and treatment of gastrointestinal anastomotic leakage, Chadi [/bib_ref].
The most important factor influencing the proper healing of intestinal anastomosis is good blood supply. Other factors remain less significant if this condition is not fulfilled [bib_ref] Integrated approach to colorectal anastomotic leakage: Communication, infection and healing disturbances, Sparreboom [/bib_ref] [bib_ref] Anastomotic leakage in rectal cancer surgery: The role of blood perfusion, Rutegård [/bib_ref]. Therefore, it seems that intraoperative imaging of intestinal anastomotic blood supply by using ICG may become a predictive test of normal intestinal blood supply, which will consequently reduce the risk of anastomotic leak [bib_ref] Intraoperative use of ICG fluorescence imaging to reduce the risk of anastomotic..., Blanco-Colino [/bib_ref].
For this reason, we performed a systematic review and meta-analysis to evaluate the efficacy and safety of ICG use in colorectal cancer surgery.
# Materials and methods
The present systematic review and meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [bib_ref] The PRISMA 2020 statement: An updated guideline for reporting systematic reviews, Page [/bib_ref]. Owing to the study design (meta-analysis), neither institutional review board approval nor patient informed consent were required.
## Study strategy
We conducted a systematic literature search of the Embase, MEDLINE, and Cochrane Central Register of Controlled Trials (CENTRAL) databases from inception to 10 November 2021. All references were saved in an EndNote (EndNote, Inc., Philadelphia, PA, USA) library used to identify duplicates. Terms related to ICG in colorectal cancer were applied: "Indocyanine Green" OR "indocyanine green-sulfo-OSu" OR "ICG" OR "Fluorescein Angiography" AND "Robotic Surgical Procedures" OR "Minimally Invasive Surgical Procedures" OR "laparosc*" OR "Rectum" OR "Rectal" OR "Colo-rect*" OR "surge*" OR "surgi*" OR "surgeo*" OR "Colorectal Surgery" OR "Ileostomy" OR "Colostomy" OR "Colectomy". Additionally, we performed a manual search and review of the references listed in the retrieved articles.
## Inclusion and exclusion criteria
The studies included in this meta-analysis met the following PICOS criteria: (1) Participants: adult patients requiring colorectal cancer surgery; (2) Intervention: surgery with ICG use; (3) Comparison: surgery without ICG use; (4) Outcomes: detailed information concerning anastomotic leak rate, occurrence of other adverse events; (5) Study design: randomized controlled trials (RCTs), retrospective trials. The exclusion criteria were as follows: (1) studies involving pediatric patients; (2) letters to the editor; (3) editorials; (4) conference abstracts; (5) guidelines.
## Data extraction
Two reviewers (M.P. and K.S.) independently extracted data from the identified eligible studies using a specifically designed data extraction form in Microsoft Excel TM (Microsoft Corp., Redmond, WA, USA). Another author cross-checked these data before analysis (L.S.). The following data were extracted from each study: intraoperative parameters (i.e., operative time, intraoperative blood loss, transfusion requirement), postoperative parameters (i.e., anastomotic leak occurrence, adverse events). When there were suspected discrepancies in the data, we contacted the relevant authors directly.
## Outcomes
The primary analysis focused on assessing anastomotic leak in patients undergoing colorectal cancer surgery with and without ICG application. Secondary outcomes included intraoperative parameters (i.e., operative time, intraoperative blood loss, transfusion requirement) and postoperative parameters (i.e., the occurrence of other adverse events, length of hospital stay, reoperation readmissions).
## Risk of bias assessment
The risk of bias was determined independently by two reviewers (K.S. and M.K.); any disagreements were resolved by a third reviewer (L.S.). We applied revised tools to assess the risk of bias: RoB 2 for randomized trials [bib_ref] RoB 2: A revised tool for assessing risk of bias in randomised..., Sterne [/bib_ref] and ROBINS-I for non-randomized trials [bib_ref] ROBINS-I: A tool for assessing risk of bias in non-randomised studies of..., Sterne [/bib_ref]. The overall RoB 2 judgment at the domain and study level was attributed in accordance with the criteria specified in the Risk-of-Bias VISualization (robvis) tool [bib_ref] Risk-of-bias VISualization (robvis): An R package and Shiny web app for visualizing..., Mcguinness [/bib_ref].
# Statistical analysis
The meta-analysis was entirely conducted with the Review Manager software, version 5.4 (Nordic Cochrane Center, Cochrane Collaboration, London, UK). The significance level for all statistical tests was p < 0.05 (two-tailed). Descriptive statistics are presented as numbers of cases (n) and percentages (%) for dichotomous and categorical variables or as means and standard deviations for continuous variables. When a continuous outcome was reported in a study as median, range, and interquartile range, we estimated means and standard deviations by using a formula described by Hozo et al. [bib_ref] Estimating the mean and variance from the median, range, and the size..., Hozo [/bib_ref]. Heterogeneity was determined with the I 2 statistic, in which the results range from 0% to 100%. Heterogeneity was interpreted as not observed with I 2 = 0%, low with I 2 = 25%, medium with I 2 = 50%, and high with I 2 = 75% [bib_ref] Measuring inconsistency in meta-analyses, Higgins [/bib_ref]. A fixed model effect was applied when I 2 < 50%, and a random model effect was used in other cases. For dichotomous data, we employed odds ratios (ORs) or risk ratios (RRs) as the effect measure with 95% confidence intervals (CIs); for continuous data, we used mean differences (MDs) with 95% CI. Subgroup analyses were performed with respect to study design (RCTs, non-RCTs.).
# Results
## Literature search and study characteristics
The detailed process of searching eligible studies is shown in [fig_ref] Figure 1: Database search and selection of studies in accordance with the PRISMA guidelines [/fig_ref]. A total of 1936 references were finally confirmed by the electronic search, and 73 potentially relevant studies were selected after screening the titles and abstracts. Among the 73 potentially relevant studies, a total of 32 met the inclusion criteria . [fig_ref] Table 1: Characteristics of the included trials [/fig_ref] summarize the study characteristics. The 32 included studies, involving 11,047 patients, were published between 2010 and 2021. The assessment of their risk of bias is provided in Figures S1-S4. Legend: ACR = atypical colonic resection; AR = anterior resection; CL = colectomy; CR = colorectal resection; HC = hemicolectomy; ICG = indocyanine green; IPAA = ileal pouch-anal anastomosis; ISR = intersphincteric resection; LAR = low anterior resection; LSH = laparoscopic supracervical hysterectomy; NS = not stated; PR = proctectomy; RCT = randomized controlled trial; SI = sigmoidectomy; SR = sigmoid resection; SSSA = stapled side-to-side anastomosis; TaTME = transanal total mesorectal excision; TME = total mesorectal excision.
## Intraoperative evaluation
Seventeen studies reported operative duration, which equaled 214.9 ± 67.5 min in the ICG group and 228.9 ± 66.1 min in the non-ICG group (MD = −0.77; 95% CI: −12.42 to 10.87; I 2 = 97%; p = 0.90).
Intraoperative blood loss was reported in eight studies and amounted to 128.7 ± 268.9 mL in the ICG group compared with 96.4 ± 135.8 mL in the non-ICG group (MD = −4.54; 95% CI: −17.43 to 8.35; I 2 = 98%; p = 0.49).
Intraoperative transfusion was required in 1.4% of both ICG and non-ICG group patients (RR = 1.00; 95% CI: 0.37-2.72; I 2 = 9%; p = 1.00). Additional sub-analyses by study design (RCTs and non-RCTs) are presented in [fig_ref] Table 2: Pooled analysis of adverse events in the included trials [/fig_ref].
## Anastomotic leak
The pooled analysis of the 32 studies showed that the overall anastomotic leak rate in the ICG and non-ICG groups varied and amounted to 3.7% vs. 7.6%, respectively (RR = 0.46; 95% CI: 0.39-0.56; p < 0.001; [fig_ref] Figure 2: Forest plot of anastomotic leak occurrence in the ICG vs [/fig_ref].
The same relationship was observed for the results as divided by grades of the anastomotic leak. The anastomotic leak rate was 2. The sub-analysis performed with respect to the type of study design showed an anastomotic leak rate of 8.1% in the ICG group compared with 12.1% in the non-ICG group (RR = 0.67; 95% CI: 0.46-0.98; p = 0.04) for RCTs [bib_ref] A study investigating the perfusion of colorectal anastomoses using fluorescence angiography: Results..., Alekseev [/bib_ref] [bib_ref] Intraoperative angiography with indocyanine green to assess anastomosis perfusion in patients undergoing..., De Nardi [/bib_ref] [bib_ref] Perfusion Assessment in Left-Sided/Low Anterior Resection (PILLAR III): A randomized, controlled, parallel,..., Jafari [/bib_ref] and a rate of 3.1% vs. 7.3%, respectively (RR = 0.43; 95% CI: 0.35-0.52; p < 0.001) for non-RCTs.
## Postoperative period evaluation
A detailed list of the most frequently observed adverse events in the analyzed studies is presented in [fig_ref] Table 2: Pooled analysis of adverse events in the included trials [/fig_ref]. Twelve studies reported the number of patients with postoperative adverse events. The pooled analysis revealed that in the ICG and non-ICG groups, postoperative adverse events incidence equaled 19.3% and 27.7%, respectively (RR = 0.80; 95% CI: 0.70-0.92; p = 0.002). However, the analyses of individual adverse events indicated no statistically significant differences between the ICG and non-ICG groups (p > 0.05). Legend: CI = confidence interval; ICG = indocyanine green; RR = risk ratio. Note: Not all outcomes were reported in every study. "No. of studies" refers to the studies included in the analysis for the particular outcome.
Adverse events classified as Clavien-Dindo grade I-II varied in the ICG and non-ICG groups and amounted to 20.3% vs. 22.5% (RR = 0.91; 95% CI: 0.79-1.04; I 2 = 50%; p = 0.15). In the case of grade III-IV, the corresponding values equaled 6.9% and 8.6% (RR = 0.88; 95% CI: 0.63-1.25; I 2 = 0%; p = 0.49).
Thirteen studies reported the length of hospital stay, which was 8.7 ± 5.2 days for the ICG group compared with 8.5 ± 5.1 days for the non-ICG group (MD = −0.39; 95% CI: −0.84 to 0.05; I 2 = 96%; p = 0.08).
The readmission rate in a 30-day follow-up was 4.6% vs. 7.2%, respectively, in the ICG vs. the non-ICG group (RR = 0.91; 95% CI: 0.48-1.70; I 2 = 14%; p = 0.76) [bib_ref] The impact of fluorescence angiography on anastomotic leak rate following transanal total..., Otero-Piñeiro [/bib_ref] [bib_ref] Indocyanine green fluorescence imaging to assess bowel perfusion during totally laparoscopic surgery..., Su [/bib_ref]. However, the readmission rate in a 60-day follow-up equaled 15.5% vs. 10.6% (RR = 0.85; 95% CI: 0.16-4.42; I 2 = 61%; p = 0.85) [bib_ref] Near-infrared fluorescence angiography for colorectal surgery is associated with a reduction of..., Impellizzeri [/bib_ref] [bib_ref] Equivocal effect of intraoperative fluorescence angiography on colorectal anastomotic leaks, Kin [/bib_ref] [bib_ref] Indocyanine green fluorescence imaging to assess bowel perfusion during totally laparoscopic surgery..., Su [/bib_ref].
The reoperation rate was reported in nine studies. It amounted to 3.4% in the ICG group compared with 5.3% in the non-ICG group (RR = 0.73; 95% CI: 0.47-1.12; I 2 = 0%; p = 0.15).
# Discussion
Disruption of anastomosis after colorectal surgery is still one of the most severe complications. Despite the progress in surgical oncology, its incidence reaches even 11-19% of patients [bib_ref] Systematic review of preoperative, intraoperative and postoperative risk factors for colorectal anastomotic..., Mcdermott [/bib_ref] [bib_ref] Hospital variation in anastomotic leakage after rectal cancer surgery in the Spanish..., Ortiz [/bib_ref] [bib_ref] Effect of robotic-assisted vs conventional laparoscopic surgery on risk of conversion to..., Jayne [/bib_ref] [bib_ref] Bioabsorbable staple line reinforcement in restorative proctectomy and anterior resection: A randomized..., Senagore [/bib_ref] [bib_ref] Multicentric study on robotic tumour-specific mesorectal excision for the treatment of rectal..., Pigazzi [/bib_ref]. According to Choi et al. [bib_ref] Leakage after resection and intraperitoneal anastomosis for colorectal malignancy: Analysis of risk..., Choi [/bib_ref] , almost a third of patients with anastomotic insufficiency die for this reason. Several patient-dependent risk factors for anastomotic insufficiency following colorectal surgery include obesity, preoperative pelvis irradiation, male sex, malnutrition, low anastomosis, and tobacco use. The technical aspects crucial for proper anastomotic healing are the tension of the rectal stump and proximal part of the colon and appropriate blood circulation within the anastomosis. Blood support assessment helps estimate the risk and impacts on the level of proximal bowel transection [bib_ref] Ileal pouch-anal anastomosis with fluorescence angiography: A case-matched study, Spinelli [/bib_ref]. Traditionally, such assessment is highly subjective and based on visible, active bleeding from the cut tissue and lack of discoloration of the bowel observed by the surgeon. A more sophisticated and effective method involves fluorescence scintigraphy, described aptly by Kudszus et al. [bib_ref] Intraoperative laser fluorescence angiography in colorectal surgery: A noninvasive analysis to reduce..., Kudszus [/bib_ref] as "bringing light" into the (dark) picture of the anastomotic leakage. The PILLAR II study revealed insufficient blood circulation as assessed by ICG in 6.5% of patients in whom sufficient circulation was indicated in macroscopic evaluation [bib_ref] Perfusion assessment in laparoscopic left-sided/anterior resection (PILLAR II): A multi-institutional study, Jafari [/bib_ref]. The observation of impaired blood supply leads to modifications of the surgery through cutting the altered tissue at the level of proper microcirculation. This enables anastomosis healing and decreases the risk of anastomotic leak. Although ICG use demands an additional procedure, and poor microcirculation assessment results in additional bowel transection, our analysis did not imply any increase in the operation time, with the mean of 214.9 ± 67.5 min in the ICG group and 228.9 ± 66.1 min in the non-ICG group.
Our analysis demonstrated lower rates of anastomotic leak in both RCTs and non-RCTs.
A study by Otero-Piñeiro et al. [bib_ref] The impact of fluorescence angiography on anastomotic leak rate following transanal total..., Otero-Piñeiro [/bib_ref] , encompassing 284 patients with rectal cancer operated on with the transanal total mesorectal excision method with a mean anastomotic height of 4.85 vs. 5.08 cm and a diverting stoma constructed in 72.1% vs. 72.5% of patients, showed a modification of the surgical intervention after ICG angiography (ICGA) in 28.7% of cases. The authors noted 2.5% of anastomotic leak cases in the ICGA group vs. 11.3% in the control one (p = 0.02). ICGA was described as "an easy-to-use, accessible and reproducible technology that allows real-time evaluation of tissue perfusion".
A meta-analysis by Blanco-Colino and Espin-Basany [bib_ref] Intraoperative use of ICG fluorescence imaging to reduce the risk of anastomotic..., Blanco-Colino [/bib_ref] , involving 554 patients, showed a significant reduction of anastomotic insufficiency rate after the use of ICG fluoroscopy compared with interventions without its application (1.1% vs. 6.1%, respectively) and an 81% anastomotic insufficiency reduction owing to analyzed circulation assessment (OR = 0.19; 95% CI: 0.05-0.75; p = 0.02). In a multicenter study by Ris et al. [bib_ref] Multicentre phase II trial of near-infrared imaging in elective colorectal surgery, Ris [/bib_ref] , fluorescein angiography performed during surgery led to a change in the operation plan in 5.8% of cases. The PILLAR II study demonstrated no anastomotic leak incidence in the cases with alteration of the surgical plan after fluorescence angiography (7.9%, 11 cases) [bib_ref] Perfusion assessment in laparoscopic left-sided/anterior resection (PILLAR II): A multi-institutional study, Jafari [/bib_ref]. Other studies also indicated a lower rate of anastomotic insufficiency in cases of ICGA use for blood supply assessment. The publication by Skrovina et al.showed an anastomotic leak incidence decrease by 8% (10% vs. 18% in the angioscintigraphy and the non-angioscintigraphy group), while fluoroscintigraphy extended the operation time by 6 min. ICG use changed the operation scenario in 12% of the patients when the resection line was moved from 2 to 5 cm proximally. In a single-surgeon retrospective study, Mizrahi et al. [bib_ref] Indocyanine green fluorescence angiography during low anterior resection for low rectal cancer:..., Mizrahi [/bib_ref] reported even 0% incidence of anastomotic insufficiency in the group of 30 patients assessed with ICG. The examination changed the intervention in four (13.3%) individuals, while there were two anastomotic insufficiency cases in the control group (n = 30). It is worth mentioning that the average anastomosis level was low-2.8 cm from the anal verge; all of the patients were treated with a loop ileostomy. Similar results were obtained by Boni et al. [bib_ref] Indocyanine green fluorescence angiography during laparoscopic low anterior resection: Results of a..., Boni [/bib_ref] : there was a reduction of anastomotic insufficiency rate to 0% in the ICG group vs. 5% in the control group.
A lower rate of reoperations following intraoperative use of fluoroscintigraphy was reported by Kudszus et al. [bib_ref] Intraoperative laser fluorescence angiography in colorectal surgery: A noninvasive analysis to reduce..., Kudszus [/bib_ref] : 3.5% vs. 7.5% in the control group. It is especially worth mentioning that the proportion was even more striking in the most vulnerable group of patients, i.e., those aged over 70 years (4.3% vs. 11.9% [p = 0.04]), in whom adverse events are associated with higher mortality. The risk of reoperation was reduced by 64% in this particular subgroup. Additionally, hospital stay was shortened in the fluorescence angiography group.
Fluorescence radiography and near-infrared angiography can help estimate blood microcirculation. However, a change of the transection line level owing to blood supply assessed as insufficient in fluorescein angiography did not prevent anastomotic leak in ther out of eighteen cases (16.7%) [bib_ref] Efficacy of intraoperative ICG fluorescence imaging evaluation for preventing anastomotic leakage after..., Yanagita [/bib_ref]. This could support the thesis by Kin et al. [bib_ref] Equivocal effect of intraoperative fluorescence angiography on colorectal anastomotic leaks, Kin [/bib_ref] , implying that success is an effect of a combination of more than one factor concerning microcirculation.
Three randomized clinical trials assessed the effectiveness of ICG fluorescein angiography as the best available evidence source. Two of these support the hypothesis of the positive role of ICG fluorescein angiography in decreasing rates of anastomotic insuffi-ciency, while one of them, despite the authors' previous findings in a former phase II study, failed to demonstrate any difference between the outcomes in the examined groups. Our sub-analysis encompassing randomized clinical trials [bib_ref] A study investigating the perfusion of colorectal anastomoses using fluorescence angiography: Results..., Alekseev [/bib_ref] [bib_ref] Intraoperative angiography with indocyanine green to assess anastomosis perfusion in patients undergoing..., De Nardi [/bib_ref] [bib_ref] Perfusion Assessment in Left-Sided/Low Anterior Resection (PILLAR III): A randomized, controlled, parallel,..., Jafari [/bib_ref] showed an anastomotic leak rate of 8.1% in the ICG group and 12.1% in the non-ICG group (RR = 0.67; 95% CI: 0.46-0.98; p = 0.04).
The single-center FLAG trial performed by Alekseev et al. [bib_ref] A study investigating the perfusion of colorectal anastomoses using fluorescence angiography: Results..., Alekseev [/bib_ref] encompassed 377 cases randomized to ICG fluorescein angiography or visual assessment of blood supply. Fluorescein angiography revealed impaired perfusion in cases. Although anastomotic leak occurred in both groups, its rate was noticeably higher in the group without angiography: 31 (16.3%) vs. 17 (9.1%) in the ICG fluorescein angiography arm. The examination decreased anastomotic insufficiency rate in the group with low colorectal anastomoses (4-8 cm from the anal verge); the result was 14.4% vs. 25.7% in the non-ICG fluorescein angiography group (p = 0.04). There was no such effect among patients with higher levels of anastomosis . ICG demonstrated impaired blood circulation in the bowel in 36 (19%) cases.
Another randomized trial, performed by De Nardi et al. [bib_ref] Intraoperative angiography with indocyanine green to assess anastomosis perfusion in patients undergoing..., De Nardi [/bib_ref] , showed the favorable role of fluorescence angiography; it was, however, not statistically significant. Among 240 patients, angiography led to extended resection of the bowel in 11% of cases (n = 13), while anastomotic insufficiency occurred in six (5%) patients in the study group and eleven (9%) in the control one.
The PILLAR III study, performed by Jafari et al. [bib_ref] Perfusion assessment in laparoscopic left-sided/anterior resection (PILLAR II): A multi-institutional study, Jafari [/bib_ref] , did not confirm the promising results of the PILLAR II study, showing the benefit of ICG application in decreasing the rate of anastomotic insufficiency after extensive bowel surgery. This RCT encompassed 347 patients in 25 centers and was concluded early owing to decreasing accrual rates. Its primary endpoint was the anastomotic leak; the secondary endpoints involved the effectiveness of perfusion assessment with ICG fluoroscopy and pelvic abscesses requiring surgical intervention. Both groups of patients had similar demographic characteristics, as well as tumor-and patient-dependent factors. Although perfusion was successfully assessed in 94.5% of cases, anastomotic insufficiency occurred in 9% of patients after ICG compared with 9.6% without the examined method usage. Surgical intervention was required in 6.9% of the perfusion group compared with 8.6% of the standard group. Postoperative abscess occurred in 5.7% of the intervention group and 4.2% of the control one. The rates of other complications were similar in both groups. The authors indicate the heterogeneity of groups assessed in the studies mentioned above compared with their material, i.e., the higher risk of anastomotic leak in their group due to preoperative radiotherapy in 65% of the patients compared with 10-20% in the other trials, a lower level of anastomosis, and a higher proportion of males. They explain the results with the multifactorial character of anastomotic insufficiency, the different surgeon experience, and the fact that the study was underpowered.
On the other hand, the distal rectal stump and the lack of its perfusion assessment appear to be the most critical factors. Since fluoroscintigraphy examines blood perfusion in the proximal part of the bowel, it does not assess blood circulation in the rectal stump. According to Vignali et al. [bib_ref] Altered microperfusion at the rectal stump is predictive for rectal anastomotic leak, Vignali [/bib_ref] , there is a positive correlation between anastomotic leak and altered blood flow in the rectal stump. On the contrary, Alexeev et al. [bib_ref] A study investigating the perfusion of colorectal anastomoses using fluorescence angiography: Results..., Alekseev [/bib_ref] noted a "bright fluorescent reflection" in all cases. In a study by Kin et al. [bib_ref] Equivocal effect of intraoperative fluorescence angiography on colorectal anastomotic leaks, Kin [/bib_ref] , ICG use did not change the rate of anastomotic leak, although it led to surgical management modification in some of the patients.
There are some weaknesses of the presented meta-analysis. The heterogeneity of the published material could impact on the results, showing the superiority of ICG angiography [bib_ref] The effects of intraoperative ICG fluorescence angiography in laparoscopic low anterior resection:..., Wada [/bib_ref]. Heterogeneous operative methods (right and left hemicolectomy) were reported by Kudszus et al. [bib_ref] Intraoperative laser fluorescence angiography in colorectal surgery: A noninvasive analysis to reduce..., Kudszus [/bib_ref] or Kin et al. [bib_ref] Equivocal effect of intraoperative fluorescence angiography on colorectal anastomotic leaks, Kin [/bib_ref] , and small sample sizes characterized the studies by Jafari et al. [bib_ref] Perfusion assessment in laparoscopic left-sided/anterior resection (PILLAR II): A multi-institutional study, Jafari [/bib_ref] or Mizrahi et al. [bib_ref] Indocyanine green fluorescence angiography during low anterior resection for low rectal cancer:..., Mizrahi [/bib_ref]. Moreover, some publications presented single-surgeon experience, and diverting stoma was formed in all patients described by Boni et al. [bib_ref] Indocyanine green fluorescence angiography during laparoscopic low anterior resection: Results of a..., Boni [/bib_ref]. Our review encompassed randomized clinical studies as the leading and most powerful evidence source, as well as retrospective, much smaller studies. The latter constituted the majority as prospective data are sparse.
# Conclusions
The publications encompassed in our meta-analysis show different patients, with different factors influencing the results. The pooled analysis revealed a lower incidence of anastomotic leak in cases with ICG use. There are several other convincing advantages: safety, simplicity, and short time of the method adjustment. Although the results in the analyzed studies vary, the presented meta-analysis demonstrates ICG perfusion assessment as a tool worth considering to decrease the rate of complications following colorectal surgery-valuable in the context of other, well-known risk factors.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14041036/s1. [fig_ref] Table 1: Characteristics of the included trials [/fig_ref] : Methodology characteristics of the included trials ; [fig_ref] Table 2: Pooled analysis of adverse events in the included trials [/fig_ref] : Pooled analysis in subgroups of randomized and non-randomized trials; : PRISMA checklist. [fig_ref] Figure 1: Database search and selection of studies in accordance with the PRISMA guidelines [/fig_ref] : A summary table of the review authors' judgements for each risk of bias item for each randomized study; [fig_ref] Figure 2: Forest plot of anastomotic leak occurrence in the ICG vs [/fig_ref] : A plot of the distribution of the review authors' judgements for each risk of bias item across randomized studies; [fig_ref] Figure 3: Forest plot of anastomotic leak occurrence in the ICG vs [/fig_ref] : A summary table of the review authors' judgements for each risk of bias item for each non-randomized study; : A plot of the distribution of the review authors' judgements for each risk of bias item across non-randomized studies.
[fig] Figure 1: Database search and selection of studies in accordance with the PRISMA guidelines. [/fig]
[fig] Figure 2: Forest plot of anastomotic leak occurrence in the ICG vs. non-ICG groups. The square centers represent the weighted risk ratios for individual trials, and the corresponding horizontal lines stand for the 95% CI. The diamonds represent pooled results. Legend: CI = confidence interval; ICG = indocyanine green. [/fig]
[fig] Figure 3: Forest plot of anastomotic leak occurrence in the ICG vs. non-ICG groups with sub-analysis with respect to the anastomotic leak grade. The square centers represent the weighted risk ratios for individual trials, and the corresponding horizontal lines stand for the 95% CI. The diamonds represent pooled results. Legend: CI = confidence interval; ICG = indocyanine green. [/fig]
[fig] Author: Contributions: Conceptualization, K.S. and L.S.; methodology, K.S. and L.S.; software, L.S.; validation, K.S., L.S. and J.S.; formal analysis, K.S. and L.S.; investigation, K.S., T.P.K., L.S., J.S. and M.P.; resources, L.S.; data curation, K.S. and L.S.; writing-original draft preparation, K.S., L.S. and R.T.; writing-review and editing, M.K., M.J., A.T., K.S., L.S., J.S., M.P. and R.T.; visualization, L.S.; supervision, L.S. and K.S.; project administration, K.S. and L.S.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research received no external funding. [/fig]
[table] Table 1: Characteristics of the included trials.Table 1. Cont. [/table]
[table] Table 2: Pooled analysis of adverse events in the included trials. [/table]
|
Early Identification, Accurate Diagnosis, and Treatment of Silicosis
Silicosis is a global problem, and it has brought about great burdens to society and patients' families. e etiology of silicosis is clear, preventable, and controllable, but the onset is hidden and the duration is long. us, it is difficult to diagnose it early and treat it effectively, leaving workers unaware of the consequences of dust exposure. As such, a lack of details in the work history and a slow progression of lung disease contribute to the deterioration of patients until silicosis has advanced to fibrosis. ese issues are the key factors impeding the diagnosis and the treatment of silicosis. is article reviews the literature on the early identification, diagnosis, and treatment of silicosis as well as analyzes the difficulties in the diagnosis and the treatment of silicosis and discusses its direction of future development.
# Introduction
Silicosis is caused by the inhalation of respirable crystalline silica (RCS) dust which, over time, leads to progressive, irreversible, and fatal inflammation and fibrosis in the lungs. Although the cause of silicosis is undisputed, millions of workers worldwide continue to be exposed to hazardous levels of RCS [bib_ref] Silicosis, Leung [/bib_ref]. e condition is preventable; however, no specific treatment exists, although a small proportion of patients may receive a lung transplant. Furthermore, the failure to recognize and control the risk associated with silica exposure in contemporary work practices, such as sandblasting denim jeans and manufacturing artificial stone benchtops, has led to the reemergence of silicosis worldwide [bib_ref] Silica-related diseases in the modern world, Hoy [/bib_ref]. e health hazards related to crystalline silica exposure are not only limited to silicosis; they include other idiopathic diseases such as tuberculosis, autoimmune diseases, lung cancer, pulmonary alveolar proteinosis, sarcoidosis, and idiopathic pulmonary fibrosis [bib_ref] Silica-related diseases in the modern world, Hoy [/bib_ref] [bib_ref] Beyond silicosis, is the world failing on silica hazards?, Cavalin [/bib_ref]. is review outlines the most recent advances in understanding the mechanisms, diagnosis, and treatment of silicosis.
## Clear causes and unresolved
Mechanisms of Silicosis
## Cytotoxic effects of silica particles.
It is well documented that the inhalation of silica particles is an environmental and occupational cause of silicosis, a type of pneumoconiosis [bib_ref] New insights into pathomechanisms and treatment possibilities for lung silicosis, Adamcakova [/bib_ref]. e diverse physiochemical properties, including size, morphology, polymorphism, porosity, and surface, determine the toxicity of the silica particles. Silica particles are defined as "respirable" when they are less than 5 μm in diameter, which is small enough to reach the distal airways and alveoli [bib_ref] New insights into pathomechanisms and treatment possibilities for lung silicosis, Adamcakova [/bib_ref]. Furthermore, crystalline silica particles are the leading cause of occupational respiratory disease and are generally considered more toxic than amorphous silica particles [bib_ref] Silica-related diseases in the modern world, Hoy [/bib_ref]. e most toxic and common form of crystalline silica is α-quartz [bib_ref] Silicosis and coal workers' pneumoconiosis, Castranova [/bib_ref]. Recently, nearly free silanols (NFS) on the surface of quartz particles were reported as the critical molecular moieties responsible for the toxicity of silica particles [bib_ref] Nearly free surface silanols are the critical molecular moieties that initiate the..., Pavan [/bib_ref]. High NFS concentrations are associated with increasing hemolytic potential and IL-1β generation, but the different toxicities of the different classes of amorphous silica particles remain poorly understood [bib_ref] Are nearly free silanols a unifying structural determinant of silica particle toxicity, Brinker [/bib_ref].
## Recognition of silica particles by macrophage scavenger
Receptors.
e recognition and internalization of inhaled silica particles by alveolar macrophages in the lungs is the first critical step in initiating lung inflammation and normal immune function, both of which are regulated by macrophage scavenger receptors (SRs) such as SR-AII and macrophage receptor with collagenous structure (MARCO) [bib_ref] Silica binding and toxicity in alveolar macrophages, Hamilton [/bib_ref]. A recent study has reported that SR-B1 specifically recognizes amorphous and crystalline silica particles, but not titanium dioxide nanoparticles, latex nanoparticles, or monosodium urate crystals, whereas SR-B1-mediated recognition of silica is associated with caspase-1-mediated inflammatory responses in mouse macrophages and human peripheral blood monocytes [bib_ref] SR-B1 is a silica receptor that mediates canonical inflammasome activation, Tsugita [/bib_ref]. NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) can activate caspase-1, which cleaves precursor cytokines that secrete interleukin (IL)-1β, IL-18, and IL-33 [bib_ref] e Nalp3 inflammasome is essential for the development of silicosis, Cassel [/bib_ref]. In macrophages and epithelial cells, silica-activated NLRP3 inflammasomes consisting of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 are essential for the development of silicosis [bib_ref] Silicainduced NLRP3 inflammasome activation in vitro and in rat lungs, Peeters [/bib_ref] [bib_ref] e NLRP3 inflammasome in pathogenic particle and fibre-associated lung inflammation and diseases, Sayan [/bib_ref]. Silica also induced stimulator of interferon genes (STING)-dependent reactive oxygen species (ROS) production, cell death, and self-DNA release, leading to the type I interferon (IFN) response [bib_ref] STINGdependent sensing of self-DNA drives silica-induced lung inflammation, Benmerzoug [/bib_ref].
Toll-like receptors (TLRs), as pattern recognition receptors of damage-associated molecular pattern (DAMP), have been identified as critical mediators of pulmonary inflammation and fibrosis [bib_ref] e interplay of DAMPs, TLR4, and proinflammatory cytokines in pulmonary fibrosis, Bolourani [/bib_ref]. Silica particle inhalation activates TLR4 and receptor activator of nuclear factor kappa-B ligand (RANKL) signaling pathways in lung macrophages, thus inducing lung inflammation and the proteolytic phenotype of macrophages and osteoclasts in the lungs and bones [bib_ref] Ac-SDKP attenuates activation of lung macrophages and bone osteoclasts in rats exposed..., Jin [/bib_ref]. Silica-induced lung inflammation, as the major cause of systemic inflammation, may disrupt the functions of extrapulmonary organs in rats exposed to longterm silica inhalation [bib_ref] Silicosis decreases bone mineral density in rats, Hui [/bib_ref] , which may partly contribute to our understanding of the relationship between silicosis and autoimmunity [bib_ref] Silica, silicosis, and autoimmunity, Pollard [/bib_ref]. e extrathoracic manifestation of silicosis in the liver, spleen, and bone marrow [bib_ref] Accelerated silicosis with bone marrow, hepatic and splenic involvement in a patient..., Hernandez [/bib_ref] can be explained by the lymphangitic spread of pulmonary macrophages; however, there is still no definite and reasonable explanation.
## Multiple omics in silicosis.
Multiple omics approaches, including genomics, epigenomics, transcriptomics, proteomics, and metabolomics, have bridged underlying molecular alterations with silicosis progression [bib_ref] Omics approaches for exploring pneumoconiosis: a review, Luo [/bib_ref]. Lung tissues, sera, peripheral blood leukocytes, and bronchoalveolar lavage fluids (BALFs) collected from silicotic patients or animals and in vitro models (such as silica-or TGF-β1-induced macrophages or fibroblasts) have been applied in omics approaches to screen diagnostic biomarkers, explore new mechanisms, and reveal potential therapeutic targets [bib_ref] Pulmonary silicosis alters MicroRNA expression in rat lung and miR-411-3p exerts antifibrotic..., Gao [/bib_ref] [bib_ref] Differential expression of lncRNAs during silicosis and the role of LOC103691771 in..., Cai [/bib_ref] [bib_ref] Transcriptomic and metabolomic profiling reveal the p53-dependent benzeneacetic acid attenuation of silica-induced..., Ju [/bib_ref] [bib_ref] Proteomic profile of TGF-beta1 treated lung fibroblasts identifies novel markers of activated..., Na [/bib_ref] [bib_ref] Multi-omics study of silicosis reveals the potential therapeutic targets PGD2 and TXA2, Pang [/bib_ref]. Single-cell RNA sequencing (scRNA-seq) provides a robust and unbiased survey of the transcriptome that is comparable to bulk RNA sequencing, while preserving cellular heterogeneity information has contributed to impressive advancements, including the discoveries of the pulmonary ionocyte and the profibrotic macrophage population in pulmonary fibrosis [bib_ref] Breathing fresh air into respiratory research with single-cell RNA sequencing, Alexander [/bib_ref]. Emerging data from scRNA-seq analysis have provided novel insights on the dysfunction of alveolar type II progenitor epithelial cells and the diversity of mesenchymal cells within the fibrotic lung [bib_ref] Insights into development and progression of idiopathic pulmonary fibrosis from single cell..., Nemeth [/bib_ref]. Spatial transcriptomics can extend and complement scRNA-seq studies and allow better characterization of the physiological interactions between cell types as well as their alterations in respiratory diseases, thereby providing key insights to understand their physiopathology [bib_ref] Insights into development and progression of idiopathic pulmonary fibrosis from single cell..., Nemeth [/bib_ref] [bib_ref] Spatial transcriptomics for respiratory research and medicine, Curras-Alonso [/bib_ref].
Unfortunately, most of these studies involve bolus exposure of mice to silica, followed by the evaluation of various pulmonary parameters and the completion of various tests, including lung function, bronchoalveolar lavage fluid (BALF) tests, serum tests, collagen deposition, and myofibroblast differentiation at selected times after exposure. However, the formation of fibrotic nodules indicates the irreversibility and progression of pulmonary fibrosis. us, there are still unsolved issues in relation to the development and the progression of silicosis. First, how can one accurately judge the line of overload of lung dust burden, which is needed to understand how much silica particle inhalation, can induce the development of silicosis? Second, why does pulmonary fibrosis continue to progress after cessation of the dust exposure, how can the lung regenerate, and can pulmonary fibrosis be reversed? ese problems should be resolved to facilitate the early identification of the disease, the monitoring of disease progression, and the targeted therapies of silicosis.
## Early identification and accurate diagnosis of silicosis.
Chest X-rays (CXRs), high-resolution computed tomography (HRCT), pulmonary function tests (PFT), and health and exposure questionnaires are the major methods for the respiratory surveillance of workers exposed to RCS. However, these methods cannot detect the disease until it has significantly progressed [bib_ref] Early detection methods for silicosis in Australia and internationally: a review of..., Austin [/bib_ref]. Although it has been reported that CXR cannot reliably detect occupational lung diseases [bib_ref] Stonemasons with silicosis: preliminary findings and a warning message from Australia, Newbigin [/bib_ref] , it is still recommended in the diagnosis of silicosis by the International Labour Organisation (ILO) Classification System [bib_ref] Comparison of the international classification of high-resolution computed tomography for occupational and..., Sener [/bib_ref]. Poor-quality chest radiographs, physicians' inability to recognize the disease, and the differential latency of various types of occupational lung diseases make it difficult to detect and estimate the burden of silicosis [bib_ref] Update of occupational lung disease, Suganuma [/bib_ref] [bib_ref] What does coal mine dust lung disease look like? A radiological review..., Mcbean [/bib_ref]. HRCT is more specific and sensitive than CXR in the early evaluation and prediction of the progression of pneumoconiosis, especially in detecting early parenchymal changes, emphysema, and pleural thickening, but it is not recommended by the International Classification of HRCT for Occupational and Environmental Respiratory Diseases (ICOERD) because of the associated high cost, radiation exposure, and low accessibility [bib_ref] Comparison of the international classification of high-resolution computed tomography for occupational and..., Sener [/bib_ref]. On the other hand, further studies of HRCT are warranted because it would enable a more comprehensive correlation between the pathologic findings and the clinically-relevant imaging results [bib_ref] Imaging diagnosis of classical and new pneumoconiosis: predominant reticular HRCT pattern, Masanori [/bib_ref].
For the rapid and early detection of pneumoconiosis, deep convolutional diagnosis approaches have been applied to a pneumoconiosis radiograph dataset to obtain high accuracy in pneumoconiosis detection [bib_ref] Balanced convolutional neural networks for pneumoconiosis detection, Hao [/bib_ref]. Support vector machine (SVM)-based computer-aided silicosis diagnosis could recognize silicosis-associated opacity in several candidate regions for the radiologist's reference [bib_ref] Automatic detection and recognition of silicosis in chest radiograph, Zhu [/bib_ref].
us, artificial intelligence (AI)-enabled radiology tools stand to fill the need for regulatory compliance in pneumoconiosis screening, while offering a labor-saving solution to physician workflow issues and enhancing patient safety [bib_ref] e B reader program, silicosis, and physician workload management: a niche for..., Gowda [/bib_ref].
Lung biopsy should be avoided unless absolutely necessary for another reason because surgical manipulation associates with unfavorable repercussions [bib_ref] Prognosis of patients with silicosis due to denim sandblasting, Akgun [/bib_ref]. However, lung biopsy is the only way to arrive at an accurate diagnosis when there is no occupational exposure history, disagreement between CXR and HRCT imaging results, and atypical presentations cause physicians to consider other differential diagnoses [bib_ref] Artificial stone silicosis: rapid progression following exposure cessation, Leon-Jimenez [/bib_ref].
ere is ongoing debate about precisely when to perform these tests and which type of biopsy should be performed in different situations [bib_ref] Point: should surgical lung biopsy still be performed for interstitial lung disease..., Ryerson [/bib_ref] [bib_ref] Counterpoint: should surgical lung biopsy still be performed for interstitial lung disease..., Tomassetti [/bib_ref]. e guidelines for idiopathic pulmonary fibrosis recommend that surgical lung biopsy (SLB) be carried out when there is no relevant clinical manifestation and low confidence in the establishment of a diagnosis [bib_ref] Diagnosis of idiopathic pulmonary fibrosis. An official ATS/ERS/JRS/ ALAT clinical practice guideline, Raghu [/bib_ref] , with the justification that it provides the most informative tissue samples when clinicians are faced with diagnostic uncertainty [bib_ref] Point: should surgical lung biopsy still be performed for interstitial lung disease..., Ryerson [/bib_ref]. Transbronchial lung cryobiopsy (TBLC), compared to SLB, appears to be safer, more accurate, and more useful because it provides meaningful information in the context of the multidisciplinary discussion of cases [bib_ref] Counterpoint: should surgical lung biopsy still be performed for interstitial lung disease..., Tomassetti [/bib_ref]. Transbronchial lung biopsy (TBLB) is not recommended because the specimen size is small and the specimen is susceptible to damage, thereby affecting the pathological diagnosis [bib_ref] e evaluation of clinical usefulness of transbrochoscopic lung biopsy in undefined interstitial..., Han [/bib_ref]. Patients undergoing percutaneous puncture biopsy have a high probability of pneumothorax, but CTguided percutaneous puncture lung biopsy also has positive significance for the auxiliary diagnosis and the differential diagnosis of pneumoconiosis [bib_ref] Percutaneous transthoracic lung biopsy: optimizing yield and mitigating risk, Azour [/bib_ref].
## Treatment for silicosis
Due to the unavailability of an early diagnostic protocol, the treatment of silicosis is mostly limited to basic research, and therapeutic interventions for silicosis are still limited. Large volume whole-lung lavage is often used at an early category of silicosis to improve chest tightness, chest pain, shortness of breath, and other related symptoms. A recent study reported significant radiological improvement in patients with artificial stone-associated silicosis that may have better longterm outcomes in terms of symptomatology, functional capacity, lung function, and mortality [bib_ref] Radiological outcomes of whole lung lavage for artificial stone-associated silicosis, Chambers [/bib_ref]. Furthermore, successful exercise progression maximizes improvements and sustained treatment effects favor those with milder interstitial lung diseases.
Pirfenidone and nintedanib, approved by the U.S. Food and Drug Administration (FDA) for idiopathic pulmonary fibrosis, have been well documented in silicotic models [bib_ref] Pirfenidone ameliorates silica-induced lung inflammation and fibrosis in mice by inhibiting the..., Cao [/bib_ref] [bib_ref] Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental..., Wollin [/bib_ref]. Tetrandrine, which has been approved to treat silicosis in China [bib_ref] Effects of tetrandrine combined with acetylcysteine on exercise tolerance, pulmonary function and..., Zhang [/bib_ref] , can inhibit lung inflammation and lung fibrosis to improve pulmonary function [bib_ref] Tetrandrine modulates Rheb-mTOR signaling-mediated selective autophagy and protects pulmonary fibrosis, Liu [/bib_ref]. In recent years, herbal compounds have been documented to trigger the generation of ROS, suppress the activation of inflammasomes, and exhibit anti-inflammatory and antifibrotic effects [bib_ref] Herbal compounds in the treatment of pulmonary silicosis, Adamcakova [/bib_ref]. Clinical trials have shown that patients' lung function, quality of life, and exercise ability have significantly improved after treatment with traditional Chinese medicines [bib_ref] Efficacy and safety of dahuang zhechong pill in silicosis: a randomized controlled..., Tang [/bib_ref].
Lung transplantation is the main modality for the treatment of end-category silicosis, and it can help patients with end-category fibrosis to live longer. However, lung transplantation is expensive, difficult, and risky, and the median survival after transplantation is relatively short (approximately 6-7 years) [bib_ref] Silica-related diseases in the modern world, Hoy [/bib_ref]. In addition, long-term oral antirejection immunosuppressants are required. Studies have shown that the 3-year survival rate of silicosis patients after lung transplantation is approximately 76% [bib_ref] Survival following lung transplantation for artificial stone silicosis relative to idiopathic pulmonary..., Rosengarten [/bib_ref].
Other potential drugs have been reported to inhibit silica-induced pulmonary inflammation and fibrosis in silicotic models such as anticytokines (anakinra, anti-IL-17 antibody, and anti-IL-9 antibody), antioxidants (N-acetylcysteine, corticosteroids, and dexamethasone), agents influencing the autophagic-lysosome system (imipramine, dioscin, and rapamycin), agents increasing cAMP levels, and microRNAs [bib_ref] New insights into pathomechanisms and treatment possibilities for lung silicosis, Adamcakova [/bib_ref]. Mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) possess immunoregulatory and reparative abilities, which modulate innate and adaptive immunity and have shown great therapeutic potential in silicosis [bib_ref] Effects of mesenchymal stromal cell-derived extracellular vesicles in lung diseases: current status..., Guo [/bib_ref]. Presently, there are no large, randomized, and placebo-controlled clinical trials to assess the safety and the efficacy of these drugs in silicosis, and thus, their safety and efficacy remain to be determined.
## Summary
When silicotic nodules and collagen deposits are visible in rodent silicotic models, treatment is likely to be ineffective. Clinicians often assess myofibroblast differentiation, inflammation, macrophage activation, and epithelial-mesenchymal transition to evaluate the antifibrotic effects of potential drugs [bib_ref] Ac-SDKP attenuates activation of lung macrophages and bone osteoclasts in rats exposed..., Jin [/bib_ref] [bib_ref] N-acetyl-seryl-aspartyl-lysylproline (Ac-SDKP) attenuates silicotic fibrosis by suppressing apoptosis of alveolar type II..., Zhang [/bib_ref] [bib_ref] A new antifibrotic target of Ac-SDKP: inhibition of myofibroblast differentiation in rat..., Xu [/bib_ref] [bib_ref] N-Acetyl-Seryl-Asparyl-Lysyl-Proline regulates lung renin angiotensin system to inhibit epithelial-mesenchymal transition in silicotic..., Li [/bib_ref] ; however, here too, treatment is likely to be ineffective. Multiomics approaches, in conjunction with an established silicotic model, can be used to study the mechanism of silicosis and to identify potential biomarkers of the disease [bib_ref] Pulmonary silicosis alters MicroRNA expression in rat lung and miR-411-3p exerts antifibrotic..., Gao [/bib_ref] [bib_ref] Differential expression of lncRNAs during silicosis and the role of LOC103691771 in..., Cai [/bib_ref] [bib_ref] Transcriptomic analysis identifies upregulation of secreted phosphoprotein 1 in silicotic rats, Cai [/bib_ref] [bib_ref] Protein expression profile in rat silicosis model reveals upregulation of PTPN2 and..., Zhu [/bib_ref] [bib_ref] Evaluation of differential serum expression of three factors and pulmonary function in..., Zhu [/bib_ref] [bib_ref] Rho GDP dissociation inhibitor alpha silencing attenuates silicosis by inhibiting RhoA/ Rho..., Wei [/bib_ref]. Unfortunately, our understanding of omics is relatively poor, and diagnostic markers should comprise a large group of biomarkers reflecting the different pathological states of the disease.
In conclusion, silicosis still poses a threat to the health of many individuals worldwide. However, there is a lack of information on effective early prevention, early diagnosis, Canadian Respiratory Journal and timely drug treatment. us, it is important to explore additional pathological mechanisms that might be associated with silicosis and to identify novel early diagnostic and therapeutic modalities to improve the prognosis of silicosis patients worldwide.
## Conflicts of interest
e authors declare that they have no competing interests.
# Authors' contributions
All authors contributed to data analysis and drafting or revision of the article. All authors gave final approval of the version to be published and agree to be accountable for all aspects of the work.
Acknowledgments is work was funded by the National Natural Science Foundation of China (no. 81972988) and National Natural Science Foundation of Hebei Province (H2020209052). |
Phenotypic plasticity in nematodes
# Introduction
Modern biology's success in discovering how genes control developmental phenotypes has been due to studying genetically defined organisms in tightly controlled laboratory conditions. The invariant cell lineage of C. elegans has particularly facilitated notable progress in understanding the control of body formation and morphogenesis. This progress in C. elegans is arguably more than has been achieved for analogous endeavors in model systems which do not have such cell lineage control. More broadly, in all model systems, studying defined wild-type genotypes in constant environments has been the bed-rock of developmental biology. Genetic analytical power comes from consistency and sameness in the object of study.
The real world is much messier, and more interesting, than the laboratory. The real world is where the traits we analyze and genetically un-pick evolve, and where the precise trait characters determine an individual's fitness. An organism's phenotype is the difference between living and reproducing, or evolutionary death. In the real world, the environment is demanding and dynamic. It varies in its quality over space and time, both within and across individual generations. Environmental quality is due Model systems, including C. elegans, have been successfully studied to understand the genetic control of development. A genotype's phenotype determines its evolutionary fitness in natural environments, which are typically harsh, heterogeneous and dynamic. Phenotypic plasticity, the process by which one genome can produce different phenotypes in response to the environment, allows genotypes to better match their phenotype to their environment. Phenotypic plasticity is rife among nematodes, seen both as differences among life-cycles stages, perhaps best exemplified by parasitic nematodes, as well as developmental choices, such as shown by the C. elegans dauer/non-dauer developmental choice. Understanding the genetic basis of phenotypically plastic traits will probably explain the function of many genes whose function still remains unclear. Understanding the adaptive benefits of phenotypically plastic traits requires that we understand how plasticity differs among genotypes, and the effects of this in diverse, different environments.
## Phenotypic plasticity in nematodes
Evolutionary and ecological significance Mark Viney 1, * and Anaid Diaz 2School of Biological Sciences; University of Bristol; Bristol, UK; 2 School of Veterinary Medicine; University of Cambridge; Cambridge, UK Keywords: caenorhabditis, parastrongyloides, pristionchus, strongyloides, reaction norms, adaptation to both abiotic factors and biotic factors, including con-specifics; the competitive environment therefore contains genes and it evolves.This environmental heterogeneity has driven, through natural selection, the evolution of phenotypes and of developmental strategies that maximise the match of a phenotype to the prevailing environment.
There is therefore a potential gap between our current genetic understanding of the control of traits and of how organismal traits contribute to fitness. Evolutionary fitness of an individual comes by surviving and reproducing in its environment and outcompeting other individuals, which depends on its phenotype being better matched to this environment. Because the environment is variable, having only fixed, invariant traits is unlikely to maximise fitness. More precisely, such a completely fixed phenotype developmental strategy would be out-competed by a genotype whose phenotype was able to better match the environment, as the environment varies. Better matching of phenotypes to environments can be achieved by phenotypic plasticity. Phenotypic plasticity is the ability of one genotype to produce different phenotypes in response to environmental conditions. These different phenotypes can be discrete (e.g., different morphs) or continuous. Thus, phenotypically plastic traits can allow a genotype to, in some way, match its phenotype to its precise environment, thereby contributing to fitness. At an extreme, a perfectly plastic phenotype (where any phenotype was possible, and the phenotype-to-environment match was perfect) would give eternal, maximal fitness. That such perfect plastic phenotypes do not exist is both because of developmental constraints (some phenotypic options are just not possible) and because of presumed costs of phenotypic plasticity (the costs of the plasticity are greater than the benefit that could have been gained by that plasticity).
While we have a very good understanding of how genes control phenotypes, this is generally only true for phenotypic traits that are not plastic (or that may be plastic, but have been studied in a single, controlled environment, such that any phenotypic plasticity is absent). The time might now be ripe for mature research fields of model organisms to turn their attention to phenotypically plastic traits.
It is remarkable, that despite our success in determining genome sequences for many species, and for understanding the function of many genes, we still actually have very little idea of what most of the genome is doing. Of course, there is some annotation for very many genes in sequenced genomes, but the vast This is very clearly seen with species of parasitic nematodes where there is an infective larva that is free-living whose destiny is to enter a host (often by penetrating the host skin).These freeliving infective larvae remain in this stage until they receive cues (usually signaling the presence of a suitable host), which brings about biochemical and behavioral changes that allow them to infect hosts and to recommence their developmental progression to the next stage, which usually also involves a moult.Thus here, the environment is the cue that controls the timing of events; this is in contrast to the dauer/non-dauer case where the environment controls the nature of the choice that is made, but not its timing.
The developmental choice as exemplified by the dauer/nondauer choice of free-living nematodes, also occurs in the life-cycle of parasitic nematodes . Most notably, in Strongyloides and Parastrongyloides (both genera that parasitise vertebrates) the free-living phase of the life-cycle has a phenotypically plastic developmental choice between (1) development as larvae only (leading directly to the development of infective larvae) and (2) development of a free-living dioecious adult generation, whose progeny (following sexual reproduction) develop into infective larvae.The developmental choice between these two routes (so called direct and indirect, respectively) is controlled by environmental conditions. In S. ratti, a natural parasite of the rat, these have been well characterized.They are an interaction of outwith-host conditions (especially environmental temperature) and the host immune response (i.e., that of the host from which the relevant transmission stage was passed).For Parastrongyloides spp, a host feces-derived factor affects this developmental choice.How Strongyloides and Parastrongyloides sense the host immune response is not known. For example, it could be a parasitic female maternal effect (which then raises the question of how these parasitic females sense the immune response), or an effect acting directly on eggs and larvae in transit in the gut, or on larvae developing outside of the host in faeces.Considering the C. elegans and S. ratti developmental decisions together shows that they both use multiple cues in making these developmental decision (C. elegans: pheromone concentration, food concentration; S. ratti: host immune response, temperature). For S. ratti, these cues are temporally and spatially separated, which implies that there is a memory of cues, to thereby allow their integration.Specifically, the host immune response will affect adult parasites that are in the host gut, and eggs that are passing along the gut to be voided in host faeces, whereas the environmental temperature is sensed later by first and second stage larvae (L1s and L2s) outside of the host, yet together these cues are used by L1s and L2s to make the developmental decision.
Another more recently described example of phenotypic plasticity is of mouth morphology in Pristionchus pacificus (a scarab beetle necromenic nematode) and among other groups of diplogastrid nematodes.In P. pacificus, there are two mouth morphologies, one wide (containing two denticles), one narrow (with just one denticle) (more formally known as eurystomatous and stenostomatous mouth morphs, respectively). The mouth morphology is a developmental choice that is controlled by starvation in early larval stages (which favors the wide mouth morphology) and by a P. pacificus pheromone. majority of these annotations are no more than identification of sequence analogs in other species, when they exist. The difficulties in understanding gene function can be seen, for example, from yeast gene knockout studies, where systematic knockouts of the approximately 5,000 genes resulted in phenotypes for only some 15% of genes.Clearly, 85% of genes could not be functionless. Indeed, deletion of many of these other genes did have phenotypic consequences, but these were only seen in other environmental conditions.Analogous findings, that phenotypic analyses require environmental space, have now been made more widely.Similar results are also, in essence, implicit in the genome-wide RNAi screens of C. elegans (e.g., ref. 5). Here, RNAi of a majority of genes had no observed effect. If it is the case that the functional effect of many genes/gene products is context-dependent, then assay of these RNAi-treated worms in different, diverse environments would increase the proportion of genes for which a phenotype could be observed, and hence a putative role assigned, which is now being observed in on-going research. Therefore, an organism's phenotypic space can only be known by looking at phenotypes in environmental space.
## Phenotypic plasticity among nematodes
Phenotypic plasticity is rife among nematodes, including C. elegans. The term phenotypic plasticity has been so widely used (e.g., refs. 6-9) that its utility is weakened; to be useful, the precise trait and the environments that affect it need to be explicit. For nematodes, plasticity of phenotype among life-cycle stages is so obvious that it is rarely commented upon. This form of plasticity is particularly apparent among parasitic nematodes. Here, different life-cycle stages can have very different morphologies, physiologies, lifestyles, etc. Most notably, within a species some life-cycle stages can be parasitic (for example living inside the gut of a vertebrate host) while other life-cycle stages are free-living (e.g., in host faeces, the soil or water) or, depending on species, a transmission stage living inside an arthropod intermediate host.In all these cases, the different phenotypes are products of the same genome. For free-living nematodes, including C. elegans, the nearest analog of this within life-cycle plasticity of parasitic nematodes is the dauer larva stage (although clearly there are substantial differences between all life-cycles stages of free-living nematodes, including C. elegans).
Considering how parasitic nematodes have different life-cycle stages, which can have different lifestyles etc., and the dauer larva stage of free-living nematodes, makes clear an important distinction. In free-living nematodes, the dauer/non-dauer switch is (1) a facultative developmental choice which is (2) based on environmental conditions (specifically, food availability, con-specific population density and temperature); therefore, the environment controls what developmental choice is made.In contrast, in the parasitic nematode examples, where life-cycle stages differ, this is not a developmental choice. In these life-cycles there is progress through larval and adult stages, and each stage has its own different phenotype, physiology and lifestyle. The environment plays no part in the sequence of this life-cycle progression. Instead, the environment cues when the next steps in the sequence are started. choice between sexual reproduction (i.e., developmental via freeliving adults, which reproduce sexually) and direct development (which is asexual, because there is no further reproduction).These examples of phenotypically plastic traits are where there are alternative developmental choices, socalled polyphenisms. There are, of course, other examples of plasticity of phenotypes among nematodes too (e.g., ref.. For example, the food that is fed (i.e., bacterial species and strain and quantity) to Caenorhabditis spp larvae alters adult body and brood size.Here the environment is food, and the plastic trait is body size and fecundity (see Testing for Adaptation). Among parasitic nematodes there are analogous effects. For S. ratti, the within-host immune environment affects the size and fecundity of parasitic female worms, such that in immune hosts parasitic adult size is approximately halved, but this is reversible if the host is immune suppressed; analogous effects are seen in other parasitic nematodes too.These may be direct effects of the host immune response that damages the worms, or this may occur via interference with feeding, or a combination of both of these effects.In all these examples, there are clearly opportunities for maternal effects more widely, for example, while maternal environment may reduce fecundity, there may be a compensatory change in offspring quality, as seen in other systems. 33
## Phenotypic plasticity and adaptation
These developmental choices of C. elegans, Strongyloides, Parastrongyloides, P. pacificus can be intuitively understood as an adaption to environmental conditions in which worms find themselves. As is oft repeated for C. elegans dauer larvae, it is obvious that assessment of environmental conditions (how much food and how many other con-specifics there are to eat it), is key information for this developmental decision.The choice is between (1) continuing developmental growth to adulthood and reproduction, both of which require food or (2) altering one's metabolism to store food, to arrest development as a dauer larvae, which is therefore adaptive when there is insufficient food available to allow choice (1). More precisely, therefore, the assessment of environmental conditions is also a prediction (presumably with some error) of future conditions too. Failure to make the correct, optimal decision will lose evolutionary fitness.
Understanding the analogous developmental choice of Strongyloides and Parastongyloides is not quite so straightforward. At the core of the developmental choice in these genera is a . The life-cycle choices of (A) C. elegans and other free-living nematodes, (B) S. ratti where gray box 1 is a sexdetermination event, and gray box 2 is a female larva-only developmental choice between direct and indirect development (larval stages, except the infective L3 stage, have been omitted for clarity) and (C) P. trichosuri as for S. ratti, except that the P. trichosuri life-cycle differs (1) by having a dioecious parasitic generation and (2) the progeny of the free-living adult generation can form multiple, secondary free-living adult generations.
For S. ratti, there appears to be a geographic effect too. Genotypes isolated from the UK (but assayed in defined laboratory conditions) have predominately asexual reproduction, whereas development via the sexual route is more common among genotypes from other geographical locations.The degree of sexual/indirect vs. asexual/direct developmental can be selected for, as can aspects of within-host development, which is likely to be due inpart to how within-host stages interact with the host immune response.That there are genotype-specific differences in plastic phenotypes is then a further challenge for developmental biology. Not only do environmentally plastic phenotypes need to be considered, but also that the nature of that plasticity may be genotypedependent. In fact this is an important, positive factor because this genotype-diversity can be used to facilitate analyses of such traits, particularly using quantitative genetic approaches.
While our understanding of these facultative traits can give an idea (though untested) of their likely adaptive value, it is rather more difficult to understand and investigate why these trait values differ among genotypes. In part this difficulty is because we do not really understand the precise niche from which these genotypes came. While a genotype's geographical location may be known, the functionally relevant niche is likely to exist on a much smaller scale, thus a micro-niche. Further, when trying to understand the adaptive value of a trait, it is likely to be inappropriate to consider one trait in isolation, because different traits will trade-off with one another. Consider the C. elegans dauer example and imagine two genotypes that have different dauer larva formation responses: one that forms a high proportion of dauer larva, and one a low proportion, under the same environmental conditions. These different phenotypes are unlikely to be the only evolved differences between the genotypes. There will be other differences too, very possibly in key life-history traits (for example, brood size or schedules of reproduction).The way to understand this is that these genotypes have evolved in their own micro-niches, where a life-history strategy has evolved, manifest in more than one trait. There may be common patterns of how different traits are related among genotypes, such that these can be characterized as trade-offs, be they positive or negative. Trade-offs can result from selection within a niche that independently affects different genetic pathways and hence traits, or they can occur directly from pleiotropic effects, for example within a genetic pathway. The term "trait" is useful for biologists, but it is not something with which an organism, or evolution, is concerned. The challenge for us seeking to understand the adaptive value and evolution of traits, is therefore to consider suites of traits and how they differ among genotypes.
## Measuring phenotypic plasticity
That phenotypically plastic traits are environmentally determined obviously requires that different environments are used to induce and assay the different phenotypes. For species in which genetic clones are available (thus individuals of inbred, homozygous lines of C. elegans or P. pacificus etc.), different individuals (A further consequence is that development of infective larvae via the sexual route takes longer, compared with direct development). Development via the sexual reproduction route has two effects: sexual reproduction and an increase in the number of worms (because two mating adults produce ca. 40 offspring).It is likely that sexual reproduction is the particular advantage that is acquired by an indirect developmental route. The reason for believing this is that given the diversity of reproductive mechanisms among nematodes it is clear that nematodes could evolve an asexual method of adult reproduction that generates more offspring (i.e., sexual reproduction is not needed for adults to produce progeny). That Strongyloides and Parastrongyloides did not do this suggests that sexual reproduction is the key functional aspect of their developmental choice. In this respect, the pattern of the induction of facultative sexual reproduction is similar to that seen in other species, for example cyclical parthenogens (Daphnia spp, aphids), where sexual reproduction occurs at times of environmental stress. For Strongyloides, the stress is presumably some aspect of the within-host conditions (signaled by the host immune response), perhaps also signaling some wider aspect of the status of the host biology and population.
The presumed adaptive value of the P. pacificus developmental choice is that the different mouth morphologies give access to different food sources. The narrow (stenostomatous) form allows worms to feed on bacteria, whereas the wide (eurystomatous) form allows worms to slice open fungal hyphae and other nematodes to use as a food source, which may be the next best food source in the absence of bacteria. Because evolution has found this solution to P. pacificus feeding, it is presumed that developing alternative, specialized mouth morphologies gives greater fitness compared with having a generalist, all-purpose mouth morphology.
The sensitivity of a genotype's switch to environmental conditions can be an evolved trait, which will have evolved in the environment in which the genotype in question is living. This has the consequence that genotypes that have evolved in different micro-niches may have different sensitivities (measured as the phenotypic response) to the same environmental signals. This is now well known for both C. elegans and Strongyloides. Specifically, different genotypes of C. elegans have quantitatively different responses to the same dauer pheromone or food concentration signals.Of parenthetical interest, the C. elegans wild type, N2, routinely seems to have the greatest sensitivity to dauer larva formation, which is presumably a result of artificial laboratory selection for survival on temporarily neglected laboratory agar plates where the formation of dauer larvae results in survival. C. elegans genotypes differ in other traits too (e.g., refs. 40-42), as does P. pacificus.For S. ratti, different genotypes also make quantitatively different responses to the same within-and, or without-host environmental conditions, such that some genotypes have almost complete asexual development, whereas others have extensive sexual development.It is likely that these differences reflect genotypes' different sensitivities to the host immune response, perhaps therefore reflecting different local within-host environments experienced by those genotypes during their evolution.proportions, that are therefore bounded by a 0 and 1 (or 0 and 100%) minima and maxima, the slope of the reaction norm can become confounded with the absolute trait value .
Of course, there can be more than two environments, and then reaction norms need to be described over this more fine-grained environmental range. If only two environments are considered, one can only ever consider a linear reaction norm across those environments . Measurement of phenotypes in more than one environment, will therefore show whether a reaction norms are, indeed, linear or not. Understanding different shapes of reaction norms is also likely to be important in considering the adaptive value of phenotypically plastic traits. 49
## Testing for adaptation
There are two meanings to the question of whether a trait is adaptive. The first is whether the trait per se is adaptive. The P. pacificus mouth morphologies is an example of this; here, is having alternative mouth morphologies adaptive compared with not having this (thus instead, presumably, a single mouth morphology)? This is a deep evolutionary question, which asks why the trait that is observed has evolved. The second version of this question is to ask when and where the extant trait value is adaptive. Continuing the P. pacificus example, this would ask under what conditions does a narrow mouth morphology give greater fitness compared with the wide mouth morphology. When we considered this before (Phenotypic Plasticity and Adaptation section) these two questions were asked together. This second question can be extended by asking why specific trait values are adaptive, of the same genotype can be placed in the different environments, such that environmental-dependent effects can be seen. Other systems also have this experimental advantage. For taxa that are parthenogenetic, clones can be readily produced, such as for Strongyloides spp as well as, for example, Daphnia spp. For dioecious sexually reproducing species, lines can be inbred to achieve homozygosity, therefore producing functional clones. If this is not possible (either because the inbreeding would take so long, or because of deleterious effects of inbreeding), then the alternative approach is to use various sib-ship experimental designs, where genetic effects among sib-ships have to be accounted for.
Using clones, while straightforward, allows greater resolution of phenotypically binary traits. Consider the C. elegans dauer/non-dauer developmental choice. Each individual developing larva has to make a binary choice between these two developmental possibilities. However, among genetically identical individuals not every individual will make the identical choice, for example 60% of the larvae may form dauer larvae, 40% non-dauer larvae. This means that the probability of an individual forming a dauer larva under the given conditions is 60%. Therefore, an apparently binary trait, is actually a continuous trait, and can be thought of as a threshold trait.This conceptual approach also applies to both the Strongyloides and Parastrongyloides developmental choices as well as to the alternative mouth morphologies of P. pacificus. Of course, for continuous traits (e.g., brood size, length), the value of the trait in the different environments can be directly measured. Therefore, there is no conceptual difference between phenotypically discrete traits and phenotypically continuous traits.
Among the phenotypic plasticity literature, the concept of reaction norms is used, particularly to compare plastic traits among different genotypes .This is useful because it clarifies different aspects of the plastic trait, each of which may separately be relevant and subject to both statistical and genetic analyses. The different aspects of the trait are the trait value in each environment and the difference in trait value between environments; this latter value is a measure of how plastic (or sensitive) a trait is to a change in the environment. From , it can be seen that different genotypes can have different trait values (i.e., the absolute elevation of the line), but the same plasticity (i.e., the slope of the line between environments). Different genotypes may also have different plasticities, thus where the slopes of the reaction norms differ. This can be summarized as the difference in trait value between environments, though there are difficulties . For traits measured on a continuous scale there are little further difficulties; however, for traits measured as . A reaction norm displays the value of a trait in a minimum of two environments. Thus, in (A), as the environment (e.g., temperature, or quantity of food) changes from E 1 to E 4 , then the value of the trait changes from T 1 to T 4 . The difference in the trait value, which is the slope of the line, is therefore the norm of reaction, more easily thought of as the sensitivity of the phenotype to the environment. This can be summarized numerically as T 4 -T 1 . Different genotypes may have different reaction norms. In (B) a second genotype (blue line) has different trait values (T 3 and T 6 ), though the sensitivity is the same, (T 4 -T 1 ) = (T 6 -T 3 ). A third genotype (red line) has different trait values (T 2 and T 3 ), and a different sensitivity, in this case less sensitive, (T 3 -T 2 ) < (T 4 -T 1 ). For traits measured in two environments (A and B), only a linear reaction norm can be interpolated. Measurement in more environments, may reveal nonlinear reaction norms (C). For binary traits measured as proportions (e.g., for C. elegans the proportion of developing larvae that form dauer larvae, and for analogous traits in Strongyloides and Parastrongyloides) the trait values have 0 and 1 minima and maxima, respectively. As a consequence, the trait value in E 1 limits the maximum trait sensitivity, because the trait has a maximum of 1 in E 2 (for the case where the slope is positive, in A or B). Such proportion data also have a non-normal error structure (errors cannot lie outside of the 0-1 range) which requires a different error structure. relevant comparisons are context dependent, in this example it being within a poor food environment. The key point is that a plastic phenotype is recognized, indeed defined, by changing as the environment changes, but with respect to questions of adaptation, the across-environment comparison might be uninformative, and rather an among-genotype comparison within one environment may be informative. It is important to remember that in this example of the C. elegans food environment, larval exposure to food conditions effects a lifelong change to size and fecundity. There are other phenotypes which can continually change through an individual's life. In these cases the relevant comparisons for questions of adaptation may well be other comparisons, such as among genotypes within and between environments. This makes the important, general point that the best test of the adaptiveness of a plastic trait depends on the nature of that traits plasticity.
## Phenotypic plasticity and noise
A challenge for natural selection driving the evolution of phenotypically plastic traits is the accuracy with which an environmental cue can be used to measure the environment (and possibly a future environment) of an individual where its fitness will be determined. There are two different problems: (1) the actual accuracy of the cue and (2) whether the relevant environment is in fact knowable (i.e., the environment may not be knowable via any cue, because stochasticity determines the environment, and there is no cue for stochasticity). One approach to addressing these difficulties that may evolve is the evolution of variation in trait(s) among individuals of a single genotype. This type of trait variation will therefore generate some phenotypes that better match the actual environment in which individuals find themselves. In effect, within-genotype trait variance widens the phenotypic space of a genotype. With this perspective, such variance in a trait could be an adaptive. Empirically it would be observed as phenotypic variation among genetically identical individuals. This trait variance is district from phenotypic plasticity, because the trait variance is independent of the environment.
Clearly, for any trait measured among genetically identical individuals in a constant environment, there is some variance due to stochastic processes of development (as well as experimental and measurement error). Adaptive within-genotype trait variation would be variation that is beyond this background level. Characteristics of adaptive trait variation are likely to be that the degree of trait variance will be subject to selection, such that different genotypes may differ in their trait variance (and that trait variance can be artificially selected). Variance among genetically identical C. elegans individuals has been noted before. In fact, the very extensive genetic analyses of C. elegans implicitly show this too. For many mutations, those mutations result in incomplete phenotypic penetrance, which means that genetically identical individuals are phenotypically different, thus there is withingenotype trait variance.A common way of thinking about this is that a trait is genetically tightly controlled, such that a reduction or loss of function mutation of a gene that contributes to that trait, relaxes the control of the trait, seen as incomplete which comes from observing that there are genotypic differences in trait values. Both of these questions (and their answers) are context-dependent.
Rigorously testing and investigating adaption of phenotypically plastic traits is key to understanding how suites of trait values contribute to fitness, especially when the trait values differ among genotypes. Studies with plants have been very powerful in this respect. Here, with the key perspective that a genotype's traits only make adaptive sense in the niche in which they evolved, reciprocal transplant, common-garden experiments have been used. In these, the genotype in question is grown both in its original environment (ideally in the wild) and in a different environment (and this is repeated for many genotypes, with a reciprocal experimental design) (e.g., ref.. Evidence in support of a genotype's trait value being adaptive, is that it will have greater fitness in its home environment compared with the other environments; specifically, this would be observed if there has been local adaptation of the genotype to its environment. More generally though, each genotype should have an environment where it has the greatest fitness. In these studies the question of adaptiveness is asked by testing this among different environmental contexts. These style of studies are relatively straight forward for plants, but much more challenging for animals especially because mark-release-recapture approaches are essentially impossible for small invertebrates. The next best approach is therefore to use microcosms, which would test different environments (especially harsh and dynamic environments), and within which different genotypes are allowed to compete.
Experimentally testing for evidence of trait adaptation to an environment is key; not testing for this only leaves 'just so' stories. This is especially important when considering plastic traits, to try and separate whether a plastic phenotype is adaptive or an inevitable physiological limit or constraint, and thus not adaptive. To think about this further, consider the example of how the feeding environment of C. elegans changes adult worm size and reproduction, i.e., less food results in smaller, less fecund worms.This is clearly phenotypically plasticity (the food environment changes the phenotype), but is it adaptive or is it an inherent physiological constraint of being a C. elegans worm (or any other invertebrate or animal), or it is both? This is a difficult question; it might actually be an uninteresting question of semantics. Notwithstanding, taking the examples above of testing for adaptiveness, key to the approach is to make comparisons: for poorly fed, and so small, low fecundity worms, then what is the appropriate comparison? The correct environment in which to test them is the environment in which they developed (i.e., a poor food environment). In such an environment in nature there are likely to only be other C. elegans genotypes, similarly poorly fed, and thus small and with a low fecundity. Therefore, among-genotype comparisons within the same environment would be the appropriate comparison. Clearly, well fed (and so large and highly fecund) worms will outcompete small poorly fed worms, but this is an uninformative comparison; it can tell us nothing about adaptation, because it is comparing what natural selection does not compare during evolution. This therefore makes clear that the that this heterogeneity can have phenotypic effects whose success differs in different environments. Importantly, this makes the point that "noise", seen as inter-individual variation in traits may have an adaptive role in organismal life in demanding, heterogeneous environments. Our challenge is to study this, with this perspective.
# Conclusions
There has been extensive theoretical study of phenotypic plasticity which has, for example, considered the fitness consequences of plasticity as well as the genetics, and hence evolution, of plastic phenotypes.The theoretical genetics studies have centered on the question of whether "plasticity" is a trait that is separate from the trait itself.The difficulties of this debate were largely due to a lack of clarity of terminology, including theoretical concepts of genetics beyond empirical knowledge. That we are now in the genomic age means that understanding the control of phenotypically plastic traits is more empirically tractable (though still difficult) and so this can inform these theoretical studies. Moreover, by combing such studies with studies of adaptation, means that the genetic basis of important components of fitness can be dissected, which is a central challenge of modern biology. It is likely that phenotypically plastic traits will have multilocus control and so understanding the structure and function of gene networks will be key. Considering, as an example, the C. elegans dauer/non-dauer developmental choice, the genetic pathways that control dauer larva development are well known, and clearly constitute a large complex network.Given that different C. elegans genotypes have quantitatively different responses to the same environmental conditions, a pressing question is where are these differences encoded in this genetic network?Conceptually, the network can be considered to have three functional components: sensing the environment, transducing that environmentally-derived information and executing the dauer larva program. Changes in any of these three functional components could alter a genotype's phenotypic response to an environmental signal. Apparently similar dauer formation phenotypes could be achieved by different genetic means. The answer to the question is unlikely to be straightforward (e.g., ref. 62). However, the answer to this question, and analogous plastic phenotypes in other systems, are examples of the next big step for organismal genetics and genomics, in which nematode studies may lead the way.
penetrance. While this isn't necessarily wrong, these observations show that (1) within-genotype trait variance can be genetically controlled and then (2) raise the more relevant question of whether such trait variance can be adaptive, and if so, when and how is it adaptive? Studies in microorganisms may show the way here. There have now been many studies that have looked at heterogeneity in gene expression in bacteria and yeast. There are two sources of the heterogeneity that have been considered. One is so-called intrinsic noise, which is the difference in expression of two genes within an individual cell (measured by the expression of identical, introduced genes, which have distinguishable reporters). The second, referred to as extrinsic noise, is how expression of these pair of genes differs among otherwise identical cells.It is this latter, extrinsic noise, which may be adaptive. The fitness effects of different degrees of heterogeneity have been measured, and have shown that this can have different advantages under different environmental conditions. For example, in Bacillus subtilis the heterogeneity in gene expression that arises from a small circuit, causes an environmental dose-dependent (i.e., environmental DNA concentration) effect on cellular competence.Comparatively noisy and non-noisy circuit phenotypes performed similarly in some environments, but the more noisy circuit phenotype had a superior phenotype in other environments. This, in principle, shows that heterogeneity has fitness effects. Analogously, strains of Saccharomyces cerevisiae were constructed that differed only in the variability of expression of a gene among individual cells.Comparing strains with high or low variation in gene expression showed that each was advantageous, but under different environmental conditions. This therefore also shows that cellular heterogeneity can have environmentally-dependent fitness effects.
Gene expression heterogeneity has been shown to underlie incomplete phenotypic penetrance in C. elegans.Specifically, in the molecular circuit which controls the fate of cells forming part of the C. elegans intestine, variation in gene expression near a key molecular threshold can alter this intestinal phenotype, such that there is phenotypic variance among genetically individual worms. Inter-individual variation in the expression of reporter genes in early C. elegans larval stages can predict a substantial proportion of the variation in adult lifespan among individuals.Other studies have shown that phenotypic penetrance of a mutation is also dependent on the activity of other genes, including the chaperone Hsp90.This more broadly shows that gene networks control phenotypic variance. Interestingly, chaperone expression, which reduces the penetrance of mutations can have a cost, because it also reduces brood size.Together, what this work shows is that there is heterogeneity among genetically identical individuals in gene expression, that this can be modified by multiple loci/genetic networks, and |
Stem cell therapy for chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD), characterized by persistent and not fully reversible airflow restrictions, is currently one of the most widespread chronic lung diseases in the world. The most common symptoms of COPD are cough, expectoration, and exertional dyspnea. Although various strategies have been developed during the last few decades, current medical treatment for COPD only focuses on the relief of symptoms, and the reversal of lung function deterioration and improvement in patient's quality of life are very limited. Consequently, development of novel effective therapeutic strategies for COPD is urgently needed. Stem cells were known to differentiate into a variety of cell types and used to regenerate lung parenchyma and airway structures. Stem cell therapy is a promising therapeutic strategy that has the potential to restore the lung function and improve the quality of life in patients with COPD. This review summarizes the current state of knowledge regarding the clinical research on the treatment of COPD with mesenchymal stem cells (MSCs) and aims to update the understanding of the role of MSCs in COPD treatment, which may be helpful for developing effective therapeutic strategies in clinical settings.Currently, the therapeutic strategies in clinical setting for COPD are relieving symptoms, reducing the frequency and Correspondence to: Prof.
# Introduction
Chronic obstructive pulmonary disease (COPD) is a common respiratory disease that is characterized by persistent respiratory symptoms and airflow limitation. The airway or alveolar abnormalities are usually caused by significant exposure to noxious particles or gases. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Rabe [/bib_ref] [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Vestbo [/bib_ref] COPD represents an important public health challenge that is preventable and treatable, but there are still many people who die prematurely from it. COPD is currently the fourth leading cause of death in the world and is projected to be the third leading cause of death by 2020. [bib_ref] Global and regional mortality from 235 causes of death for 20 age..., Lozano [/bib_ref] [bib_ref] A nomogram for predicting severe exacerbations in stable COPD patients, Chen [/bib_ref] The Global Initiative for Chronic Obstructive Lung Disease (GOLD) defines and classifies COPD based on the severity of airflow obstruction. First, the patients are featured by forced expiratory volume in the first second (FEV 1 )/forced vital capacity (FVC) <0.70 and the airflow limitation is not fully reversible. [bib_ref] Standards for the diagnosis and treatment of patients with COPD: a summary..., Celli [/bib_ref] Then, according to the percentage of FEV 1 in the estimated value, COPD was classified into four stages. If FEV 1 is ≥80% of the predicted value, the stage is defined as mild; if FEV 1 is ≥50% and <80% of the predicted value, the stage is defined as moderate; if FEV 1 is ≥30% and <50% of the predicted value, the stage is defined as severe; if FEV 1 is <30% of the predicted value, the stage is defined as very severe. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive lung..., Vogelmeier [/bib_ref] The pathogenesis of COPD was extremely complicated, which mainly includes airway inflammation, alveolar structure destruction, and excessive expansion mediated by a variety of causes. [bib_ref] Pathogenesis of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke, Hikichi [/bib_ref] In general, cigarette smoke and other inhaled particles stimulate the epithelial cells to produce reactive oxygen species, which may induce inflammatory cells, including lymphocytes, neutrophils, macrophages, and eosinophils, [bib_ref] The nature of small-airway obstruction in chronic obstructive pulmonary disease, Hogg [/bib_ref] to infiltrate around the airway and cause the imbalance of protease/antiprotease. [bib_ref] Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung..., Rahman [/bib_ref] Given that elastin is the main component of connective tissue in the lung parenchyma, [bib_ref] Elastin in lung development and disease pathogenesis, Mecham [/bib_ref] the imbalance between protease and antiprotease will further cause lung overinflation, expansion, and loss in lung elasticity, thus resulting in emphysema. [bib_ref] Alpha1-antitrypsin deficiency, Strnad [/bib_ref] [bib_ref] Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus..., Chillappagari [/bib_ref] severity of exacerbations, and improving exercise tolerance. Although the standard pharmacological therapies, including bronchodilators, inhaled corticosteroids and the phosphodiesterase 4 inhibitor, roflumilast, showed modest efficacy in improving pulmonary function, [bib_ref] COPD: analysing factors associated with a successful treatment, Duarte-De-Ara Ujo [/bib_ref] to date, no conclusive clinical evidence was found to show that any existing medications for COPD could modify the longterm decline in pulmonary function as well as the mortality. Therefore, the development of novel effective treatments to reverse the decline in pulmonary function and reduce the clinical symptoms of the COPD patients is urgently needed.
Stem cells are a class of cells with the ability to self-renew repeatedly, and produce at least one type of highly differentiated progeny. [bib_ref] Multilineage potential of adult human mesenchymal stem cells, Pittenger [/bib_ref] [bib_ref] The ability of inner-cell-mass cells to self-renew as embryonic stem cells is..., Boroviak [/bib_ref] The most important function of stem cells is to maintain cell regeneration. Stem cells exist in most tissues of the body from early embryogenesis all the way throughout adult life and are thought to contribute to tissue maintenance and repair. [bib_ref] Evidence for human lung stem cells, Suzuki [/bib_ref] In particular, stem cells could give rise to subsequent generations with variable degrees of differentiation capacities, which offers significant potential for the generation of tissue that could potentially replace diseased and damaged areas in the human body. [bib_ref] Role of stem cells in the pathogenesis of chronic obstructive pulmonary disease..., Coppolino [/bib_ref] [bib_ref] Biological effects and mechanisms of action of mesenchymal stem cell therapy in..., Jin [/bib_ref] According to different differentiation potential, stem cells can be divided into totipotent stem cells, pluripotent stem cells, and unipotent stem cells. [bib_ref] Mesenchymal stem cells and immunomodulation: current status and future prospects, Gao [/bib_ref] Totipotent stem cells are a kind of cells that have the ability to self-renew and differentiate into any cell types. They have the potential to differentiate into any of the components of a complete individual, such as embryonic stem cells (ESCs). [bib_ref] Glycomics of human embryonic stem cells and human induced pluripotent stem cells, Furukawa [/bib_ref] Pluripotent stem cells have the ability to differentiate into many types of cells of a specific organ system, without the ability to develop into complete individual. Unipotent stem cells are unidirectionally differentiated stem cells in many tissues that normally produce only one type of cell. [bib_ref] Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent..., Terada [/bib_ref] Currently, pluripotent stem cells are most widely used in clinical research due to their broadly acting anti-inflammatory and regenerative properties, [bib_ref] MSC based therapies-new perspectives for the injured lung, Behnke [/bib_ref] [bib_ref] Pluripotent stem cells in adult tissues: struggling to be acknowledged over two..., Bhartiya [/bib_ref] such as hematopoietic stem cells, mesenchymal stem cells (MSCs), and human lung stem cells (hLSCs). [bib_ref] Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Takahashi [/bib_ref] Among them, MSCs are the most widely studied. MSCs exist in a variety of tissues, such as bone marrow, adipose tissue, and umbilical cords. According to the different sources, they are respectively named as bone marrowderived MSCs (BM-MSCs), adipose-derived MSCs (AD-MSCs), and umbilical cords-derived MSCs (UC-MSCs). [bib_ref] Epigenetic regulation of mesenchymal stem cell homeostasis, Sui [/bib_ref] Theoretical Basis At present, stem cells therapies have been applied to numerous diseases like cardiovascular and cerebrovascular diseases, endocrine system diseases, autoimmune system diseases, malignant tumors, hematopoietic system diseases, neurological diseases, and medical cosmetology industry. [bib_ref] Myeloid-derived suppressor cells as regulators of the immune system, Gabrilovich [/bib_ref] [bib_ref] Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures..., Takahashi [/bib_ref] [bib_ref] Stem cells, cancer, and cancer stem cells, Reya [/bib_ref] [bib_ref] Modeling neurological disorders with human pluripotent stem cell-derived astrocytes, Suga [/bib_ref] Of course, many latest findings of stem cells research have also provided new insights into the potential of stem cells to treat a variety of lung diseases, and the stem cell therapy for COPD has gradually become a hot spot.
In recent years, the therapeutic effect of stem cells in animal models of COPD has been demonstrated by many preclinical studies, which mainly focus on BM-MSCs, AD-MSCs, and UC-MSCs. [bib_ref] Role of stem cells in the pathogenesis of chronic obstructive pulmonary disease..., Coppolino [/bib_ref] [bib_ref] Improving effects of mesenchymal stem cells on symptoms of chronic obstructive pulmonary..., Karaoz [/bib_ref] People treat the experimental animals with different sources of stem cells, different methods, doses, and times of administration. Stem cell therapy might exert its effects through the following mechanisms: First, stem cell therapy can shorten the mean linear interception, reduce the apoptosis of epithelial cells in the lungs and improve the structure of the damaged lung tissue. [bib_ref] Bone marrow cells repair cigarette smoke-induced emphysema in rats, Huh [/bib_ref] [bib_ref] Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol alleviates elastase-induced emphysema..., Chen [/bib_ref] Second, stem cell therapy can promote the proliferation of a variety of cells in the lung and facilitate the self-repair of lung tissue. [bib_ref] Autologous transplantation of adipose tissue-derived stromal cells ameliorates pulmonary emphysema, Shigemura [/bib_ref] [bib_ref] Bone marrow mesenchymal stem cell transplantation for treatment of emphysemic rats, Zhao [/bib_ref] Third, stem cell therapy can improve pulmonary function to some extent. [bib_ref] Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via..., Guan [/bib_ref] [bib_ref] Effects of different mesenchymal stromal cell sources and delivery routes in experimental..., Antunes [/bib_ref] Fourth, stem cell therapy can reduce systemic inflammatory response and promote the secretion of a variety of anti-inflammatory mediators. [bib_ref] Mesenchymal stem cells alleviate airway inflammation and emphysema in COPD through down-regulation..., Gu [/bib_ref] Meanwhile, the mechanism of stem cells in the regulation of COPD has been extensively studied. [bib_ref] Premature lung aging and cellular senescence in the pathogenesis of idiopathic pulmonary..., Chilosi [/bib_ref] First of all, stem cells are cells with multidirectional differentiation potential. Studies have shown that MSCs can differentiate into type I and/or type II alveolar epithelial cells and participate in the repair of lung tissue structure. [bib_ref] The antifibrotic effects of mesenchymal stem cells on irradiated lungs via stimulating..., Dong [/bib_ref] In addition to promoting lung structural repair by differentiating into alveolar epithelial cells, stem cell transplantation also inhibits the apoptosis of alveolar epithelial cells. [bib_ref] Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway, Kim [/bib_ref] [bib_ref] Mesenchymal stem cell therapy for the treatment of chronic obstructive pulmonary disease, D'agostino [/bib_ref] Specially, cytokines secreted by MSCs interfere the expression level of apoptotic gene Bax, cleaved-caspase 3, and the antiapoptotic gene Bcl-2 in alveolar epithelial cells. [bib_ref] Mesenchymal stem cells transplantation protects against rat pulmonary emphysema, Zhen [/bib_ref] [bib_ref] Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage, Kim [/bib_ref] It is noted that COPD is the result of an abnormal and persistent inflammatory process that damages the lung architecture. [bib_ref] Chronic obstructive pulmonary disease biomarkers and their interpretation, Stockley [/bib_ref] Especially, cigarette smoke activates macrophages, neutrophils, and lymphocytes in the lung, causing the release of a variety of inflammatory cytokines that result in COPD progression. [bib_ref] Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in..., Gualano [/bib_ref] [bib_ref] Innate immune processes are sufficient for driving cigarette smoke-induced inflammation in mice, Botelho [/bib_ref] MSCs have shown the ability to slow the progression of COPD by effectively decreasing the inflammatory response with attenuated classic activated macrophage cytokine release including interleukin (IL)-1b, IL-6, tumor necrosis factor alpha and monocyte chemotactic protein 1 and promoting the release of antiinflammatory mediators, like IL-10, transforming growth factor-b, indoleamine 2,3 dioxygenase 1. [bib_ref] Biological effects and mechanisms of action of mesenchymal stem cell therapy in..., Jin [/bib_ref] [bib_ref] Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via..., Guan [/bib_ref] Another equally important factor for the pathogenesis of COPD is the balance of proteases and antiproteases. The imbalance of protease/antiprotease will cause the degradation of extracellular matrix, [bib_ref] Absence of plasma protease-antiprotease imbalance in the formation of saccular cerebral aneurysms, Sakai [/bib_ref] promote the apoptosis of alveolar wall structure cells, increase the high secretion of mucus and finally lead to the destruction of alveolar wall and the expansion of air space. [bib_ref] A novel zebrafish model to emulate lung injury by folate deficiency-induced swim..., Lee [/bib_ref] Previous data have shown that stem cells reversed the up-regulation of matrix metalloproteinases induced by cigarette smoke. [bib_ref] Extracellular regulated kinase/mitogen activated protein kinase is up-regulated in pulmonary emphysema and..., Mercer [/bib_ref] Indeed, MSCs can effectively inhibit the progression of COPD by regulating the balance between proteases and antiproteases. [bib_ref] The therapeutic effects of optimal dose of mesenchymal stem cells in a..., Kim [/bib_ref] Additionally, stem cell transplants can also reduce oxidative stress in the lung tissue. [bib_ref] Mesenchymal stem cells attenuate diabetic lung fibrosis via adjusting Sirt3-mediated stress responses..., Chen [/bib_ref] Excessive oxidative stress will cause cell damage and further aggravate the inflammatory response in the lung by inducing the release of inflammatory cytokines. [bib_ref] Flavonoids isolated from loquat (Eriobotrya japonica) leaves inhibit oxidative stress and inflammation..., Jian [/bib_ref] Clinical Research
On the basis of the previous preclinical research, the results of clinical trials for stem cells are also being gradually integrated. This paper provides an overview of clinical trials in the treatment of COPD with stem cells, which is crucial for researchers to get clearer understanding of the current research situation and achieve the ultimate goal of curing patients with COPD.
MSCs are pluripotent stem cells that share all the characteristics of stem cells: self-renewal, immunomodulatory, and multipolarity. [bib_ref] Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation, Prockop [/bib_ref] [bib_ref] Immunomodulatory properties and therapeutic application of mesenchymal stem cells, Shi [/bib_ref] MSCs were first described in the bone marrow where they constitute a small fraction of cells (0.001%-0.01%) that closely interact with hematopoietic cells to support hematopoiesis and skeletal homeostasis. [bib_ref] Multilineage potential of adult human mesenchymal stem cells, Pittenger [/bib_ref] Since then, it has become evident that MSCs reside in many tissues, including mesenchymal tissues (bone, adipose tissue, connective tissue), umbilical cord, and several organs including the liver, spleen, and lung. [bib_ref] Concise review: multifaceted characterization of human mesenchymal stem cells for use in..., Samsonraj [/bib_ref] [bib_ref] Quantitative proteomic characterization of lung-MSC and bone marrow-MSC using DIA-mass spectrometry, Enes [/bib_ref] [bib_ref] Stem cell-derived polarized hepatocytes, Dao Thi [/bib_ref] There are no specific markers for MSCs, therefore, they are identified by their expression of a range of markers and their functional characteristics. Nowadays in most clinical trials, the MSCs were derived from bone marrow.
## Bone marrow-derived stem cells
The first clinical trial of cell therapy in COPD patients was an uncontrolled phase I clinical trial (ClinicalTrials.gov identifier: NCT01110252) carried out in Brazil from May 2009 to October 2009. [bib_ref] Mesenchymal stromal cells: a novel therapy for the treatment of, Broekman [/bib_ref] [bib_ref] Unicentric study of cell therapy in chronic obstructive pulmonary disease/pulmonary emphysema, Ribeiro-Paes [/bib_ref] The purpose of this study was to evaluate the safety of bone marrow-derived monocytes (BM-MCs) infusion procedure in patients with advanced COPD (GOLD stage IV). With a single intravenous infusion, each patient received a total of 1 Â 10 8 cells. Unlike other subsequent studies using BM-MSCs, the cells used in this study were BM-MCs, which were isolated directly from bone marrow without subsequent in vitro culture. The 12-month follow-up after the BM-MCs infusion showed that there were no adverse reactions. Therefore, the researchers claimed that this treatment was quite safe. The laboratory analysis reported a slight improvement in pulmonary function in all patients, chiefly in the first 30 days after the procedure was carried out. In addition, the results showed that their clinical conditions also improved to some extent. However, because of the small size (only four patients) and lack of statistical analysis in this design, the results did not support definite conclusions. [bib_ref] Unicentric study of cell therapy in chronic obstructive pulmonary disease/pulmonary emphysema, Ribeiro-Paes [/bib_ref] It should be noted that this study was the first clinical trial of cell therapy in COPD patients, and it provided meaningful guidance for the clinical cell therapy of COPD in the future.
Five years after the first clinical trial using BM-MCs to treat COPD, a prospective, randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov identifier: NCT00683722) of BM-MSCs in COPD was conducted by Weiss et al [bib_ref] A placebo-controlled, randomized trial of mesenchymal stem cells in COPD, Weiss [/bib_ref] in the United States. The goals of this study were to evaluate safety and efficacy of MSCs treatment and the effect of MSCs on circulating inflammatory mediators. In this study, 62 patients with moderate to severe COPD (GOLD stage II or III) were randomized to double-blinded infusions of either allogeneic MSCs or vehicle control. Patients received four monthly infusions (1 Â 10 8 cells/infusion) and were subsequently followed for 2 years after the first infusion. There were several significant improvements in this clinical trial, compared to the first one. First, the number of enrolled cases increased significantly, from 4 to 62. Second, the input cells were improved from BM-MCs to BM-MSCs. After in vitro culture, the BM-MSCs, which were isolated from BM-MCs through further screening of growth mode, would have better proliferation capacity. Third, the authors made a change in the frequency of administration, increased from a single dose to four, monthly. Fourthly, the source of the cells was also changed, from autogenous to allogeneic. The researchers in this clinical trial found that allogeneic MSCs administration seemed also safe in patients with moderate to severe COPD. They did not observe some infusion related toxicities and serious clinically relevant adverse events. No statistically significant differences were observed in pulmonary function and quality of life. There were also no significant differences in the frequency of COPD exacerbations or worsening of disease in this study. It was exciting to observe a significant decrease in Creactive protein (CRP) levels up to 1 month after the first infusion with MSCs in patients who had elevated CRP levels at study entry, indicating that the systemic inflammatory response in these patients was reduced by MSCs to some extent. Although no significant improvements in clinical symptoms and pulmonary function were observed in this study, sufficient cases provide more confidence about the safety of MSCs in the treatment of COPD.
In another study, Stolk et al [bib_ref] A phase I study for intravenous autologous mesenchymal stromal cell administration to..., Stolk [/bib_ref] finished a phase I, prospective open-label trial (ClinicalTrials.gov identifier: NCT01306513) to observe the safety and feasibility of BM-MSCs intravenous administration. They selected ten patients with severe COPD who would undergo lung volume reduction surgery (LVRS). The BM-MSCs therapy was performed 8 weeks after the first LVRS, with (1-2) Â 10 6 cells/kg BM-MSCs injected intravenously once a week for 2 weeks. A second LVRS was performed one month after the stem cell treatment. Finally, the study showed that there were no serious adverse events observed and the lung tissue showed no fibrotic responses. FEV 1 and body weight increased in all patients but showed no difference between two groups. Considering that these patients all had experienced LVRS, the changes in FEV 1 and body weight were more likely to be resulted from LVRS than from BM-MSCs infusion. [bib_ref] Weight gain after lung reduction surgery is related to improved lung function..., Kim [/bib_ref] The current study would only demonstrate that LVRS itself had a substantial effect on FEV 1 , but would not be further enhanced by the BM-MSCs treatment. [bib_ref] Densitometry for assessment of effect of lung volume reduction surgery for emphysema, Stolk [/bib_ref] The most prominent feature of this clinical trial was that the patients included were about to undergo LVRS, thus, the researchers were able to obtain lung tissue before and after the stem cell treatment. Through the comparative study of lung histopathological sections before and after BM-MSCs treatment, the researchers can more intuitively understand the effect of stem cells on lung tissue at the pathological and microscopic levels. Thus, for safety evaluation, this study not only focused on the occurrence of adverse events, but also intuitively demonstrated that BM-MSCs treatment would not cause any adverse changes in lung structure. Surprisingly, after BM-MSCs infusion, alveolar septa showed a three-fold increased expression of the endothelial marker CD31, which suggested that BM-MSCs therapy might promote endothelial repair. In addition, the dosage Chinese Medical Journal 2021;134 [bib_ref] COPD: analysing factors associated with a successful treatment, Duarte-De-Ara Ujo [/bib_ref] www.cmj.org of BM-MSCs was also adjusted. Instead of fixing the total number of the cells, the researchers gave the patients different amounts of cells depending on their body weight.
The above three clinical trials all transplanted MSCs into patients via intravenous infusion. However, whether the stem cells would circulate throughout the body or migrate directly to damaged lung tissue is unknown. Armitage et al [bib_ref] Mesenchymal stromal cell infusion modulates systemic immunological responses in stable COPD patients:..., Armitage [/bib_ref] conducted a single site, phase I clinical trial (Australian clinical trials identifier: 12614000731695) to observe the distribution of stem cells in vivo and the systemic inflammatory response after intravenous infusion. Nine patients received two infusions of allogeneic BM-MSCs of 2 Â 10 6 cells/kg once a week for 2 weeks. BM-MSCs used for the first infusion were labeled with indium-111 to monitor its location in the body. The results showed that BM-MSCs infusion had no attributable adverse effects and was well tolerated. BM-MSCs were detected in the lung within 30 min and remained detectable after 24 h, after which, BM-MSCs were mainly distributed in the liver. After the injection of BM-MSCs, the number of hospitalized patients with acute exacerbations of COPD decreased, but there was no significant improvement in pulmonary function. However, BM-MSCs tended to migrate to normal lung tissue rather than emphysema areas, which may be a reason why stem cell therapy has no significant effect on pulmonary function. Many inflammatory mediators, such as F2-isoprostanes, IL-6, and CD163, showed a decreasing trend 1 to 7 days after treatment. It is worth noting that CRP exhibited a transient elevated state 1 to 2 days after stem cell intervention, which contrasts with data from another clinical trial where systemic administration of allogeneic BM-MSCs in COPD patients reduced CRP levels 1 to 3 months after infusion. [bib_ref] Combined bone marrow-derived mesenchymal stromal cell therapy and one-way endobronchial valve placement..., De Oliveira [/bib_ref] Considering that the stem cells, which were injected intravenously, did not stay in the lung long enough to exert its role, researchers began to try new ways of administration. Compared with intravenous administration, intratracheal administration is a more direct and accurate mode of drug application. A phase I, prospective, patientblinded, randomized, placebo-controlled study (Clinical-Trials.gov identifier: NCT01872624), which was implemented by de Oliveira et al, [bib_ref] Combined bone marrow-derived mesenchymal stromal cell therapy and one-way endobronchial valve placement..., De Oliveira [/bib_ref] attempted to treat COPD by intratracheal administration of BM-MSCs combined with one-way endobronchial valves (EBV) insertion for the first time. In this trial, ten patients (GOLD stage III or IV) were divided into two groups, randomly receiving either allogeneic BM-MSCs (1 Â 10 8 cells) or 0.9% saline solution bronchoscopically, just before insertion of oneway EBVs. In the EBV + MSCs group, no patient experienced adverse events and the serum CRP levels were significantly reduced in 30 and 90 days compared to EBV + saline group. No statistically significant betweengroup differences in pulmonary function indicators were observed. EBV + MSCs group had a significant lower body mass index, airflow obstruction, dyspnea, and exercise capacity index, lower modified medical research council (mMRC) scores, and decreased Saint George's respiratory questionnaire (SGRQ) scores. Consistent with above data, in this study, intrabronchial administration of MSCs in severe COPD patients was relatively safe and was able to reduce systemic inflammation by reducing the level of CRP and to improve life quality of COPD patients.
All in all, above clinical trials indicated that administration of allogeneic or autologous BM-MSCs is safe and no adverse side-effects are observed. Additionally, the therapeutic effects of BM-MSCs need more clinical trials to confirm.
## Adipose tissue-derived stem cells
Adipose tissue is another major source of MSCs. Compared to bone marrow, adipose tissue contains a much higher percent of MSCs. [bib_ref] Adipose-derived mesenchymal stem cells attenuate rejection in a rat lung transplantation model, Watanabe [/bib_ref] [bib_ref] Multilineage cells from human adipose tissue: implications for cellbased therapies, Zuk [/bib_ref] In addition, AD-MSCs have higher proliferative capability, [bib_ref] Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood,..., Kern [/bib_ref] retain differentiation potential for a longer period and have increased immunomodulating capacity compared to BM-MSCs. [bib_ref] Adipose tissuederived multipotent stromal cells have a higher immunomodulatory capacity than their..., Melief [/bib_ref] Adipose stromal cells can be readily separated from the adipocyte population by methods which require less than 2 h of processing time and yield a concentrated cellular preparation termed the stromal vascular fraction (SVF). [bib_ref] A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal..., Traktuev [/bib_ref] The SVF, which is easy to obtain, contains all cellular elements of fat excluding adipocytes and can be used as an option for stem cell therapy. [bib_ref] Concise review: human adiposederived stem cells: separating promise from clinical need, Locke [/bib_ref] A non-randomized, phase I, open label trial (ClinicalTrials. gov identifier: NCT02041000) was performed by Comella et al. [bib_ref] Autologous stromal vascular fraction in the intravenous treatment of end-stage chronic obstructive..., Comella [/bib_ref] Twelve patients with COPD participated in the clinical trial. The researchers isolated SVF from the patients' adipose tissue and infused (1.5-3) Â 10 8 cells back to the patients intravenously. The primary purposes of this study were to evaluate the feasibility and safety of SVF infusion in COPD patients. During the infusion and 12 months of follow-up, no adverse events were observed. Unlike other studies, this trial focused specifically on patients' subjective feelings such as the attitudes towards the procedure and the willingness to undergo next procedure of stem cells treatment. Attitudes toward the study were predominantly positive, three stated that there was no effect, four noted a subjective sense of benefit within a day, and five noted a gradual improvement, with maximal improvement noted at approximately 1 month following infusion. Surprisingly, the SGRQ score was decreased from 73 units at baseline to 45 at 3 months (P = 0.005) and to 44 at 6 months (P = 0.008) after treatment. The clinical trial relied on subjective patient feedback, but lacked objective clinical evidence such as pulmonary function tests. Therefore, infusion of autologous SVF was safe and might improve the quality of life for patients with COPD, but it was necessary to determine whether it has a positive impact on pulmonary function within further studies.
Umbilical cord-derived stem cells UC-MSCs have been reported as promising MSCs sources for treating various diseases in humans, including heart failure, [bib_ref] Safety and efficacy of the intravenous infusion of umbilical cord mesenchymal stem..., Bartolucci [/bib_ref] [bib_ref] Functional characterization of human umbilical cord-derived mesenchymal stem cells for treatment of..., Fang [/bib_ref] ankylosing spondylitis, [bib_ref] Infusion of umbilical cord mesenchymal stem cells alleviates symptoms of ankylosing spondylitis, Li [/bib_ref] type 2 diabetes mellitus, [bib_ref] Umbilical cord mesenchymal stem cell transfusion ameliorated hyperglycemia in patients with type..., Kong [/bib_ref] and angioplasty for diabetic feet. [bib_ref] Clinical evaluation of human umbilical cord mesenchymal stem cell transplantation after angioplasty..., Qin [/bib_ref] BM-MSCs are most commonly used in clinical trials of stem cell therapies. However, with the development of stem cell research, the shortage of BM-MSCs is gradually exposed. Human BM-MSCs from aged patients would highly Chinese Medical Journal 2021;134(13) www.cmj.org express senescence-related genes, have shorter telomere length, low proliferation, and low differentiation capacity. [bib_ref] Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem..., Cheng [/bib_ref] This will inevitably lead to obstacles in the treatment of autologous stem cell transplantation. Through comparative analysis, the researchers found that UC-MSCs exhibit strong modulation capacity. In addition, under the same conditions, UC-MSCs inhibited allogeneic lymphocytes more strongly than BM-MSCs and AD-MSCs did. [bib_ref] Contribution of bone marrow-derived mesenchymal stem cells to the morphological changes in..., Kanno [/bib_ref] UC-MSCs also had higher proliferation rates and exhibited better potential to differentiate into other cells due to its better primitiveness. [bib_ref] Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison..., Taléns-Visconti [/bib_ref] Therefore, when allogeneic stem cell transplantation is needed (such as in elderly patients), UC-MSCs is a better choice.
Le Thi Bich et al [bib_ref] Allogeneic umbilical cord-derived mesenchymal stem cell transplantation for treating chronic obstructive pulmonary..., Bich [/bib_ref] finished a pilot clinical trial (ISRCTN70443938) of treating COPD with UC-MSCs. In this study, 20 patients with COPD at stage C or D (GOLD) were enrolled. UC-MSCs were isolated from umbilical cord samples of donors during childbirth. All patients were intravenously infused with expanded allogeneic UC-MSCs (1.5 Â 10 6 cells/kg) and followed for 6 months after the first infusion. Interestingly, no serious or clinically significant adverse events were observed for all patients during the study. Unfortunately, the pulmonary function showed no statistically significant differences before and after the treatment of UC-MSCs. It was satisfactory to find that the mMRC score, COPD assessment test score, and number of exacerbations decreased significantly after 1, 3, and 6 months compared with those before treatment, which suggested that UC-MSCs can improve the patient's quality of life. And there was an interesting phenomenon that stage D COPD patients exhibited a stronger medical response after UC-MSCs transplantation than stage C COPD patients did, which was in contrast to another study, in which researchers found COPD patients with mild disease retained MSCs in the pulmonary vasculature longer than those with more severe disease did. [bib_ref] Mesenchymal stromal cell infusion modulates systemic immunological responses in stable COPD patients:..., Armitage [/bib_ref] Perhaps this difference can be attributed to the use of different sources of MSCs, but further research is really needed. The trial was the first clinical trial to use MSCs from umbilical cord tissue to treat COPD patients. Conclusions can be drawn that UC-MSCs administration appears to be safe in patients with moderate-to-severe COPD and can significantly improve their quality of life.
In the latest research, Karaoz et al [bib_ref] Improving effects of mesenchymal stem cells on symptoms of chronic obstructive pulmonary..., Karaoz [/bib_ref] finished an openlabel, single-armed study carried out in LIV hospital in Istanbul. After the pre-treatment measurements, all the patients were administered a total of four doses of UC-MSCs (1-2 Â 10 6 cells/kg) by intravenous infusion at 2week intervals. Respiratory function tests, SGRQ scores, and 6 min walk test were next examined. Surprisingly, UC-MSCs therapy not only improved patients' quality of life but also improved pulmonary function to some extent. The mean pre-treatment FEV 1 /FVC ratios were only 66.90% while the mean FEV 1 /FVC value raised to 69.58% after the treatment. The greatest difference between this clinical trial and the clinical trial conducted by Le Thi Bich et al [bib_ref] Allogeneic umbilical cord-derived mesenchymal stem cell transplantation for treating chronic obstructive pulmonary..., Bich [/bib_ref] is the schedule of administration. Single dose was adopted by Le Thi Bich et al, [bib_ref] Allogeneic umbilical cord-derived mesenchymal stem cell transplantation for treating chronic obstructive pulmonary..., Bich [/bib_ref] while Karaoz et al [bib_ref] Improving effects of mesenchymal stem cells on symptoms of chronic obstructive pulmonary..., Karaoz [/bib_ref] increased the number of doses to double, with an interval of 2 weeks, which is likely to be one of the reasons for the difference in the result of pulmonary function. Certainly, the shortcomings of this clinical trial exist, such as the small number of cases (only five) and the short follow-up time (only 3 months). However, it still offered a glimmer of hope for future stem cell treatment of COPD.
hLSCs hLSCs refer to the cells that can differentiate into functional lung tissues under specific conditions and play an important role in maintaining lung tissue renewal and repairing lung injury. [bib_ref] Lung-derived mesenchymal stromal cell post-transplantation survival, persistence, paracrine expression, and repair of..., Hoffman [/bib_ref] They can be isolated from lung tissue and have similar cell surface markers with other stem cells. The difficulty of obtaining human lung tissue significantly limited the study of this type of cells. Nevertheless, lung stem cells (LSCs) may be involved in alveolar homeostasis and post-injury repair and may need to be considered as a potential tool or target when referring to stem cell therapy.
In animal experiments, the effect of LSCs has been confirmed. Injecting LSCs into the airway of emphysema model mice can effectively reduce the severity of emphysema and improve the survival of mice. [bib_ref] Lung mesenchymal stem cells ameliorate elastaseinduced damage in an animal model of..., Cappetta [/bib_ref] [bib_ref] Bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema, Ishizawa [/bib_ref] However, it is difficult to transform the animal experiments into clinical practice because of the difficulty to obtain human lung tissue. Moreover, rejective reaction of host-vs.-graft is still a troubling aspect needed to consider after the LSCs injection therapy. Although it is impossible to directly treat COPD with exogenous LSCs infusion, it is possible to activate endogenous LSCs with specific drugs. For example, studies have shown that all-trans-retinoic acid (ATAR) may activate the endogenous stem/progenitor cells in the lung that result in lung structural regeneration. [bib_ref] A pilot study of all-trans-retinoic acid for the treatment of human emphysema, Mao [/bib_ref]
## Summary
In the above eight clinical trials (the trial of ATAR was not included in the discussion), the researchers used different kinds of stem cells, different cell sources, different modes, and dosages of administration to treat COPD. There are both similarities and differences in the final results. In the following part, we will analyze the possible improvement methods of stem cell treatment for COPD by comparing the differences in each trial design and the different results [ [fig_ref] Table 1: Clinical trials on the stem cell therapy for COPD with published results [/fig_ref] ].
## Cell types
In the current clinical trials of stem cell therapy for COPD, the main cell types used were MSCs because other kind of stem cells all have their own limitations. ESCs have potentials for forming teratoma and immune rejection, and there are ethical concerns for application of ESCs; induced pluripotent stem (iPS) cells can form teratoma, and the current technique cannot produce reliable amount of clinical-grade iPS cells [bib_ref] Road to future: iPSC clinical application in Parkinson's disease treatment, Xu [/bib_ref] [bib_ref] The promise of induced pluripotent stem cells in research and therapy, Robinton [/bib_ref] ; LSCs are also a good research direction, but clinical trials are not easy to carry out because of the difficulty in obtaining lung tissue. The most common sources of MSCs are bone marrow, fat and umbilical cord, which are respectively named as BM-MSCs, AD-MSCs, and UC-MSCs. Some studies pointed out that BM-MSCs have decreased differentiation potential and maybe suboptimal for this line of therapy. [bib_ref] Stem cell therapies for chronic obstructive pulmonary disease: current status of pre-clinical..., Sun [/bib_ref] At Chinese Medical Journal 2021;134 [bib_ref] COPD: analysing factors associated with a successful treatment, Duarte-De-Ara Ujo [/bib_ref] www.cmj.org the same time, many studies have shown that AD-MSCs and UC-MSCs are very promising cells because the source is quite available. In addition, the process of collecting UC-MSCs is non-invasive, and there has been no tumorigenicity reported up to date. [bib_ref] Biological effects and mechanisms of action of mesenchymal stem cell therapy in..., Jin [/bib_ref] [bib_ref] Experimental basis and new insights for cell therapy in chronic obstructive pulmonary..., De Faria [/bib_ref] Trends in clinical trials are also consistent with this conclusion, the early clinical trials were mainly focused on BM-MSCs, while in recent years, the clinical trials related to AD-MSCs and UC-MSCs gradually increased. Until there is a better choice, AD-MSCs and UC-MSCs may be the mainstream research targets for the treatment of COPD with stem cells.
## Cell sources
The source of cells is either from autologous or allogeneic.
In the early days, people were afraid to use allogeneic stem cells for clinical trials mainly for fear of immune transplant rejection. However, it has been proved that the safety of allogeneic MSCs transplanted into COPD patients is good, and there are no related adverse reactions. [bib_ref] A placebo-controlled, randomized trial of mesenchymal stem cells in COPD, Weiss [/bib_ref] [bib_ref] Mesenchymal stromal cell infusion modulates systemic immunological responses in stable COPD patients:..., Armitage [/bib_ref] [bib_ref] Allogeneic umbilical cord-derived mesenchymal stem cell transplantation for treating chronic obstructive pulmonary..., Bich [/bib_ref] On the premise of good safety, we can further compare the advantages and disadvantages of autologous stem cells and allogeneic stem cells. For UC-MSCs, it is not practical to choose cells from autogenous source, so cells from allogeneic source would be the only choice. For BM-MSCs and AD-MSCs, we can use either MSCs from autogenous or allogeneic. Studies have shown that human BM-MSCs from aged patients would highly express senescence-related genes, with shorter telomere length, low proliferation, and low differentiation capacity. [bib_ref] Investigation of telomerase/telomeres system in bone marrow mesenchymal stem cells derived from..., Antoniou [/bib_ref] In addition, the patients will suffer a certain degree of pain in the process of bone marrow or fat acquisition. Some adverse reactions related to puncture and liposuction may occur at the same time. Therefore, for elderly patients, allogeneic stem cells may be a better choice. However, allogeneic stem cells also have their own problems. The biggest one is in the storage of the cells, because liquid nitrogen cryopreservation will cause a decrease in cell activity, and they cannot play the maximum therapeutic effect. [bib_ref] Comparison of different cryopreservation systems for peripheral blood stem cells, Huang [/bib_ref] Whether to use autologous or allogeneic stem cells will be up to the researchers to make a judgment based on the actual situation.
## Mode, schedule, and dosage of administration
The research showed that after the stem cells were injected into the body intravenously, they concentrate in the lungs for the first half hour and then gradually migrate to the liver. [bib_ref] Mesenchymal stromal cell infusion modulates systemic immunological responses in stable COPD patients:..., Armitage [/bib_ref] The inability of stem cells to stay in the lung for longer time may affect the therapeutic effects of stem cells. There are two ways to solve this problem, that is by adjusting the schedule of administration or mode of administration. By comparing the two UC-MSCs trials, we found that multiple doses may have a better therapeutic effect than single dose. [bib_ref] Improving effects of mesenchymal stem cells on symptoms of chronic obstructive pulmonary..., Karaoz [/bib_ref] [bib_ref] Allogeneic umbilical cord-derived mesenchymal stem cell transplantation for treating chronic obstructive pulmonary..., Bich [/bib_ref] Therefore, increasing the number of doses is an ideal improvement for future stem cell research. In addition, airway injection by bronchoscope is a good way to transfer the stem cells directly to the patient's lungs. Of the eight completed clinical trials, there was only one trial which directly transplanted stem cells into patients via airway injections. Although the result of this experiment was negative, it is one of the directions of our future development. In terms of the dosage of [bib_ref] COPD: analysing factors associated with a successful treatment, Duarte-De-Ara Ujo [/bib_ref] www.cmj.org Chinese Medical Journal 2021;134 [bib_ref] COPD: analysing factors associated with a successful treatment, Duarte-De-Ara Ujo [/bib_ref] www.cmj.org administration, the eight clinical trials have maintained a high level of consistency, basically at (1-3) Â 10 8 cells or (1-2) Â 10 6 cells/kg. Most researchers are conservative in this aspect because the schedule of administration can be adjusted, and too many cells per dose may cause unintended consequences.
# Experimental results
No matter what type of MSCs, which would be transplanted into the lungs of COPD patients, in what mode of administration, it has been proved to be safe. There were no adverse events associated with stem cell transplantation. In terms of the effect of stem cells on pulmonary function, only 2 clinical trials reported that MSCs could improve pulmonary function (autologous BM-MCs 1 Â 10 8 and allogeneic UC-MSCs [1-2] Â 10 6 /kg), and the remaining six clinical trials all showed that MSCs had no effect on it. In view of the small number of patients (4 and 5, respectively) enrolled in the two clinical trials that showed a therapeutic effect of stem cells on pulmonary function, further research is needed to see whether MSCs can improve it. In eight clinical trials, six studies suggested that MSCs transplantation could improve patients' quality of life, while the other two studies on BM-MSCs showed no effect on it. We believe that stem cell transplantation may have the ability to improve patients' quality of life, perhaps because of the placebo effect, the inhibition of systemic inflammatory response or other extra-cognitive effects. In addition, stem cell transplantation would disrupt the CRP level, which rises briefly 1 to 2 days after transplantation, followed by persistent low expression for several months.
At present, 17 clinical trials on the treatment of COPD with stem cells are registered at ClinicalTrials.gov. The relevant information is summarized in [fig_ref] Table 2: Clinical trials registered at ClinicalTrials [/fig_ref]. In future clinical trials, the following suggestions may be effective in improving the experimental design: (1) expand the sample size; (2) extend the follow-up time to 2 years or even longer; (3) select patients with different grades of COPD to determine the most suitable subjects for MSCs treatment; (4) AD-MSCs and UC-MSCs are more inclined to be used in future research compared with BM-MSCs; (5) multiple injections to enhance the treatment effect; (6) teams with appropriate clinical conditions may attempt to perform MSCs transplantation through bronchoscope; (7) assess lung function and quality of life comprehensively to obtain more accurate research data; [bib_ref] The nature of small-airway obstruction in chronic obstructive pulmonary disease, Hogg [/bib_ref] further explore the effects of MSCs on changes in other inflammatory, immune, and metabolic indicators. It is believed that stem cell therapy may play a revolutionary role in the treatment of COPD and other respiratory diseases in the near future.
[table] Table 1: Clinical trials on the stem cell therapy for COPD with published results. [/table]
[table] Table 2: Clinical trials registered at ClinicalTrials.gov on stem cell therapy for COPD. [/table]
|
Reliable brain morphometry from contrast‐enhanced T1w‐MRI in patients with multiple sclerosis
Brain morphometry is usually based on non-enhanced (pre-contrast) T1-weighted MRI. However, such dedicated protocols are sometimes missing in clinical examinations. Instead, an image with a contrast agent is often available. Existing tools such as FreeSurfer yield unreliable results when applied to contrast-enhanced (CE) images.Consequently, these acquisitions are excluded from retrospective morphometry studies, which reduces the sample size. We hypothesize that deep learning (DL)-based morphometry methods can extract morphometric measures also from contrastenhanced MRI. We have extended DL+DiReCT to cope with contrast-enhanced MRI. Training data for our DL-based model were enriched with non-enhanced and CE image pairs from the same session. The segmentations were derived with Free-Surfer from the non-enhanced image and used as ground truth for the coregistered CE image. A longitudinal dataset of patients with multiple sclerosis (MS), comprising relapsing remitting (RRMS) and primary progressive (PPMS) subgroups, was used for the evaluation. Global and regional cortical thickness derived from non-enhanced and CE images were contrasted to results from FreeSurfer. Correlation coefficients of global mean cortical thickness between non-enhanced and CE images were significantly larger with DL+DiReCT (r = 0.92) than with FreeSurfer (r = 0.75). When comparing the longitudinal atrophy rates between the two MS subgroups, the effect sizes between PPMS and RRMS were higher with DL+DiReCT both for non-enhanced (d = À0.304) and CE images (d = À0.169) than for FreeSurfer (non-enhanced d = À0.111, CE d = 0.085). In conclusion, brain morphometry can be derived reliably from contrast-enhanced MRI using DL-based morphometry tools, making additional cases available for analysis and potential future diagnostic morphometry tools.K E Y W O R D S brain morphometry, cortical thickness, deep learning, MRI, post-contrast imaging
# | introduction
Brain morphometry is usually derived from high-resolution, 3D T1-weighted (T1w) MRI, owing to the good gray/white-matter contrast of protocols such as MP-RAGE (van der [bib_ref] Brain morphometry with multiecho MPRAGE, Van Der Kouwe [/bib_ref].
Morphometry tools such as FreeSurferusually expect non-enhanced (pre-contrast) MR images of this type as input data.
For diagnostic purposes, T1w images after administration of gadolinium-based contrast agents (GBCAs) are often acquired [bib_ref] Revised recommendations of the consortium of MS centers task force for a..., Traboulsee [/bib_ref] , either as a replacement for or in addition to the non-enhanced image. MRI acquired for clinical purposes sometimes include only contrast-enhanced 3D T1w MRI, leaving those data inaccessible for retrospective morphometric analysis. For example, the latest MR imaging guidelines for multiple sclerosis recommend a contrast-enhanced T1w acquisition, but not necessarily a corresponding image without contrast-agent [bib_ref] 2021 MAGNIMS-CMSC-NAIMS consensus recommendations on the use of MRI in patients with..., Wattjes [/bib_ref].
Few studies have investigated the influence of contrast agents on morphometry. A significant impact was reported on thalamic volumes [bib_ref] Gadolinium effect on thalamus and whole brain tissue segmentation, Hannoun [/bib_ref] and tissue relaxation times [bib_ref] Effects of gadolinium contrast agent administration on automatic brain tissue classification of..., Warntjes [/bib_ref]. Good results using FreeSurfer were recently reported for volumes and thickness on a small and pre-selected sample [bib_ref] The effect of gadolinium-based contrast-agents on automated brain atrophy measurements by FreeSurfer..., Lie [/bib_ref].
In multiple sclerosis (MS), quantification of the global gray-matter (GM) loss is already used as a secondary outcome measure in clinical trials [bib_ref] Brain atrophy: An in-vivo measure of disease activity in multiple sclerosis, Radü [/bib_ref] [bib_ref] MAGNIMS consensus recommendations on the use of brain and spinal cord atrophy..., Sastre-Garriga [/bib_ref]. With the adoption of No evidence of disease activity (NEDA) in its updated version NEDA-4 [bib_ref] Inclusion of brain volume loss in a revised measure of 'no evidence..., Kappos [/bib_ref] , brain atrophy quantification has become increasingly important for the assessment of individual patients. For research purposes, studying the distribution of MStypical atrophy patterns over the cortex (like observable in regional GM volume or cortical thickness) in addition to the global GM volume might help improve the understanding of the disease [bib_ref] Cortical atrophy patterns in multiple sclerosis are nonrandom and clinically relevant, Steenwijk [/bib_ref]. Beyond these univariate approaches, network properties of cortical reorganization due to aging or neurodegenerative processes can be studied by investigating coordinated structural changes of different subregions of the cortex using the concept of structural covariance networks (SCN; [bib_ref] Imaging structural co-variance between human brain regions, Alexander-Bloch [/bib_ref] [bib_ref] Networks of anatomical covariance, Evans [/bib_ref]. In the context of MS, SCNs have been used to study the impact of this reorganization on physical and cognitive impairment [bib_ref] Disruption of structural and functional networks in longstanding multiple sclerosis, Tewarie [/bib_ref] , to reveal differences between conversion vs. non-conversion from clinically isolated syndrome to MS , or to show early longitudinal reorganization processes in patients with relapsing remitting [bib_ref] Longitudinal cortical network reorganization in early relapsingremitting multiple sclerosis, Fleischer [/bib_ref] or primary progressive MS [bib_ref] Clinical relevance of cortical network dynamics in early primary progressive MS, Tur [/bib_ref] as compared to healthy controls. Importantly, none of these findings were noticeable in simpler univariate analyses of global or regional cortical thickness.
Cortical thickness is a frequently used measure in surface-based morphometric analysis [bib_ref] Measuring the thickness of the human cerebral cortex from magnetic resonance images, Fischl [/bib_ref] , employed by many of the largest brain morphometry studies . Being largely independent of head size [bib_ref] A largescale comparison of cortical thickness and volume methods for measuring Alzheimer's..., Schwarz [/bib_ref] and less correlated to the corresponding volume measures [bib_ref] Cortical thickness or grey matter volume? The importance of selecting the phenotype..., Winkler [/bib_ref] makes it an interesting independent quantitative marker. Its self-explanatory nature is another advantage for potential clinical applications.
The urgent need to make large amounts of additional T1w MRI accessible to morphometric analysis, particularly retrospective analysis of CE images acquired in clinical routine, motivated us to explore the feasibility of deriving global and regional cortical thickness measures from contrast-enhanced MRI. We hypothesized that CE images are a challenge for processing with Freesurfer, resulting in an increased runtime and degraded quality of the reconstructed surface as indicated by the number of surface holes [bib_ref] Quality control strategies for brain MRI segmentation and parcellation: Practical approaches and..., Monereo-Sánchez [/bib_ref].
In this study, we propose an extension for DL+DiReCT [bib_ref] Direct cortical thickness estimation using deep learningbased anatomy segmentation and cortex parcellation, Rebsamen [/bib_ref] to cope with contrast-enhanced input data by .
Patients with RRMS were all under treatment with Natalizumab (Tysabri, Biogen Corp., Cambridge MA, USA), and therefore underwent regular MRI examinations at an interval of $6 months due to their increased risk of progressive multifocal leukoencephalopathy (PML). PPMS patients were examined on an annual interval on average. All images were acquired at the Bern University Hospital (Inselspital) on 1.5 T and 3 T scanners from Siemens (Siemens, Erlangen, Germany) with an MP-RAGE protocol [bib_ref] Brain morphometry with multiecho MPRAGE, Van Der Kouwe [/bib_ref]. Owing to the retrospective nature of the images acquired in clinical routine, sequence parameters were variable as detailed in Supplementary Figure S1. Contrast-enhanced images were acquired using an MP-RAGE sequence with a water excitation pulse [bib_ref] Optimizing 3D EPI for rapid t1-weighted imaging, Norbeck [/bib_ref] after administration of a contrast agent. Patients received an intravenous bolus injection of gadobutrol (0.1 ml/kg body weight Gadovist ® 1 mmol/ml, Bayer) or, in case of contraindications, gadoterate meglumine (0.2 ml/kg DOTAREM ® 0.5 mmol/ml, Guerbet).
## | mri processing
## | dl+direct
DL+DiReCT is an existing morphometry tool to derive global and regional volumes and cortical thickness measures from T1w MRI [bib_ref] Direct cortical thickness estimation using deep learningbased anatomy segmentation and cortex parcellation, Rebsamen [/bib_ref]. Its main processing steps include a deeplearning (DL) based segmentation of tissue classes and cortical parcellations followed by a diffeomorphic registration of the GM/WM boundary to the pial surface to derive a voxel-wise cortical thickness map (DiReCT) [bib_ref] Registration based cortical thickness measurement, Das [/bib_ref] [bib_ref] The ANTs cortical thickness processing pipeline, Tustison [/bib_ref].
A new model for the segmentation was added to DL+DiReCT by enriching the training data with CE images. Initially, DL+DiReCT was trained using a total of 840 non-enhanced T1w MRI, among them 128 images of patients with MS, and auxiliary labels from FreeSurfer, as detailed in [bib_ref] Direct cortical thickness estimation using deep learningbased anatomy segmentation and cortex parcellation, Rebsamen [/bib_ref]. These training data were now enriched with the corresponding 128 same-session CE MRI of these MS patients. Tissue class labels for the CE images were derived from the non-enhanced images by co-registering the image pairs with an affine transformation using FSL flirt [bib_ref] Improved optimization for the robust and accurate linear registration and motion correction..., Jenkinson [/bib_ref] [bib_ref] A global optimisation method for robust affine registration of brain images, Jenkinson [/bib_ref] The tool is publicly available (https://github.com/SCAN-NRAD/ DL-DiReCT) including the new models with the "--model v6" option for the Desikan-Killiany [bib_ref] An automated labeling system for subdividing the human cerebral cortex on MRI..., Desikan [/bib_ref] and "--model v7" for the Destrieux atlas [bib_ref] Automatic parcellation of human cortical gyri and sulci using standard anatomical nomenclature, Destrieux [/bib_ref]. Regional cortical thickness and GM volume estimates for the evaluation were generated from the original T1w images. After brain extraction using HD-BET [bib_ref] Automated brain extraction of multisequence MRI using artificial neural networks, Isensee [/bib_ref] , the new model of DL+DiReCT was applied using default settings (running on one GPU and four CPU cores on Linux).
## | freesurfer (fs)
All MRI were processed with FreeSurfer 6.0using the recon-all pipeline with default settings without manual interventions. Global and regional mean cortical thickness values were extracted from the surface statistics (lh.aparc.stats, rh.aparc.stats) for regions of interest (ROI) as defined by the Desikan-Killiany atlas [bib_ref] An automated labeling system for subdividing the human cerebral cortex on MRI..., Desikan [/bib_ref].
## | evaluation
For the evaluation, we processed all non-enhanced and CE images with FreeSurfer and DL+DiReCT as described above and extracted global and regional mean cortical thickness measures. Pearson correlation coefficients (r) were calculated between image pairs, both across all pairs (n = 454) as well as on the subset of pairs with identical sequence parameters (TI/TR, n = 213). To allow direct comparison with previous work [bib_ref] The effect of gadolinium-based contrast-agents on automated brain atrophy measurements by FreeSurfer..., Lie [/bib_ref] , intra-class correlation coefficients (ICC) were calculated based on mean-rating (k = 2), consistency, 2-way mixed-effects model [bib_ref] A guideline of selecting and reporting intraclass correlation coefficients for reliability research, Koo [/bib_ref] with the Rpackage irr.
Leveraging the longitudinal nature of the data, we calculated annual atrophy rates in mm/year by fitting a linear model to the measures of all time points for each patient with age as the covariate.
Patients with less than three time points were discarded, resulting in rates from n = 73 patients (eight PPMS and 65 RRMS). Group differences in the atrophy rates between the two MS cohorts were quantified with effects sizes using Cohen's d.
Finally, structural covariance networks (SCN) were constructed [bib_ref] Networks of anatomical covariance, Evans [/bib_ref] using the "brain connectivity toolbox" for Python [bib_ref] Complex network measures of brain connectivity: Uses and interpretations, Rubinov [/bib_ref]. ROI-wise cortical thickness measures were corrected for age by fitting linear models to each measure with age as co-variate. The resulting residuals were then used to calculate correlation matrices. Binary undirected graphs were derived by thresholding the correlation matrix at 0.01 intervals. Confidence intervals were determined with random sampling by repeatedly constructing SCNs from 80% of the images 1000 times. Given multiple sclerosis has been associated with SCN disruption [bib_ref] Disruption of structural and functional networks in longstanding multiple sclerosis, Tewarie [/bib_ref] , and since this work is not intended as a complete network study, we focused on the example of global efficiency [bib_ref] Efficient behavior of small-world networks, Latora [/bib_ref] as a metric of network integration to compare the two subgroups of MS patients. In contrast to the mean shortest path length, the global efficiency has a finite value also for disconnected graphs. Based on very recent findings in [bib_ref] Longitudinal cortical network reorganization in early relapsingremitting multiple sclerosis, Fleischer [/bib_ref] [bib_ref] Clinical relevance of cortical network dynamics in early primary progressive MS, Tur [/bib_ref] we speculated that SCNs could be differently affected in PPMS and RRMS.
Statistical analyses were performed using R with the stats package version 3.6.2 (R Core Team, 2019). A significance level α = 0.05 was used.
# | results
The median processing runtime of FreeSurfer was 9.3 h (range: 5.8-
## | annual atrophy rates
Both methods revealed an increased mean global annual atrophy rates for PPMS patients compared to the RRMS group, except FreeSurfer from CE images (see . The effect sizes were more pronounced with DL+DiReCT (non-enhanced = À0.304, CE = -0.169) than Free-Surfer (non-enhanced = À0.111, CE = 0.085). While these group effects were consistently larger than the differences between the two image types for DL+DiReCT, the group effects from FreeSurfer were similar or smaller in magnitude than between the image types (cf. columns d vs. last row in . Increased atrophy rates were also observed regionally for the PPMS group .
## | structural covariance networks (scn)
Analysis using SCN revealed group differences, reflecting a higher global efficiency metric for the PPMS group compared to RRMS . This observation was present for the networks constructed with DL+DiReCT from both image types, as well as with FreeSurfer from the non-enhanced images. In contrast, no such difference could be observed when calculated with FreeSurfer from the CE images.
Confidence intervals of the measures from DL+DiReCT were generally smaller than from FreeSurfer, as indicated for the networks constructed at a threshold of 0.5 (see right). F I G U R E 4 Mean annual regional atrophy rates in mm/year for the two MS subroups derived from non-enhanced and contrast-enhanced (CE) images with FreeSurfer and DL+DiReCT F I G U R E 5 Global efficiency metric for the PPMS and RRMS subgroups calculated from structural covariance networks derived from nonenhanced and contrast-enhanced (CE) images with FreeSurfer (FS) and DL+DiReCT (DL). Left: Global efficiency as a function of threshold applied on the correlation matrix. Right: Results corresponding to the threshold at 0.5. Error bars indicate one standard deviation without premature termination (with the exception of 17/454 images, i.e., 3.7%), we observed a substantially higher number of surface holes (Euler number) than for non-enhanced images. The Euler number can serve as a proxy for the input image quality [bib_ref] Common brain disorders are associated with heritable patterns of apparent aging of..., Kaufmann [/bib_ref] and higher values are generally considered an indicator for a lower quality of the reconstructed surface [bib_ref] Quality control strategies for brain MRI segmentation and parcellation: Practical approaches and..., Monereo-Sánchez [/bib_ref].
Consequently, the correlations of cortical thickness estimates between non-enhanced and the corresponding CE images were weaker (r = 0.75).
Previous work on morphometry from contrast-enhanced T1w
MRI is rare, especially for global and regional cortical thickness mea- This systematic bias was reduced to 0.06 mm using DL+DiReCT.
More pronounced global annual atrophy rates were observed for the PPMS patients compared to the RRMS group under treatment with Natalizumab, in line with expectations [bib_ref] Natalizumab may reduce cognitive changes and brain atrophy rate in relapsing-remitting multiple..., Portaccio [/bib_ref] [bib_ref] Effects of natalizumab and fingolimod on clinical, cognitive, and magnetic resonance imaging..., Preziosa [/bib_ref]. The rates of the RRMS group were similar to previously reported age-related atrophy of À0.004 mm/year in healthy cohorts [bib_ref] Normal agerelated brain morphometric changes: Nonuniformity across cortical thickness, surface area and..., Lemaitre [/bib_ref]. However, only the results from
DL+DiReCT consistently yielded larger effect sizes between those two groups than between the image types, likely due to the lower robustness of FreeSurfer on the CE images. Regional atrophy rates revealed a better consistency of the patterns between the two image types for DL+DiReCT than for FreeSurfer (cf. . In the RRMS group, the pattern suggested accelerated atrophy rates fronto-parietal as compared to temporal regions. For cortical thickness, less reliable measurement has been reported for the cingulate cortex [bib_ref] Influence of processing pipeline on cortical thickness measurement, Kharabian Masouleh [/bib_ref] [bib_ref] Direct cortical thickness estimation using deep learningbased anatomy segmentation and cortex parcellation, Rebsamen [/bib_ref]. We attribute the regions with a putative increase of cortical thickness over time to this kind of uncertainties, especially in the results derived with FreeSurfer from CE images and accentuated in the cingulate cortex and temporobasal and temporo-polar regions.
For SCNs, sizable uncertainties for edge weights and derived graph measures have recently been demonstrated, particularly when estimated from cortical thickness using FreeSurfer [bib_ref] Reliability and comparability of human brain structural covariance networks, Carmon [/bib_ref]. Our findings of smaller error bars when using DL+-DiReCT instead of FreeSurfer together with larger group separation capabilities strongly suggest DL-based input for SCN estimation as an alternative. Enhanced robustness of DL+DiReCT compared to Free-Surfer has been reported before [bib_ref] Direct cortical thickness estimation using deep learningbased anatomy segmentation and cortex parcellation, Rebsamen [/bib_ref] [bib_ref] A quantitative imaging biomarker supporting radiological assessment of hippocampal sclerosis derived from..., Rebsamen [/bib_ref]. SCN analysis revealed clear separation between the two subgroups of MS, with DL+DiReCT from both non-enhanced and CE images. Whether this finding generalizes remains to be investigated since our PPMS subgroup was small and literature about network efficiency in various phenotypes of MS is sparse [bib_ref] Longitudinal cortical network reorganization in early relapsingremitting multiple sclerosis, Fleischer [/bib_ref] [bib_ref] Structural cortical network reorganization associated with early conversion to multiple sclerosis, Tur [/bib_ref] [bib_ref] Clinical relevance of cortical network dynamics in early primary progressive MS, Tur [/bib_ref].
# | limitations
FreeSurfer failed to process 17 contrast-enhanced MRIs, and we have made no attempts to re-process these cases after manual interven- [bib_ref] Natalizumab may reduce cognitive changes and brain atrophy rate in relapsing-remitting multiple..., Portaccio [/bib_ref] [bib_ref] Effects of natalizumab and fingolimod on clinical, cognitive, and magnetic resonance imaging..., Preziosa [/bib_ref] , interpretation of regional atrophy patterns requires a critical appraisal of the results.
Due to the retrospective nature of this study and the fact that images were acquired in clinical routine over a period of 9 years including protocol and scanner upgrades, the data consist of gradientecho sequences with variations of sequence parameters and sources from different scanners. These variations likely result in lower correlations. However, even when comparing only pairs of MRI with identical parameters (TI/TR), the effect of considerably higher correlations with DL+DiReCT (r = 0.96) than for FS (r = 0.87) remained.
## | outlook
The ability to derive brain morphometry reliably from contrastenhanced T1w MRI will make additional data accessible for quantitative analysis. As a consequence, studies on retrospective clinical data might benefit from a larger sample size. In datasets with both nonenhanced and contrast-enhanced images, one could run the analysis twice with both image types, increasing statistical power. For future applications of morphometry on individual patients in clinical routine, running an analysis twice might increase confidence in the results.
While correlations between non-enhanced and CE images were excellent, there remains a systematic bias in their absolute values.
Whether mixing measures from non-enhanced and CE images in the same analysis is feasible, e.g., by applying a correction factor, remains to be investigated.
# | conclusions
With the proposed deep learning-based morphometry tool (DL+DiReCT), brain morphometry can be derived reliably from contrast-enhanced T1-weighted MRI. The main findings (effect sizes of atrophy rates between groups and network effects from SCN) in the analyzed cohorts were consistent between the non-enhanced and contrast-enhanced images.
# Acknowledgments
# Data availability statement
The morphometry tool is publicly available, including trained models
## Orcid
## Michael rebsamen
https://orcid.org/0000-0002-8441-1485
Christian Rummel https://orcid.org/0000-0003-2345-7938 |
An innate IL-25-ILC2-MDSC axis creates a cancer-permissive microenvironment for Apc-mutation-driven intestinal tumorigenesis
[fig] Figure S1: High tumor IL25 but not IL33 expression is associated with poor survival in human CRC patients. (A) Overall survival of CRC patients from the TCGA Firehose Legacy microarray dataset, stratified into two groups by tumor IL25 expression. IL25-high and IL25-low groups are defined as patients with CRC that had tumor IL25 expression above and below the mean of the total samples, respectively. (B) Graph shows relapse-free survival of CRC patients from the large publicly available RNA dataset by Marisa et al. 2013 (20), analyzed through the R2 platform (http://r2.amc.nl), and stratified into two groups defined as above (IL25-high) or below (IL25-low) the overall cohort tumor IL25 mean expression. (C) Weight and size measurements of CRC tumor samples from the TCGA Firehose Legacy microarray dataset, in the IL25-high and IL25-low groups defined as above and below the mean cohort tumor IL25 expression respectively. (D) Overall survival of the IL25-high group (above cohort mean) from the TCGA dataset, further segregated into top and bottom half based on tumor IL25 gene expression. (E and F) Overall (E) and disease-free (F) survival of CRC patients from the TCGA dataset, stratified into IL33-high or IL33-low group based on tumor IL33 expression (IL33-high, above cohort mean; IL33-low, below cohort mean). (G) Gating strategy of CD8 + T cells and Th1 cells in human CRC and adjacent normal gut. SB645, Super Bright 645 fluorochrome. Statistical significance determined by log-rank test on cBioPortal (A, D, E and F) or R2 platform (B), and Wilcoxon test on cBioPortal (C). [/fig]
[fig] Figure S2: Immunosuppressive tumor microenvironment in Apc 1322T/+ mouse intestinal tumors. (A) Representative images (left, 10x magnification, scale bar 150 µm) of intestinal tumor (dotted line) and tumor infiltrating ILC2s (right, 40x magnification, scale bar 20 µm) from Apc 1322T/+ mice. (B) FACS plots show gating strategy of ILC2s in Apc 1322T/+ mice. (C) Plot shows ST2 (using antibody clone DJ8) expression on total ILCs in intestinal tumors and lung from Apc 1322T/+ mice. (D) FACS plots show gating strategy (top) and representative immunofluorescence images (bottom, 40x magnification, scale bar 20 µm) of tumor M-MDSCs and G-MDSCs. (E) Percentage MHC-II expression on adjacent lamina propria (Adj LP) and tumor M-MDSCs in Apc 1322T/+ mice (n = 7). (F) FACS plots show gating strategy of gd, CD4 + and CD8 + T cells while graph shows Th1 cell frequency in tumor and adjacent epithelium (Adj epi) (n = 8), and immunofluorescence images show tumor infiltrating CD8 T cells (40x magnification, scale bar 20µm). (G and H) gating strategy (G) and frequency (H) of NK cells in indicated tissues from Apc 1322T/+ mice (n = 7). (I) Frequency of gd T cells in and tumor in Apc 1322T/+ mice (n = 5). (J) Granzyme, perforin and IFNg expression in CD8 + T cells in tumor and Adj epi from Apc 1322T/+ mice (n = 8). (K) Granzyme, perforin and IFNg expression in gd T cells in tumor and Adj epi from Apc 1322T/+ mice (n = 6). Data pooled from two or more independent experiments and error bars show mean ± SEM. Statistical significance determined by paired two-tailed t-test, except in (H) by one-way ANOVA with Tukey's post hoc. n.s. non-signifiacnt, [/fig]
[fig] Figure S3: IL-25 promotes tumors in Apc 1322T/+ mice. (A to C) Number of tumors per centimeter intestinal tissue (A) and average tumor size (B) (vehicle, n = 7; rIL-25, n = 9), and frequency of Ki67 + EpCAM + tumor cells (C) (vehicle, n = 7; rIL-25, n = 8), in vehicle and rIL-25 treated Apc 1322T/+ mice. (D) Representative FACS plots showing tumor ILC2s in vehicle and rIL-25 treated Apc 1322T/+ mice. (E and F) Frequency of Th1 cells (E), M-MDSCs and G-MDSCs (F) in vehicle and rIL-25 treated Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 8). (G to J) Gating strategy (G) and frequency of macrophages and conventional dendritic cells (cDC) (H), plasmacytoid dendritic cells (pDC) (I), and mast cells (J), in vehicle and rIL-25 treated Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 8). (K) Frequency of tumor gd T cells in vehicle and rIL-25 treated Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 8). (L) Gating strategy of tumor Th1 cells, Th2 cells and Tregs, and frequency of Tregs, in vehicle and rIL-25 treated Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 8). (M) Frequency of tumor Th2 cells in vehicle and rIL-25 treated Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 8). Data pooled from two or more independent experiments and error bars show mean ± SEM. Statistical significance determined by unpaired two-tailed t-test. n.s. non-signifiacnt, *P < 0.05. [/fig]
[fig] Figure S4: IL-25-deficient Apc 1322T/+ mice show prolonged survival. (A) Schematic of Il25-tomato construct and Southern analysis. (B) Representative FACS plots showing DCLK1 + tuft cell frequency within Il25-tomato-expressing CD45 -EpCAM + tumor epithelial cells in Apc 1322T/+ mice. (C) Survival of Il25 +/+ and Il25 tom/tom Apc 1322T/+ male mice. (D to K) Frequency of tumor gd and CD8 + T cells (Il25 +/+ , n = 20; Il25 tom/tom , n = 11) (D), Th1 cells and Tregs (Il25 +/+ , n = 10; Il25 tom/tom , n = 11) (E), macrophages (F) and conventional dendritic cells (cDC) (Il25 +/+ , n = 8; Il25 tom/tom , n = 9) (G), plasmacytoid dendritic cells (pDC) (Il25 +/+ , n = 15; Il25 tom/tom , n = 10) (H), mast cells (Il25 +/+ , n = 8; Il25 tom/tom , n = 9) (I), NK cells (Il25 +/+ , n = 8; Il25 tom/tom , n = 9) (J), and Th2 cells (Il25 +/+ , n = 10; Il25 tom/tom , n = 11) (K) in Il25 +/+ and Il25 tom/tom Apc 1322T/+ mice. (L) Frequency of tumor ILC2s in control (Il33 +/+ ) and IL-33-deficient (Il33 cit/cit ) Apc 1322T/+ mice (Il33 +/+ , n = 11; Il33 tom/tom , n = 16). (M) Number of tumors and average tumor size in control (Il33 +/+ ) and IL-33-deficient (Il33 cit/cit ) Apc 1322T/+ mice (Il33 +/+ , n = 8; Il33 tom/tom , n = 15). (N and O) Frequency of tumor Tregs (N) (Il33 +/+ , n = 10; [/fig]
[fig] Figure S5: Ablation of ILC2s protects from intestinal tumors and prolongs survival. (A) Representative FACS plots (left) and frequency (right) of ILC2s in ILC2-replete Cre-control (Rora +/+ Il7r Cre/+ or Rora +/f Il7r Cre/+ ) and ILC2-deficient (Rora f/f Il7r Cre/+ ) Apc 1322T/+ mice (Cre-control, n = 6; ILC2-deficient, n = 11). (B) Survival of ILC2-deficient and Cre control Apc 1322T/+ male mice. (C) Frequency of tumor CD4 + T cells and Tregs in Rora f/f Cd4 Cre/+ (n = 6) and control Rora +/f Cd4 Cre/+ (n = 7) Apc 1322T/+ mice. (D) Number of tumors and average tumor size of Rora f/f Cd4 Cre/+ (n = 9) and control [/fig]
[fig] Figure S6: ILC2 deficiency does not affect tumor B cell infiltration. (A) UMAP plot of single-cell RNAseq (scRNAseq) analysis showing expression of indicated genes (encoded protein shown in brackets). (B) UMAP plot of scRNAseq analysis of tumor epithelial cells (C1; CD45 -EpCAM + ), M-MDSCs (C3; CD45 + CD11b + Ly6C + Ly6G -) and total immune population (remaining clusters; CD45 + ) sorted from intestinal tumors from ILC2-deficient and Cre control Apc 1322T/+ mice. (C) Frequency of tumor B cells in Cre control and ILC2-deficient Apc 1322T/+ mice from flow cytometry analysis (Cre control, n = 6; ILC2-deficient, n = 12). (D) Representative FACS plots showing tumor B cells in individual Apc 1322T/+ mouse tumors. (E and F) Heatmap (E) and KEGG pathway analysis (F) of differentially expressed genes identified through scRNAseq, between tumor epithelial cells from ILC2-deficient and Cre control Apc 1322T/+ mice. (G) Plot shows differentially expressed genes identified through scRNAseq, by tumor CD8 + T cells (C2) from ILC2-deficient Apc 1322T/+ mice relative to Cre-control Apc 1322T/+ mice. Data representative of, or pooled from, two or more independent experiments and error bars show mean ± SEM (C). Statistical significance determined by unpaired two-tailed t-test (C), and (E and F) using the10x Genomics Loupe Browser and Enrichr. n.s. non-signifiacnt. [/fig]
[fig] Figure S7: Ablation of ILC2s improves anti-tumor immunity. (A) Frequency of tumor Th1 cells (Cre control, n = 6; ILC2-deficient, n = 8) in ILC2-deficient and Cre control Apc 1322T/+ mice. (B and C) Frequency (Cre control, n = 6; ILC2-deficient, n = 12) (A), and granzyme, perforin and IFNg expression (Rora +/+ Il7r +/+ control, n = 7; ILC2-deficient, n = 6) (B), in tumor gd T cells from ILC2deficient and control Apc 1322T/+ mice. (D) Frequency of tumor Th2 cells (Cre control, n = 6; ILC2deficient, n = 8) in ILC2-deficient and Cre control Apc 1322T/+ mice. (E to I) Frequency of macrophages (E), conventional dendritic cells (cDC) (F), plasmacytoid dendritic cells (pDC) (G), mast cells (H), and NK cells (I), in ILC2-deficient and control Apc 1322T/+ mice (Cre control, n = 6; ILC2-deficient, n = 12). (J) Frequency of tumor Tregs in ILC2-deficient and control Apc 1322T/+ mice (Cre control, n = 6; [/fig]
[fig] Figure S8: IL-33 treatment in ILC2-deficient mice increases intestinal tumorigenesis. (A) Representative FACS plots showing (lack-of) ILC2s in vehicle and rIL-25 treated ILC2-deficient Apc 1322T/+ mice. (B to I) Frequency of tumor CD8 + , gd and Th1 T cells (B), Th2 cells (C), macrophages (D), conventional dendritic cells (cDC) (E), plasmacytoid dendritic cells (pDC) (F), mast cells (G), Tregs (H), and M-MDSCs and G-MDSCs (I), in vehicle and rIL-25 treated ILC2deficient Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 7). (J) Number of tumors and average tumor size in vehicle and rIL-33 treated ILC2-deficient Apc 1322T/+ mice (vehicle, n = 7; rIL-25, n = 6). (K)Frequency of tumor Tregs and mast cells in vehicle and rIL-33 treated ILC2-deficient Apc 1322T/+ mice(vehicle, n = 7; n = 6). Data pooled from two or more independent experiments and error bars show mean ± SEM. Statistical significance determined by unpaired two-tailed t-test. n.s. nonsignifiacnt, *P < 0.05, **P < 0.01. [/fig]
[fig] Figure S9: ILC2-derived IL-4 and IL-13 promotes M-MDSC-mediated CD8 + T cell suppression. (A) Representative FACS plot and quantification (relative gMFI) of IL-4Ra receptor expression on G-MDSCs from Apc 1322T/+ mice (n = 5). Relative gMFI, geometric mean fluorescent intensity relative to isotype control. (B) Representative FACS plot showing IL-13Ra1 receptor staining on G-MDSCs from Apc 1322T/+ mice. (C) Representative FACS plots showing Arginase 1 (Arg1) expression in tumor M-MDSCs from ILC2-deficient and Cre control Apc 1322T/+ mice. (D) Representative FACS plots showing IL-4 and IL-13 expression in rIL-25-stimulated cultured intestinal ILC2s. (E) Representative FACS plots showing IFNg (top) and granzyme B (bottom) expression in CD8 + T cells cultured alone or with tumor M-MDSCs, in the presence of conAb-ILC2-SNT. (F) TNFa and perforin expression in CD8 + T cells cultured alone or with tumor M-MDSCs, in the presence of aIL-4/13Ab-ILC2-SNT or conAb-ILC2-SNT (n = 6). (G) Quantification of CD8 + T cell proliferation measured by average number of divisions, fold expansion, and percentage undivided, when cocultured with tumor M-MDSCs treated with aIL-4/13Ab-ILC2-SNT or conAb-ILC2-SNT (n = 6). aIL-4/13Ab-ILC2-SNT and conAb-ILC2-SNT, ILC2-supernatent (ILC2-SNT) pre-treated with anti-IL-4 and anti-IL-13 [/fig]
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VEGF and VEGFR2 bind to similar pH-sensitive sites on fibronectin, exposed by heparin-mediated conformational changes
# Results
## Equilibrium binding by enzyme-linked immunoassay (elisa)based experiments
To study the interactions between fibronectin and the angiogenic factors VEGF 165 (termed VEGF for simplicity) and ECD in a simple and fast way that would not require protein labeling, we developed an ELISA-based solid-phase binding assay. Fibronectin was adsorbed on 96-well plates and allowed to interact with the ligand (VEGF or ECD) until equilibrium was reached. The fraction of the ligand that was specifically bound to fibronectin was then extracted with a solution of high ionic strength (5 M NaCl), adsorbed onto a second 96-well plate, and detected using a primary antibody against the ligand and a corresponding HRP-labeled secondary antibody. The signal was detected by luminescence. The ligand extraction step was necessary because of the high levels of VEGF nonspecific binding, which could not be reduced by blocking agents either added in the binding buffer or adsorbed on the plate. The steps for the assay optimization are described in Figs. S1-S6.
Using this assay, we evaluated the binding of VEGF and ECD to surface-immobilized fibronectin. VEGF binding followed the same pattern as previously reported, with higher binding occurring at acidic pH only after fibronectin was treated with heparin . The measured values reflect the VEGF fraction specifically bound to fibronectin, since no VEGF was extracted from the naked substrate or from adsorbed bovine serum albumin (BSA). Interestingly, a very similar binding pattern was observed with ECD . For both ligands, significant binding was observed at pH 5.5 after incubating the surface-immobilized fibronectin with heparin. Thus, we chose this condition to further analyze the protein interactions.
Dose-response experiments showed that ECD binding to surface-immobilized fibronectin reached saturation at significantly lower concentrations than those for VEGF, suggesting that ECD binds to fibronectin with higher affinity than VEGF . This conclusion is further reinforced if we consider that the binding was performed with equal masses for the two ligands, which, given the difference in molecular weight between VEGF (45 kDa) and ECD (83 kDa), means lower molarity for ECD. We did not attempt to calculate affinities at this stage, because of the many intermediate assay steps between the binding event and the final measurement. However, we observed significantly lower luminescence values for ECD than for VEGF. These differences could originate from different recognition of VEGF and ECD by the primary antibody and/or different degrees of adsorption of the two proteins. Indeed, calibration curves revealed different signals for the same amount of VEGF and ECD, which depended on both the ionic strength of the buffer and the protein concentration. Taken all this into account, it appears that ECD possesses higher affinity for fibronectin than VEGF, but the absolute amount of VEGF and ECD bound to fibronectin cannot be determined by this experimental setup.
Since VEGF and ECD bind to fibronectin following a very similar pattern, we wanted to elucidate whether they share the same binding sites and if their binding is mutually exclusive. To do so, we compared the combined binding of VEGF and ECD to fibronectin when they were added simultaneously or sequentially, using an anti-His antibody that can recognize both ligands (as they both contain a His-tag; see Experimental Procedures) or an anti-VEGFR2 antibody that recognizes specifically ECD. We confirmed that both the anti-His and anti-VEGFR2 antibodies could detect their respective ligands when bound individually to fibronectin. When both VEGF and ECD were added to fibronectin, the signal detected Added ECD (μg/ml) FN BSA . Acidic pH and pretreatment of fibronectin with heparin increase VEGF and ECD binding. A and B, Fibronectin (dark green/blue bars) or bovine serum albumin (BSA) (light green/blue bars) was adsorbed on 96-well polystyrene plates (20 μg/ml; 50 μl/well) overnight at 4 C. The plate was incubated for 1 h on ice with 5 μg/ml VEGF (A) or ECD (B) in binding buffer (150 mM NaCl, 25 mM Hepes) at pH 7.5 or 5.5, in the absence (control) or presence (heparin) of pretreatment of the adsorbed proteins with 100 μg/ml heparin in PBS (1 h on ice). Bound VEGF or ECD was extracted with 5 M NaCl, 25 mM Hepes, pH 7.5 for 1 h on ice, readsorbed on a second plate, and detected by ELISA with an anti-His primary antibody (1:1000) and an HRP-labeled secondary antibody . Both antibody incubations were performed in ELISA blocking buffer (1 mg/ml BSA + 0.05% Tween20 in PBS). Samples were measured in quadruplicate, and the data are presented as mean values ± standard deviation. C and D, Fibronectin (dark green/blue lines) or BSA (light green/ blue lines) was adsorbed on 96-well polystyrene plates (20 μg/ml; 50 μl/well) overnight at 4 C. The plate was incubated for 1 h on ice with increasing concentrations of VEGF (C) or ECD (D) in binding buffer (150 mM NaCl, 25 mM Hepes) at pH 5.5 in the presence of pretreatment of the adsorbed proteins with 100 μg/ml heparin in PBS (1 h on ice). VEGF and ECD extraction and detection were performed as in (A) and (B). Samples were measured in quadruplicate, and the data are presented as mean values ± standard deviation.
by the anti-His antibody was always higher than if VEGF or ECD was added alone, irrespectively of the order of ligand addition, suggesting that their binding to fibronectin is not mutually exclusive. The anti-VEGFR2 antibody detected the presence of ECD under all conditions, except when ECD was added before VEGF. Since no binding scenario predicts the complete absence of ECD under this condition, this result suggests that when fibronectin-bound ECD is allowed to interact with VEGF as well, the epitope for this particular antibody is masked. The only possibility consistent with all these observations is that VEGF and ECD are likely to share binding sites on fibronectin, and when bound to fibronectin, they retain their ability to recognize each other and lead to the formation of a VEGF/ECD/fibronectin triple complex. Both VEGF and ECD could be released from fibronectin by changing the pH from 5.5 to 7.5, even if the ionic strength of the extraction solution was not increased . Indeed, gradually increasing the ionic strength of the extraction buffer did not result in higher release . This suggests that the interaction depends on pH-sensitive amino acids. The release was very fast, occurring within a few minutes after buffer change. Interestingly, longer extraction times led to a decrease in the amount of released VEGF or ECD , which may be the result of ligand readsorption .
## Structural determinants of the interactions
Circular dichroism (CD) spectra of VEGF and ECD were acquired at pH 7.5 and pH 5.5. The ionic strength of the buffer prevented data collection below 205 nm, and at low ionic strength buffers, the structure of VEGF was altered, suggesting significant unfolding . Therefore, the spectra could not be subjected to structure deconvolution with certainty, especially, since both VEGF and ECD are beta-sheet dominated and the CD spectra of beta-sheet proteins show significant variability, rendering their deconvolution Fibronectin was adsorbed on 96-well polystyrene plates (20 μg/ml; 50 μl/ well) overnight at 4 C. The plate was incubated for 1 h on ice with 50 μg/ml of VEGF or ECD alone or in combination (VEGF + ECD). Additionally, some wells were treated first with 50 μg/ml of VEGF or ECD for 1 h, followed by a second 1 h incubation with 50 μg/ml ECD or VEGF, respectively (conditions labeled as '1) VEGF 2) ECD' and '1) ECD 2) VEGF', respectively). Ligand binding was studied in binding buffer (150 mM NaCl, 25 mM Hepes) at pH 5.5 after pretreatment of the adsorbed fibronectin with 100 μg/ml heparin in PBS (1 h on ice). Bound VEGF and/or ECD were extracted with 5 M NaCl, 25 mM Hepes, pH 7.5 for 1 h on ice, readsorbed on a second plate, and detected by ELISA with an anti-His or an anti-VEGFR2 primary antibody (1:1000) and an HRP-labeled secondary antibody . Both antibody incubations were performed in ELISA blocking buffer (1 mg/ml bovine serum albumin + 0.05% Tween20 in PBS). Samples were measured in quadruplicate, and the data are presented as mean values ± standard deviation. . Switching the pH from acidic (5.5) to neutral (7.5) is sufficient to release fibronectin bound VEGF and ECD. A, Fibronectin was adsorbed on 96-well polystyrene plates (10 μg/ml; 50 μl/well) overnight at 4 C. The plate was incubated for 1 h on ice with 50 μg/ml of VEGF (green) or ECD (blue) alone or in combination (VEGF + ECD) in binding buffer (150 mM NaCl, 25 mM Hepes) at pH 7.5 or 5.5 after pretreatment of the adsorbed fibronectin with 100 μg/ml heparin in PBS (1 h on ice). Bound VEGF and ECD were extracted with binding buffer (150 mM NaCl, 25 mM Hepes) at pH 7.5 or 5.5, for 1 h on ice, readsorbed on a second plate, and detected by ELISA with an anti-His primary antibody (1:1000) and an HRP-labeled secondary antibody . Both antibody incubations were performed in ELISA blocking buffer (1 mg/ml bovine serum albumin + 0.05% Tween20 in PBS). Samples were measured in quadruplicate, and the data are presented as mean values ± standard deviation. B, Fibronectin was adsorbed on 96-well polystyrene plates (10 μg/ml; 50 μl/well) overnight at 4 C. The plate was incubated for 1 h on ice with 50 μg/ml of VEGF (green) or ECD (blue) in binding buffer (150 mM NaCl, 25 mM Hepes) at pH 5.5 after pretreatment of the adsorbed fibronectin with 100 μg/ml heparin in PBS (1 h on ice). Bound VEGF and ECD were extracted with binding buffer (150 mM NaCl, 25 mM Hepes) at pH 7.5 or 5.5, or with 5 M NaCl, 25 mM Hepes, pH 7.5 for 5, 15, 30 or 60 min (each time interval in a different set of wells) on ice, readsorbed on a second plate, and detected by ELISA with an anti-His primary antibody (1:1000) and an HRP-labeled secondary antibody . Both antibody incubations were performed in ELISA blocking buffer (1 mg/ml bovine serum albumin + 0.05% Tween20 in PBS). Samples were measured in quadruplicate, and the data are presented as mean values ± standard deviation.
VEGFR2 binding to fibronectin problematic. However, the spectra at pH 7.5 and 5.5 were very similar, suggesting that the pH does not cause a major change in the protein secondary structure. If there is a pHsensitive conformational change of VEGF and ECD, it should be slight or local. Therefore, it is more likely that the increased binding observed at pH 5.5 would depend on the protonation state of critical amino acids in the binding site rather than large conformational changes. We decided to further study this point, by computing the pH dependance of the protonation state of surface amino acids for both the ligands (VEGF and ECD) and the domain of fibronectin where the binding is likely to happen (FNIII 12-14) (7).
Based on the known structures of VEGF and ECD, we calculated the pKa values of surface-exposed amino acids, and we identified several residues with a pKa value between 5.5 and 7.5, whose protonation state would change between these two pH valuesand thus, could belong to the fibronectin-binding sites on each molecule. In the case of ECD, most of the candidate residues were His residues, which often function as pH sensors. Whereas for ECD almost all candidate residues seem to be free of interactions with the ligand, the majority of the pH-sensitive residues on VEGF were on the receptor-binding domain and were involved in interactions in the VEGF dimer or between VEGF and the Structure-based predictions of pKa values of individual amino acid residues at the surface of VEGF and ECD were performed with the DelPhiPKa web server, using the known high-resolution structures of VEGF and ECD fragments (pdb files used: 2VPF and 2VGH for VEGF and 3V2A, 2X1W, 2X1X, 3S35, 3S36, 3S37, 5OYJ, and 3KVQ for ECD). It is also indicated whether the side chains of the candidate amino acids are free from known interactions (hydrogen bonds and salt bridges) with other parts of the protein or holding together the VEGF/ECD complex.
VEGFR2 binding to fibronectin receptor, leaving free of interactions only a His residue at the C-terminal domain of VEGF. However, since full-length structures are lacking for both VEGF and ECD, these results should be interpreted with caution since prediction of pKa values is very sensitive to the local environment of the amino acid. Because of the pH sensitivity of His residues, we would like to mention that experiments with nontagged VEGF conducted in an earlier study showed the same binding profile. Moreover, His-tagged VEGF-E showed no binding to heparin-treated fibronectin at acidic pH (data not shown). These observations support the conclusion that the His tag in the recombinant proteins used in this study is unlikely to affect the enhanced binding observed at acidic pH.
As mentioned above, we performed a similar analysis on the C-terminal 40 kDa fragment of fibronectin encompassing domains FNIII 12-14, which was identified earlier as the VEGF-binding site on fibronectin. Interestingly, no pHsensitive residues with a pKa value between 5.5 and 7.5 were identified in this region. We run the analysis using two different conformations reported for this fibronectin domain (pdb codes: 1FNH and 3R8Q), obtaining almost identical results: 22 acidic residues (pKa < 4), 32 basic residues (pKa > 10), and no exposed pH-sensitive residues. This suggests that, after being exposed by the heparin action, this binding site on fibronectin is not regulated further by changing the local pH. Instead, it remains always available for binding, which occurs only when key amino acids in the ligand become protonated at acidic pH.
## Mechanistic insights on the interactions by spr kinetic experiments
To further understand mechanistically the interactions between fibronectin and VEGF/ECD, we followed the kinetics of the interactions by surface plasmon resonance (SPR). Fibronectin was immobilized via N-hydroxysuccinimide (NHS) chemistry on the surface of the carboxymethyldextran hydrogel of the biosensor chip . VEGF or ECD was added in the mobile phase under flow. Based on the results of the equilibrium studies, all experiments were performed at pH 5.5. Binding in the absence of any heparin treatment of the surface-immobilized fibronectin was minimal, even at a high fibronectin density . We tested the effect of injecting 1 mg/ml BSA into both flow cells, which could act as a blocking protein, reduce nonspecific binding of VEGF to both flow cells, and unmask specific associations between VEGF and the immobilized fibronectin. However, the low levels of VEGF binding persisted. Interestingly, BSA injection stabilized the sensogram values for background VEGF binding after repeated injection and regeneration cyclesand was, therefore, employed in all subsequent experiments. Heparin treatment of the immobilized fibronectin was thus necessary in order to observe any appreciable binding. Based on the resulting resonance units (RU) values following heparin treatment, we chose an intermediate fibronectin density (2 μg/ ml) to get values within the recommended range for SPR experiments .
To test the action of heparin in the SPR setup, 10x molar excess of heparin was injected prior to the binding assay, was allowed to associate with the surface-immobilized fibronectin for 1 min and then dissociate for increasing periods of time (0, 10, or 30 min) by changing accordingly the flow rateand B). This experimental design was based on a previous study, describing the catalytic mechanism by which heparin mediates the conformational change on fibronectin that exposes the VEGF-binding sites. According to this model, fibronectin-heparin interactions are transient and characterized by multiple rebinding and release events, resulting in a low amount of bound heparin at equilibrium. In the SPR setup, heparin rebinding could occur during the long dissociation phase, increasing the contact time between heparin and fibronectin and resulting in maximal conversion of fibronectin to the open form, exposing the binding sites. At the same time, the longer dissociation times would allow for a more complete heparin release, thus, minimizing the amount of heparin present on the surface-immobilized fibronectin during the binding assay with VEGF/ECD. Unfortunately, an estimation of the amount of heparin bound to fibronectin directly from the SPR sensograms was not possible, because of the low signal generated by this amount of heparin (Figs. S13 and S14), further demonstrating the low levels of heparin binding to fibronectin. However, the VEGF sensograms show that the duration of the dissociation step following heparin treatment did not affect VEGF binding. This supports the idea that a 1-min association time was sufficient for this amount of heparin (1 μg/ml) to convert the immobilized fibronectin to the open conformation (this structural rearrangement has been reported earlier to be fastand that the subsequent VEGF binding occurs on fibronectin and not on any residual heparin remaining bound to fibronectin. Interestingly, ECD binding stabilized only after at least 10 min of heparin dissociation. If no dissociation was performed following heparin treatment, there was no ECD binding. This would be explained if ECD shared binding sites with heparin, and in order to observe appreciable binding, heparin must be completely released. Based on these results, we performed all further experiments after heparin treatment of the surfaceimmobilized fibronectin by 1 min association, followed by 10 min dissociation.
To extract kinetic rates, we collected association and dissociation data with a concentration series of VEGF or ECD. Already a visual inspection of the binding curves, prior to any attempt to fit a model for the macromolecular interaction, reveals certain characteristics. First, there was a very fast initial burst during the association phase, especially evident at higher VEGF/ECD concentrations, followed by a slower increase. Second, dissociation was slow, resulting in a significant amount of ligand remaining bound at the end of the experiment, which was increased with VEGF/ ECD concentration.
A conventional one-site modelfailed to fit the data (section 6 in Experimental Procedures; Figs. S15 and S16), even when considering mass transfer effects. Given the VEGFR2 binding to fibronectin observed slow dissociation, we considered a model whereby the ligand upon dissociation from one receptor molecule performs a random walk across the surface and rebinds to neighboring receptor molecules multiple times before diffusing back to the bulk phase. However, this model also failed to capture the binding behavior, as it could not account for the fact that the dissociation rate depended on the VEGF/ ECD concentration. We considered also models whereby each monomeric chain of fibronectin possesses two binding sites for VEGF or ECD, which could act independently (parallel reactions model) or affect each other (consecutive reactions model). Although the consideration of two binding sites improved somewhat the fitting, there were still significant deviations from the experimental data (Figs. S15 and S16). The model that generated the best fit was the twosites model, assuming two populations of fibronectin molecules, each possessing a single binding site per monomeric chain that interacts with the ligand (VEGF or ECD) with distinct association/dissociation rate constants, C and D;. Interestingly, only VEGF binding required consideration of mass transfer phenomena for better fitting, which may be related to the faster association rates for VEGF than ECD, rendering the process diffusion limiting. The kinetic parameters governing fibronectin-VEGF/ECD interactions extracted from the two-sites model are summarized in. Instead of single values, we report intervals, which were either calculated during fitting for the parameters extracted directly from the model or derived from these values for the K d estimates.
Statistical analysis indicates that the discrepancies observed between experimental and fitted data can be ascribed to experimental error (section Statistical analysis of curve fitting in Experimental Procedures; Figs. S18 and S19;. Additional uncertainties in parameter estimation could originate from the limited number of experimental points during the initial association burst. However, it is possible that a simple two-sites model fails to describe certain aspects of the macromolecular interactions, such as rebinding events, and it should be considered only as an approximation of the experimental observations. . Mean values from triplicate measurements are shown, with the shaded area representing the standard deviation. Following association, VEGF and ECD were allowed to dissociate for 720 s. The different VEGF/ ECD concentrations were injected following a random order in order to ensure that the measurements were free of systematic errors, and the progressive increase in VEGF/ECD binding with increasing concentration reflected the true binding. The surface was regenerated with 2 M NaCl and 0.05 N NaOH after every VEGF/ECD binding cycle. The experimental data (solid lines) were fitted to a two-sites model (dashed lines) considering mass transfer for VEGF (C) and rapid mixing for ECD (D).
## Vegfr2 binding to fibronectin
Discussion VEGF binding to the extracellular matrix has been recognized as an important event regulating angiogenesis. Previous studies have shown that fibronectin, a major component of the extracellular matrix, possesses cryptic binding sites for VEGF, which become available when fibronectin undergoes a conformational change, catalyzed by heparin and heparan sulfate chains within the extracellular matrix. VEGF binding to these sites is enhanced at acidic pH. Here, we report for the first time a similar class of heparin-sensitive binding sites on fibronectin that interact with the ECD of VEGFR2, also at acidic pH. VEGFR2 is a major cell-surface receptor for VEGF, and it is possible that interactions between VEGF, VEGFR2, and fibronectin are important for the regulation of VEGF/VEGFR2 signaling, which is critical for angiogenesis. In this study, we developed assays in order to characterize these protein-protein interactions by equilibrium and kinetic studies in vitro. (2) a narrow volume slit (V i ) in the proximity of the surface (S) where receptors (fibronectin) have been immobilized at an initial concentration R T . Ligand molecule exchange occurs between the flowing bulk phase and V i (mass transfer), as well as between V i and S (binding/unbinding events). For simplicity, the two monomeric chains of a fibronectin molecule are considered to act independently, and the surface-immobilized receptors depicted correspond to each monomeric chain and not to the complete fibronectin dimer. Four different models were used to describe ligand-receptor interactions, generating ligand-bound (B) receptor molecules: the one-site model, assuming one ligand binding site per receptor; the two-sites model, assuming a heterogeneous receptor population composed of two types of molecules, each with a single binding site with distinct association/dissociation kinetics; the parallel reactions model, assuming that each receptor contains two ligand-binding sites that act independently; and the consecutive reactions model, assuming that each receptor contains two allosterically linked ligand binding sites, where a second ligand can bind only after the first site has already been engaged. From a modeling perspective, we consider the concentration of binding sites for the one-site (B), two-sites (B 1 and B 2 ), and parallel reactions (B 1 and B 2 ) models, and the concentration of the various receptor molecular species (R, R A , and R 2A ) for the consecutive reactions model. The experimental data presented on, C and D for the interaction between fibronectin and VEGF or ECD were fitted with the models shown on, and the fits that generated well-defined solutions (Figs. S15 and S16 andwere evaluated with measures for absolute fit (RMS and σ 2 ) and model parsimony (AIC), as described in the section. Statistical analysis of curve fitting' under Experimental Procedures. The number of data points is lower for ECD than for VEGF, since the curve corresponding to 1000 nM ligand was not considered for the fitting in the case of ECD because the interaction reached saturation already at 500 nM ligand and additionally, the data associated with 1000 nM ECD displayed a large experimental error. The number of parameters for each model is derived from, and the corresponding parameter estimates are shown in.
## One-site model
## Vegfr2 binding to fibronectin
For equilibrium studies, we followed an ELISA-based approach, which does not require ligand labeling. This not only increases the versatility and simplicity of the assay, but also circumvents the need for testing the potential effects of labeling on the structure, activity, and stability of the ligand. However, we noticed high levels of nonspecific VEGF binding to the plate surface, which masked the specifically bound VEGF to fibronectin and prevented its direct detection. Several blocking protein-based agents (BSA, egg white albumin, betalactoglobulin, gelatin, hemoglobin, and milk) were tested, either by coadsorption on the substrate or by inclusion in the binding buffer, but none could suppress VEGF nonspecific binding consistently. Therefore, we used an indirect approach, whereby we extracted the bound ligand by either increasing the ionic strength of the buffer or changing the pH back to neutral, readsorbed it on another assay plate, and detected it with a typical ELISA. Negative controls with the uncoated substrate or with adsorbed BSA instead of fibronectin confirmed that the extraction step released only the fraction of the ligand that interacted specifically with fibronectin .
In our experiments, we used recombinant VEGF and ECD proteins carrying a His-tag, which allowed the detection of both proteins by the same anti-His primary antibody, rendering the assay uniform. However, the assay worked well also with ligand-specific antibodies. A drawback of this assay design is the number of intermediate steps before ligand detection (ligand binding, extraction, readsorption, and ELISA), which makes it difficult to extract equilibrium constants for the interactions. Nonetheless, our ELISA-based binding assay can be readily applied to compare the specific binding of different VEGF/VEGFR isoforms, fragments, and mutants, and potentially additional growth factors and cytokines, to fibronectin.
The results of our ELISA-based binding assay show that binding of both VEGF and ECD to surface-adsorbed fibronectin requires opening up of cryptic binding sites on the fibronectin molecules through the catalytic action of heparin and is enhanced at acidic pH. The heparin-exposed VEGFbinding sites have been localized on the 40 kDa C-terminal domain of fibronectin, comprising domains FNIII 12-14. Although it is possible that the heparin-catalyzed structural changes may affect more than one region of fibronectin, our binding experiments with sequential ligand addition suggest that VEGF and ECD may share binding sites on fibronectin, and binding of either one can bring the entire VEGF/VEGFR2 complex in contact with fibronectin. This is consistent with previous studies showing the ability of fibronectin-bound VEGF to interact with VEGFR2.
Mechanistic insights on the interactions between fibronectin and VEGF/VEGFR2 were gained by SPR kinetic experiments. Similar to the results of the equilibrium studies, binding of either VEGF or ECD to fibronectin at acidic pH was minimal in the absence of any heparin treatment. To achieve the maximum effect of heparin on the conformation of the immobilized fibronectin, while minimizing VEGF or ECD binding to any residual heparin remaining bound to fibronectin, we allowed fibronectin to interact with 10x molar excess heparin during a 1-min association phase, followed by a 10-min dissociation phase. Previous research has shown that heparin-fibronectin interactions are transient and governed by repeated rebinding and release events whereby a heparin molecule dissociating from a fibronectin molecule will rebind to neighboring fibronectin molecules multiple times before diffusing into the bulk. Accordingly, the combined heparin association and dissociation phases should offer sufficient time for heparin to interact with the majority of the immobilized fibronectin layer and be almost completely released prior to VEGF or ECD binding. Interestingly, the presence of the dissociation phase during heparin pretreatment had no effect on VEGF binding. This suggests that the 1-min association phase at that heparin concentration was sufficient to expose all available VEGFbinding sites on the immobilized fibronectin layer. Indeed, the heparin-catalyzed structural rearrangement of fibronectin is very fast and able to reach completion within 1 min. At the same time, the fact that VEGF binding remained the same, even in the absence of heparin dissociation, confirms that, in this experimental setup, VEGF interactions with fibronectin-bound heparin were negligible. For ECD, however, heparin dissociation was necessary in order to observe any binding. In the absence of heparin dissociation, we observed a first fast initial burst of binding, but the RU returned quickly to the baseline levels. There was no difference in ECD binding after a 10-min or 30-min heparin dissociation phase. These data suggest that heparin interferes and competes with ECD for binding to fibronectin, possibly because the ECDbinding sites on fibronectin overlap with one of the heparin-binding sites. On the contrary, the heparin presence The experimental data presented on, C and D were fitted with the two-sites model (considering mass transfer for VEGF and rapid mixing for ECD). Association and dissociation rates (k a and k d ) for each site, the site occupancy α (Site A/Site B), the density of total binding sites (R T ), and the mass transfer rate (k m ), when applicable, were extracted from the model. Instead of single values, the lower and upper boundaries of the confidence intervals for each parameter, calculated during fitting, are given. Dissociation affinity constants for each site were calculated by dividing k d by k a . The intervals for the K d values were derived from those associated with the k d by k a values according to the formula: for Z = A/B, (ΔZ/Z) 2 = (ΔA/A) 2 + (ΔB/B) 2 , where ΔA, ΔB, and ΔZ are the errors associated with quantities A, B, and Z, respectively. had no effect on VEGF binding to fibronectin, suggesting that despite the similarities in VEGF and ECD binding to fibronectin, the sites for the two ligands are not identical. It is interesting to notice that the 40 kDa domain of fibronectin, which contains the VEGF-binding sites, can also bind a multitude of other growth factors and cytokines, suggesting the existence of mechanisms that regulate the availability of such a great number of binding sites within a limited domain and orchestrate the binding events. If the ECD-binding sites are also located in this domain, the presence of heparin chains (and not only their catalytic action) may be part of such a regulatory mechanism, determining which binding events take place. In future studies utilizing fibronectin fragments and mutants, we plan to map precisely the VEGF and ECD-binding sites on fibronectin.
The model that described best the SPR experimental data revealed the presence of two populations of fibronectin molecules (site A and site B) that can bind VEGF and ECD with different affinities. Although the two-sites model described the data better than the one-site model, there were still discrepancies between the experimental data and the fitted values, especially for VEGF. Statistical analysis suggests that these discrepancies could be explained by the levels of experimental error. However, it is possible that they reflect aspects of the interaction not captured adequately by a simple two-sites model, such as rebinding events as have been reported for one-site models. We did not incorporate this term in the fitting model to avoid overparameterization, especially since the system of differential equations describing the two-sites interaction does not have an analytical solution. We are currently developing a mathematical model to describe this phenomenon for two-site interactions, which will be presented in a future study. According to the model, site A, which is the minority of the total population (20-30%), is characterized by high association and dissociation rates, whereas site B (70-80% of the total population) has association and dissociation rates lower than those for site A by one or more orders of magnitude. Since the binding of VEGF and ECD to fibronectin was negligible in the absence of heparin treatment, this heterogeneity cannot be explained by the presence of fibronectin molecules on which the binding sites have not yet been opened by heparin. Furthermore, the action of heparin is very fast and even if the time of contact with the immobilized fibronectin was increased, the levels of VEGF and ECD binding remained the same. Therefore, it is also unlikely that the two binding populations reflect an incomplete action of heparin. Instead, these results suggest that not all fibronectin molecules respond equally to the action of heparin. Indeed, previous studies by atomic force microscopy (AFM) have identified structural heterogeneity on single fibronectin molecules, with approximately 70% of the visualized molecules adopting a similar configuration. The origin of this heterogeneity is not known, but may be related to the inherent variability in the amino acid sequence of the two fibronectin chains generated by alternative splicingand/or the presence of diverse covalently bound glycans (GlyGen database: P02751-15). Irrespectively of the source of this structural heterogeneity, it is possible that when these molecules respond to the heparin action generate binding site B, whereas the rest will generate site A. According to our model, site A is more accessible than site B for ligand binding, leading to higher association rates, but it can also be freed more quickly (higher dissociation rates). The actual values for the kinetic rates result VEGFR2 binding to fibronectin in site A having higher affinity for VEGF, and site B exhibiting higher affinity for ECD, and since site B represents the majority of binding sites, the overall affinity of ECD for the fibronectin matrix would be higher than that of VEGF, which is consistent with the results of the equilibrium studies. Consequently, high affinity binding of VEGF to fibronectin would be characterized by a high turnover rate, whereas ECD binding would be more stable. Moreover, VEGF binding would occur mostly on a small number of sites, whereas ECD could more readily interact with the majority of fibronectin sites within the extracellular matrix. These differences may have an impact on the dynamics of the interactions between the three molecules and fine-tune their functionalities. A model describing VEGF/VEGFR2/fibronectin interactions occurring in vivo, extrapolated from our in vitro observations, is shown in.
The enhanced binding of VEGF and ECD on fibronectin at acidic pH may have significant consequences for angiogenesis. It is known that hypoxia, signalizing the need for angiogenesis, leads to anaerobic metabolism and consequently, decreases locally the extracellular pH. In tumors, which also require angiogenesis to sustain their growth and metastasize, there are additional mechanisms stimulating glycolysis, even under normoxic conditions, contributing to the low levels of extracellular pH. There have been cases of tumors with extracellular pH as low as 5.8. Thus, fibronectin would bind the angiogenic factor VEGF and its receptor primarily in areas requiring active angiogenic signaling. Such sequestration of VEGF within the matrix may increase its bioavailability and help create gradients to guide the growth of new vessels. On the other hand, fibronectin-bound VEGF can alter VEGFR2 signaling. It is possible that VEGFR2 binding to fibronectin may alter its residence time on the cell membrane, altering its signaling output. Finally, such interactions may facilitate the formation of higher-order complexes, including integrins and neuropilins, fine-tuning the cell response to an angiogenic stimulus. It is interesting that upon switching the pH back to neutral, both fibronectin-bound VEGF and ECD can be quickly released . This behavior would act as a regulatory mechanism that can potentially terminate VEGF/ VEGFR2 angiogenic signaling when the conditions in the extracellular microenvironment cease to be acidic.
CD spectra of VEGF and ECD acquired at neutral (pH 7.5) and acidic (pH 5.5) pH revealed that the pH alteration did not cause major conformational changes on either molecule. Therefore, the pH dependency of the interactions might rely completely on pH-sensitive amino acids within the binding sites. Using structure-based algorithms to calculate pKa values of individual amino acids, we could identify several residues, especially His residues, on both VEGF and ECD that could act as pH sensors. Control experiments with nontagged VEGF or His-tagged VEGF isoforms that do not interact with fibronectin suggest that the His residues of the tag used for recombinant protein production are not involved in the enhanced binding observed at acidic pH. In ECD, most of the candidate residues were found on domains 3, 4, and 5, and their side chains were not involved in interactions with VEGF in the VEGF-VEGFR2 complex, or in stabilizing the secondary and tertiary structure of the protein. In VEGF, on the other hand, most of the candidate residues were involved in interactions either with VEGFR2 or with the second monomer in the biologically active dimeric form of VEGF, except for one His residue in the Cterminal domain of VEGF (His125), which is free from interactions and could participate in interactions with fibronectin. However, this residue is missing in VEGF 121 , another VEGF isoform that can also bind to fibronectin in a pH-dependent manner. Therefore, His125 cannot be the only pHsensitive residue involved in VEGF-fibronectin interactions. It is known that pKa values are very sensitive to the microenvironment of the residue, and electrostatics, conformational fluctuations, and solvent thermodynamics can cause pKa shifts (49). Moreover, structural information is fragmentary for both VEGF and ECD. High-resolution structures exist only for individual domains and not for the full-length proteins. Therefore, the prediction of the pKa values of individual residues based on the known structures should be interpreted with caution. Nonetheless, His residues often act as pH sensors, modulating protein structure and protein-protein interactions.
Decreasing the pH may also affect the binding sites on fibronectin. Several studies have shown that fibronectin undergoes conformational changes in response to pH changes. It is possible that these conformational changes also affect the VEGF and ECD-binding sites on fibronectin. We did calculate pKa values on surface-exposed residues in the FNIII 12-14 domains of fibronectin, which contains the VEGF and possibly the ECDbinding sites, based on two alternative conformations that have been reported. Interestingly, we identified a large number of basic (pKa > 10) and acidic (pKa < 4), but none that could act as pH sensors (5.5 < pKa < 7.5). This suggests an elegant mechanism whereby the action of heparin regulates fibronectin conformation to expose VEGF/ECD-binding sites, whereas the local pH regulates ligand binding. However, given the size and flexibility of fibronectin, we cannot exclude the possibility that in the fulllength protein different amino acid residues are exposed in the FNIII 12-14 domains or that they have different pKa values. Furthermore, as already discussed, additional regions of fibronectin may be involved in ECD binding.
Understanding VEGF/VEGFR2 interactions with fibronectin within the extracellular matrix may have implications for cancer therapy. Interestingly, some of the His residues in ECD that may be involved in the interactions with fibronectin are found mutated in several cancer cases (unpublished data). Understanding how fibronectin may affect the angiogenic potential of VEGF may help design novel drugs that are more specific, increase their efficacy, and decrease their side effects. Future studies identifying the exact binding sites on all molecular partners, and exploring the consequences of fibronectin binding to the structure and angiogenic signaling of VEGF/VEGFR2 complexes, can open up possibilities for novel therapeutic approaches.
# Experimental procedures materials
The pFastBac vector (cat. no 10360-014) and the CellFectin transfection reagent were purchased from Invitrogen.
## Vegfr2 binding to fibronectin
Ampicillin, kanamycin, chloramphenicol, tetracyclin, and X-Gal were purchased from VWR. Gentamicin and IPTG were purchased from Sigma. The Sf21 TiterHigh AC free cell line (European Collection of Authenticated Cell Culture, cat. no 05030202) was obtained from Sigma. The High Five (BTI-Tn-5B1-4; Hi5) cell line was kindly provided by prof. Ballmer-Hofer at the Paul Scherrer Institute, Switzerland. The insect cell culture medium SF-4 Baculo Express (cat. no 9-00F38-K) was purchased from BioConcept. The 100x Gibco Antibiotic/Antimycotic supplement (containing 10,000 units/ml penicillin, 10,000 μg/ml streptomycin, and 25 μg/ml amphotericin B) was purchased from Thermo Fisher Scientific (cat. no 15240062). The HisTrap Excel, Hi Trap G HP, and S200 10/30 Superdex chromatography columns were from GE Healthcare. Sephadex PD10 MiniTrap G-25 1-ml columns were purchased from Sigma. Glass-bottom 96well plates were obtained from Life Systems Design (cat. no 324001), and hydrophobic polystyrene F-bottom 96-well plates were from Greiner (cat. no 655 101). The 1-step Turbo TMB substrate was from VWR, and the Clarity ECL chemiluminescence substrate was from BioRad. TGX (4-20% gradient) Stain-Free electrophoresis gels were purchased from BioRad. The Cy3-NHS ester used for protein fluorescence labeling was obtained from Lumiprobe (cat. no 11020). Heparin sodium salt from porcine intestinal mucosa was purchased from Sigma (cat. no H3393). The mouse anti-His (cat. no H1029-2 Ml) and anti-VEGFR2 (V3003-2 Ml) antibodies were purchased from Sigma. The HRP-conjugated goat anti-mouse secondary antibody (from Jackson Immunolabs) was purchased from MILAN Analytica (cat. no 115-035-003). Human plasma fibronectin was obtained from Millipore (cat. no FC010). The recombinant VEGF 165 protein, the ECD cDNA, and the DH10beta Embac YFP cells were kindly provided by prof. Ballmer-Hofer at the Paul Scherrer Institute, Switzerland. All other reagents were obtained from Sigma.
## Cell culture
The Sf21 Spodoptera frugipedra and High Five (Hi5) Trichoplusia ni insect cell lines were cultured at 27 C in the serum-free SF-4 Baculo Express medium, supplemented with 1000 units/ml penicillin, 1000 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. The cultures were maintained in suspension at a density of no more than 2.0 * 10 6 cells/ml.
## Recombinant protein expression and purification
ECD, encompassing the seven domains of the extracellular part of human VEGFR2 and carrying a 6x His Tag at the Cterminus, was cloned in the pFastBac plasmid. DH10beta Embac YFP electrocompetent bacteria were transformed with the ECD/pFastBac plasmid by electroporation and positive colonies were isolated after ampicillin, kanamycin, chloramphenicol, tetracycline, gentamicin, IPTG, and X-Gal selection for bacmid generation. Sf21 cells grown in 6-well plates at a density of 0.5 * 10 6 cells/ml were transfected with the ECD bacmid using CellFectin according to the manufacturer's instructions, and the secreted virus was harvested from the medium after 48 h incubation at 27 C (V 0 ). The virus was amplified by infection of suspension Sf21 cell cultures (3 ml of V 0 in a 500-ml culture) at a density of 0.5 * 10 6 cells/ml. The V 1 viral stock (1.5 * 10 7 pfu/ml) was harvested 9 days after infection, when 90% of the cells exhibited YFP fluorescence. The virus was amplified a second time by infection of suspension Sf21 cell cultures (10 ml of V 1 in a 1-L culture) at a density of 1.0 * 10 6 cells/ml. The V 1 viral stock (2.2 * 10 8 pfu/ ml) was harvested 4 days after infection, when more than 90% of the cells exhibited YFP fluorescence. Aliquots of the viral stocks were stored at -80 C.
The V 2 viral stock was used for protein production. Suspension Hi5 cultures (4 * 1 L) were infected with 10 ml/L of the V 2 viral stock at a density of 1.0 * 10 6 cells/ml. The culture was monitored daily for cell viability and infection (by fluorescence). When more than 90% of the cell population exhibited YFP fluorescence (usually 4 days post infection), the culture medium was collected by centrifugation (5000 x g for 15 min at 10 C), filtered, and loaded on 4-ml HisTrap Excel columns. The His-tagged protein was eluted by applying a linear 0-100% buffer B gradient in 20-column volumes (buffer A: 50 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8.0; buffer B: 50 mM Tris, 300 mM NaCl, 500 mM imidazole pH 8.0). The fractions of the peak were collected, dialyzed against 20 mM Tris, pH 8.0, and loaded on an ion exchange 1-ml HiTrap Q HP column. The proteins bound to the column were eluted applying a linear 0-100% buffer B gradient in 20column volumes (buffer A: 20 mM Tris, pH 8.0; buffer B: 20 mM Tris, 300 mM NaCl, pH 8.0). The fractions corresponding to the ECD (based on molecular weight by SDS-PAGE analysis) were collected, dialyzed against SEC buffer (50 mM Hepes, 150 mM NaCl, 5% glycerol, pH 7.5), concentrated to 0.5 ml, and loaded on an S200 10/30 Superdex size exclusion column. The proteins were eluted with onecolumn volume SEC buffer and 0.5 ml fractions were collected. The fractions corresponding to monomeric ECD were pooled, concentrated, and stored at -80 C. Protein storage at 4 C for 15 days led to slight fragmentation. Protein yields ranged from 10 to 30 μg/L of culture. Protein purity during the sequential chromatographic steps was monitored by SDS-PAGE.
VEGF 165 containing an N-terminal 6x His tag, expressed in Pichia Pastoris, and purified by immobilized metal affinity and size-exclusion chromatography, was kindly provided by prof. Ballmer-Hofer (Paul Scherrer Institute, Switzerland). Human plasma fibronectin was purchased from EMD Millipore. The purity of both VEGF and fibronectin was assessed by SDS-PAGE.
## Elisa-based binding assay
Fibronectin was added on glass-bottom or hydrophobic polystyrene 96-well plates (20 μg/ml in PBS; 50 μl/well) and was allowed to adsorb overnight at 4 C. Subsequently, the plate was placed on ice, the solution was aspirated, the adsorbed layer was washed with phosphate-based saline (PBS) (three times; 50 μl/well each time), and was allowed to interact with VEGF or ECD in binding buffer (150 mM NaCl, 25 mM Hepes) at pH 7.5 or 5.5, for 1 h at 4 C. The solution was then aspirated, the plate was washed with binding buffer (three VEGFR2 binding to fibronectin times; 50 μl/well each time), and the bound ligand was released with the appropriate extraction buffer. After incubation in the extraction buffer (1 min-1 h), the solution was transferred into a second 96-well polystyrene plate. To ensure complete transfer, an additional 50 μl of the extraction buffer was added to the original wells, pipetted twice up and down, and mixed with the material from the first extraction. The extracted ligand was allowed to adsorb on a new plate for 1 h on ice. Then, the solution was aspirated, the plate was washed with assay buffer (three times; 50 μl/well each time), and was incubated with an anti-His or an anti-VEGFR2 primary antibody for 1 h on ice. The solution was aspirated, the plate was washed with assay buffer (three times; 50 μl/well each time), and was incubated with an HRP-labeled secondary antibody for 1 h on ice. The solution was aspirated, the plate was washed with assay buffer (three times; 50 μl/well each time), and the signal was developed accordingly, for absorbance or chemiluminescence measurements. For absorbance, 50 μl of TMB substrate was added on the plate and incubated for 10 min at room temperature. The reaction was stopped by adding 50 μl 1 M H 2 SO 4 and absorbance was measured at 450 nm and 570 nm using a Microplate reader Infinite 200 PRO (Tecan Group AG, Switzerland). The signal at 570 nm, corresponding to light scattering, was subtracted from the signal at 450 nm, corresponding to TMB absorbance, in order to correct for any features from the well bottom interfering with the measurement. For chemiluminescence, 100 μl of ECL substrate was added on the plate, and the luminescence signal was measured after 10 min, applying 1000 ms integration time and automatic gain using a Microplate reader Infinite 200 PRO. The luminescence signal was corrected by subtracting the sum of the signal from the surrounding eight wells multiplied by the correction factor 0.0454, which was determined by repeated measurements of the blanc in different wells, surrounded by samples of different luminescence values.
## Determination of fibronectin desorption
Fibronectin was fluorescently labeled with Cy3 according to standard amine chemistry procedures. Briefly, fibronectin (0.5 mg in 0.1 M NaHCO 3 , pH 8.4) was mixed with 20x molar excess of Cy3-NHS dye and was incubated for 1.5 h on ice protected from light. The labeled protein was separated from free dye by sizeexclusion column chromatography using 1-ml Sephadex G-25 columns. The protein was eluted in PBS and its concentration and labeling ratio were determined by measuring the absorbance at 280 nm and 550 nm (NanoDrop). Cy3-labeled fibronectin in PBS was added on black polystyrene or glass-bottom 96-well plates and was allowed to adsorb overnight at 4 C. After the total fluorescence was measured using a Microplate reader Infinite 200 PRO (excitation wavelength = 550 nm; emission wavelength = 595 nm), the solution was aspirated, the adsorbed layer was washed three times with PBS, and then was subjected to a series of incubations (1 h each, on ice) with different buffers (PBS containing 1 mg/ml BSA and 0.05% Tween20 or 5 M NaCl, 25 mM Hepes, pH 7.5). After each incubation, the solution was aspirated, the protein layer was washed three times with PBS, and fluorescence was measured as above to determine how much fibronectin remained adsorbed on the well surface.
## Surface plasmon resonance (spr) measurements
SPR measurements were performed with a BiaCore X instrument (GE Healthcare) using low-density carboxymethyldextran hydrogel (500 nm) biosensor chips with two flow cells (CDM500 L, Xantec bioanalytics GmbH, Germany). Fibronectin was immobilized on flow cell 2 according to the manufacturer's instructions, while flow cell 1 was used as a reference. Briefly, the chip surface in both flow cells was activated by injecting 70 μl of a 1:1 mixture of 100 mM NHS and 400 mM N-ethyl-N'(dimethylaminopropyl)carbodiimide hydrochloride (EDC) at a flow rate of 5 μl/min. Within 3 min after the surface activation, 70 μl fibronectin was injected on flow cell 2 only (0.2, 2, or 20 μg/ml in 10 mM sodium acetate buffer, pH 5.0) at a flow rate of 5 μl/min. The remaining free carboxyl groups on the surface of both flow cells were inactivated by injecting 70 μl 1 M ethanolamine, pH 8.0 at a flow rate of 5 μl/min. The interaction between immobilized fibronectin and VEGF or ECD was studied in running buffer (150 mM NaCl, 25 mM Hepes, pH 5.5) that had been autoclaved, filtered, and degassed. Different injection volumes (30 or 60 μl) and flow rates (10, 25, 50, and 100 μl/min) were tested, and the final measurements were performed with 60 μl VEGF or ECD (20 nM, 50 nM, 100 nM, 200 nM, 500 nM, and 1000 nM) at 50 μl/min. For assay optimization, only the association part of the sensogram was recorded. For complete analysis of the interaction and model fitting, the dissociation phase was monitored for a total of 720 s. A pretreatment step of the immobilized fibronectin with 1 μg/ml heparin in running buffer (50 μl at 50 μl/min) was included immediately before the VEGF or ECD injections. After each VEGF or ECD injection, the surface was regenerated with 2 M NaCl and 0.05 N NaOH (50 μl each at 50 ml/min). All measurements were performed at room temperature.
## Kinetic modeling
The SPR data were analyzed with multiple models to extract kinetic parameters for the interactions between fibronectin and VEGF or ECD. Interestingly, we observed a drop in RU values prior to the nominal end of the association phase (59 ± 12 s for VEGF and 65 ± 10 s for ECD instead of 72 s), which, however, showed no correlation with VEGF/ECD concentration . Although we cannot exclude the possibility that this may reflect some aspect of the binding interactions, we believe that it is caused by experimental error. Strengthening this conclusion is the fact that such time discrepancies were observed for both flow cells and were seemingly random. In the absence of any solid mechanistic justification for ligand dissociation during the association phase, the kinetic parameters reported here have been extracted using variable dissociation time in all cases. Before describing the individual models, we outline the mass conservation equations on which all further considerations are based. We assume that all VEGF or ECD molecules introduced VEGFR2 binding to fibronectin in the system either remain in the flow phase or bind to fibronectin that is immobilized on the surface; as a consequence, neglecting all other possible interactions, the total number of VEGF or ECD molecules is conserved.
The mass conservation equations that we used are derived from a two-compartment model developed previously, based on the complete description of the kinetic/mass transfer phenomena in an SPR experiment through partial differential equations (PDE). This model, although simpler than the original PDEs, is able to recapitulate accurately the key parameters describing the time evolution of the system. Briefly, the model takes into consideration two compartments: a flowing bulk phase with a time-invariant concentration of ligands (C T ) and a narrow volume slit (V i ) in the proximity of the surface (S) where receptors have been immobilized at an initial concentration R T. Ligand molecule exchange occurs between the flowing bulk phase and V i (mass transfer), as well as between V i and S (binding/unbinding events). Owing to these phenomena, the ligand concentration in the volume V i (C), as well as the amount of free (R) and receptor-bound (B) ligand molecules at S, varies with time. Applying the principle of mass conservation to the volume V i and to the surface S, we obtain the following system of equations:
[formula] 8 > > > > > > > > > < > > > > > > > > > : V i dC dt ¼ _ R in;V i − _ R out;V i þk m SðC T −CÞ S dB dt ¼ _ R in;S − _ R out;S Cðt ¼ 0Þ ¼ 0 Bðt ¼ 0Þ ¼ 0 [/formula]
In the system above, _ R in;V i and _ R out;V i are, respectively, the rates of ligand molecule gain and loss in the volume V i , as a consequence of binding/unbinding reactions. _ R in;S and _ R out;S refer to the same quantities with respect to the surface S. k m is the convective mass-transfer coefficient for the exchange of ligand molecules between the flowing bulk and the volume V i .
It is possible to express the quantities _ R in;V i , _ R out;V i , _ R in;S and _ R out;S in a way that is specific for the surface S:
[formula] _ R in;V i ¼ S b R in;V i ; _ R out;V i ¼ S b R out;V i ; _ R in;S ¼ S b R in;S ; _ R out;S ¼ S b R out;S The newly defined quantities b R in;V i , b R out;V i , b RV i dC dt ¼ S b R in;V i − b R out;V i þk m ðC T −CÞ ! S dB dt ¼ S b R in;S − b R out;S ! Cðt ¼ 0Þ ¼ 0 Bðt ¼ 0Þ ¼ 0 [/formula]
It is possible to divide the first equation by V i and the second by S. The ratio between S and V i is the reciprocal of the characteristic height (h i ) of the volume slit, where it is assumed that the mass transfer and binding/unbinding phenomena occur. Accordingly, the system of equation is transformed as follows:
[formula] 8 > > > > > > > > > < > > > > > > > > > : dC dt ¼ 1 h i b R in;V i − b R out;V i þk m ðC T −CÞ ! dB dt ¼ b R in;S − b R out;S Cðt ¼ 0Þ ¼ 0 Bðt ¼ 0Þ ¼ 0 [/formula]
In the case of multiple-site reactions, the second expression in the system can be split into multiple equations, each describing the behavior of a specific reaction site.
Below, we present in detail the different models that have been used for fitting the experimental data. In all models, the term receptor corresponds to the monomeric chain of each fibronectin dimer and the term ligand to VEGF or ECD.
## One-site model
This is the simplest model, assuming one population of receptor molecules, with each receptor molecule possessing one ligand-binding site. We keep the same nomenclature outlined before for the binding sites of the free (R) and ligandbound (B) receptor molecules, and we describe the ligandreceptor interactions using single association (k a ) and dissociation (k d ) kinetic constants. Consequently:
[formula] b R in;V i ¼ b R out;S ¼ k d B b R in;S ¼ b R out;V i ¼ k a CR ¼ k a CðR T − BÞ [/formula]
The system of equation becomes:
[formula] 8 > > > > > > > > > < > > > > > > > > > : dC dt ¼ 1 h i ½−k a CðR T −BÞþk d Bþk m ðC T −CÞ dB dt ¼ þk a CðR T −BÞ−k d B Cðt ¼ 0Þ ¼ 0 Bðt ¼ 0Þ ¼ 0 [/formula]
## Two-sites model
This model assumes two populations of receptor molecules with surface concentrations R T ;1 ¼ αR T and R T ;2 ¼ ð1 − αÞR T , respectively, with α ranging from 0 to 1. Each receptor molecule possesses a single ligand-binding site, as in the one-site model, but with distinct association (k a;1 and k a;2 ) and dissociation (k d;1 and k d;2 ) rates. Ligand binding to receptor molecules belonging to either of the two populations (R 1 and R 2 ) occurs independently, generating molecular species B 1 and B 2 . Thus, the second equation of the system that describes the evolution of the ligand-bound receptors is split into two different equations, one for each receptor population. The kinetic terms can be described as follows:
[formula] _ R in;V i ¼ k d;1 B 1 þk d;2 B 2 _ R out;V i ¼ k a;1 CR 1 þ k a;2 CR 2 ¼ k a;1 C À R T;1 − B 1 Á þk a;2 C À R T;2 − B 2 Á _ R in;S;1 ¼ k a;1 CR 1 ¼ k a;1 C À R T;1 − B 1 Á _ R in;S;2 ¼ k a;2 CR 2 ¼ k a;2 C À R T;2 − B 2 Á _ R out;S;1 ¼ k d;1 B 1 _ R out;S;2 ¼ k d;2 B 2 [/formula]
Therefore, the system of equations becomes:
[formula] 8 > > > > > > > > > > > > > > > > > < > > > > > > > > > > > > > > > > > : dC dt ¼ 1 h i  −k a;1 C À R T ;1 −B 1 Á −k a;2 C À R T;2 −B 2 Á þk d;1 B 1 þk d;2 B 2 þk m ðC T −CÞ Ã dB 1 dt ¼ k a;1 C À R T;1 −B 1 Á −k d;1 B 1 dB 2 dt ¼ k a;2 C À R T;2 −B 2 Á −k d;2 B 2 Cðt ¼ 0Þ ¼ 0 B 1 ðt ¼ 0Þ ¼ 0 B 2 ðt ¼ 0Þ ¼ 0 [/formula]
## Parallel reactions model
This model assumes that each receptor molecule possesses two ligand-binding sites that act independently. From a modeling perspective, this scenario can be treated as a special case of the two-sites model, where R T ;1 ¼ R T ;2 ¼ R T =2 and α = 0.5. Therefore, we used the same system of equations as for the two-sites model under these restrictions.
## Consecutive reactions model
This model also assumes that each receptor molecule possesses two ligand-binding sites, but in contrast to the parallel reactions model, the binding sites do not act independently; instead, the first binding event is required before the second binding can occur. In this case, we do not consider the concentration of binding sites (B 1 and B 2 ) as was done in the other cases, but instead, the concentration of the various molecular species of the receptor: free (R), bound to the first ligand (R A ), and bound to the second ligand (R 2A ), with
[formula] R T ¼ R þ R A þ R 2A . [/formula]
We consider the most general case, whereby the two binding events occur with distinct association and dissociation rates: k a;R and k d;R A for the first reaction (R to R A ), and k a;R A and k d;R 2A for the second (R A to R 2A ). As for the two-sites model, the second equation of the system has to be split in multiple equations to describe the behavior of each molecular species. Then, the kinetic terms can be computed as follows:
[formula] _ R in;V i ¼ k d;R A R A þk d;R 2A R 2A _ R out;V i ¼ k a;R CRþk a;R A CR A _ R in;S;R ¼ k d;R A R A _ R out;S;R ¼ k a;R CR _ R in;S;R A ¼ k a;R CRþk d;R 2A R 2A _ R out;S;R A ¼ k a;R A CR A þk d;R A R A _ R in;S;R 2A ¼ k a;R A CR A _ R out;S;R 2A ¼ k d;R 2A R 2A [/formula]
Accordingly, the final system of equations becomes: 8 > > > > > > > > > > > > > > > > > > > > > > > > > < > > > > > > > > > > > > > > > > > > > > > > > > > :
[formula] dC dt ¼ 1 h i  k d;R A R A þk d;R 2A R 2A −k a;R CR−k a;R A CR A þk m ðC T −CÞ Ã dR dt ¼ −k a;R CRþk d;R A R A dR A dt ¼ k a;R CRþk d;R 2A R 2A −k a;R A CR A −k d;R A R A dR 2A dt ¼ k a;R A CR A −k d;R 2A R 2A Cðt ¼ 0Þ ¼ 0 Rðt ¼ 0Þ ¼ R T R A ðt ¼ 0Þ ¼ 0 R 2A ðt ¼ 0Þ ¼ 0 [/formula]
## Solution of the system of ordinary differential equations (odes) and optimization of the parameter values
The free parameters to be determined for each model are shown in. For the parameter h i , we used a fixed value of 10 −5 m, which is a reasonable approximation based on the characteristic size of the SPR microfluidics chip. Moreover, results from a previous studyshow that the solutions of the equations describing an SPR experiment are insensitive to the value of h i , which was also true here (data not shown). The values of the free parameters were determined by fitting the experimental data. To avoid overfitting, the experimental curves collected at different values of C T were fitted simultaneously (global fitting). During the association phase, the values of C T were considered equal to the experimental ones, VEGFR2 binding to fibronectin whereas at the beginning of the dissociation phase was set to zero. The units of the experimental data (RU) were converted to mol=m 2 , based on previously reported assumptions (51), using the following equation:
[formula] LðRUÞ ¼ L mol m 2 , 10 −4 m 2 cm 2 , MW g mol , 10 10 RU g = cm 2 [/formula]
MW represents the molecular weight of the ligand and is equal to 45 or 83 g/mol for VEGF or ECD, respectively. L represents the total ligand surface concentration and is calculated for the different models as follows:
[formula] L ¼ B ðOne − site modelÞ L ¼ B 1 þB 2 ðTwo − sites model; Parallel reactions modelÞ L ¼ R A þ2R 2A ðConsecutive reactions modelÞ [/formula]
The ordinary differential equations were discretized and solved using the explicit Euler method, with a time interval of dt ¼ 10 −2 s. Given an initial set of parameter estimates, the ordinary differential equations system was solved and the output was compared with the experimental results. Unconstrained fitting was performed in Matlab using the function lsqcurvefit, which minimizes the following quantity:
[formula] F ¼ X C T X t À L exp ðC T ; tÞ−L mod ðC T ; tÞ Á 2 [/formula]
L exp and L mod represent the total ligand surface concentration as derived from the experimental data or the fitted model, respectively. The summation is performed over the time points of a single data set (inner sum) and over different data sets collected at different values of C T (outer sum). The solution of the system can be performed according to two different scenarios. The first scenario consists in considering mass transfer limitations. In such a case, the systems of equations are identical with the ones explained above. The second scenario assumes mass transfer not to be limiting (rapid mixing): ligand exchange between the flowing bulk and the volume V i is faster that the characteristic binding/unbinding timescales. Under such an assumption, C ¼ C T replaces the first equation in all the systems discussed in the previous paragraphs.
## Statistical analysis of curve fitting
The experimental data were fitted with all four models (onesite, two-sites, parallel reactions, and consecutive reactions model), under conditions of rapid mixing or mass transfer. The beginning of the dissociation phase was set to the maximum RU value achieved during the association phase (variable dissociation time). To ensure as much as possible that the solution did not represent a local minimum, the following strategy was adopted for all cases: the fitting was repeated multiple times, with sets of initial values for the free parameters that differed by several orders of magnitude. Considering all curves in the data set (with different C T values), the residual sum of squares (RMS) was calculated for each solution as the sum of the squared residuals (difference between experimental and fitted value), and the solution with the lowest RMS value was selected. Confidence intervals were then calculated for each of the estimated parameters based on the residual values and the Jacobian matrix, using the nlparci Matlab function. Solutions for which the confidence intervals for one or more of the parameters were as large as to include negative values were rejected, indicating overfitting, and that the corresponding model could not be used to fit the data. For each model that gave a solution with welldefined confidence intervals for all estimated free parametersTo assess the fitting, the absolute and relative values of these indices were examined. Traditionally, the residual variance (corresponding to what is often termed as Chi square in the SPR literature) is used to assess the goodness of fit in SPR experiments. However, it is difficult to define absolute cutoff values, primarily because the residual variance depends on the level of the average signal. Usually, the fit can be considered acceptable when the square root of the residual variance is comparable to the level of experimental noise. To estimate the levels of experimental noise, the doseresponse experiments were repeated three times using the same chip and injecting the different VEGF/ECD concentrations in a random order to ensure that the data were free of any systematic errors. The patterns were very similar in replicate experiments, but the absolute values were associated with an experimental error of 6-39% (corresponding to 3-24 RU) for ECD and 15-49% (corresponding to 7-18 RU) for VEGF . This level of experimental error, significantly higher than that of the baseline fluctuations (4%), might have originated from pipetting errors during dilution and injection, as well as stochastic events during binding in both flow cells. Considering this level of noise, all fits, despite their discrepancies with the experimental data, could be acceptable based According to the models presented on, describing the interactions between fibronectin and VEGF or ECD, the listed parameters should be extracted from fitting the SPR dose responses to each of the proposed models.
on the residual variance values. However, not all fits could be considered equivalent. According to information theory, the Akaike's information criterion (AIC) can be used for model selection among different models that can fit a certain data set, combining absolute fit with model parsimony. In other words, it penalizes for the addition of parameters to the model in order to improve absolute fit. The model with the lower AIC is considered the best describing the data in question. According to this criterion, the models generating the best overall fit for the data were the consecutive reactions model for VEGF and the two-sites model for ECD. However, the twosites model generated curves that resembled more closely the shape of the experimental curves for VEGF; therefore, the two-sites model was also chosen to describe VEGF binding to fibronectin, even though, according to AIC, it was the second best model regarding overall fit.
## Circular dichroism spectroscopy
CD spectra of VEGF, ECD, and complexes between VEGF and ECD were acquired with a J-815 spectrometer (JASCO) with a quartz cuvette and with a path length of 10 mm. Data were collected at 4 C in the 250-205 nm wavelength range with a 0.2 nm data pitch, standard sensitivity (100 mdeg), 1 s digital integration time (D.I.T.), 1 nm bandwidth, and 20 nm/ min scanning speed under a continuous scanning mode. The average spectrum of five sequential measurements (accumulation = 5) was used for processing. The baseline of each buffer was acquired using the same measurement parameters and was subtracted from each spectrum. The data were converted from mdeg to mean residue ellipticity using the molecular weight of each protein or protein complex: 45 kDa for VEGF, 83 kDa for ECD, and 211 kDa for the ECD/VEGF complex. CD measurements were performed in 150 mM NaCl, 25 mM Hepes, pH 7.5 or 5.5 (binding buffer) or in 10 mM sodium phosphate buffer, pH 7.4 or 5.8 (low ionic strength buffer).
# Bioinformatics analysis
Structure predictions of pKa values of individual amino acid residues at the surface of VEGF, ECD, and the FNIII 12-14 domains of fibronectin were performed with the DelPhiPKa web server, using the known high-resolution structures of VEGF, ECD, and FNIII 12-14 fragments (pdb codes: 2VPF and 2VGH for VEGF, 3V2A, 2X1W, 2X1X, 3S35, 3S36, 3S37, 5OYJ, and 3KVQ for ECD, and 1FNH and 3R8Q for the FNIII 12-14 fibronectin domains). The parameters used for the prediction were those that gave the best results on a benchmark study against an extensive database of experimentally determined pKa values: σ = 0.70, ε ref = 8, ε ext = 80. Three different force fields were tested: AMBER, CHARMM, and PARSE, yielding very similar results (data not shown). The pKa values reported were calculated using the AMBER force field. According to the calculated pKa values, titration simulations were performed to calculate the probability of ionization of each residue at pH 7.5 and 5.5 assuming two microstates: protonated and deprotonated.
## Data availability
All data used for this study are presented in this article in the form of graphs or tables. Raw data, as well as the Matlab source code used for fitting the various models on the SPR data, will be available upon request to the corresponding author: Maria Mitsi, [email protected], current affiliation: Ectica Technologies, Switzerland.
Supporting information-This article contains supporting information. |
Programmed Death-1/Programmed Death-Ligand 1-Axis Blockade in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma Stratified by Human Papillomavirus Status: A Systematic Review and Meta-Analysis
Background: Programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors have provided clinical benefit to head and neck squamous cell carcinoma (HNSCC) patients in recent clinical trials. However, it remains unclear as to whether human papillomavirus (HPV) status is associated with improved clinical outcome of anti-PD-1 or anti-PD-L1 immunotherapy in HNSCC.Methods: PubMed, EMBASE, Cochrane Library, and Web of Science were systematically searched up to February 28, 2021. Published clinical trials of HNSCC patients treated with only PD-1 or PD-L1 inhibitors were selected. The primary or secondary outcome of these studies included objective response rate (ORR) stratified by HPV status. The pooled odds ratio (OR) and hazard ratio (HR) were estimated using a fixed-effect model.Results: A total of seven eligible studies comprising 814 patients were included. The ORR of HPV positive HNSCC patients was significantly higher than that of HPV negative HNSCC patients (OR = 1.77; 95%CI = 1.14-2.74; P = 0.01), and this favorable effect occurred in pooled anti-PD-L1 trials (OR = 2.66; 95%CI = 1.16-6.11; P = 0.02). In comparison, the pooled OR was 1.51 in anti-PD-1 trials (95%CI = 0.90-2.54; P = 0.12). Survival analysis indicated that HPV positive HNSCC patients had a lower risk of overall Frontiers in Immunology | www.frontiersin.org death as compared to HPV negative HNSCC patients (HR = 0.77; 95%CI = 0.60-0.99; P = 0.04).Conclusions: HPV positive HNSCC patients display improved outcomes with PD-1/PD-L1 axis blockade as compared to HPV negative HNSCC patients. These improved outcomes are likely driven to a greater extent by anti-PD-L1 inhibitors. However, randomized controlled trials with greater numbers of patients are needed for validation of these early findings.Keywords: human papilloma virus, immune checkpoint blockade, head and neck squamous cell carcinoma, anti-PD-1, anti-PD-L1 Xu et al.PD-1/PD-L1-Axis Blockade in HPV+ HNSCC Frontiers in Immunology | www.frontiersin.org
# Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, with 600,000 cases diagnosed annually and mortality rates as high as 40%-50%. The vast majority of head and neck cancers are squamous cell carcinomas, which arise within different anatomical subsites. Therapeutic strategies for HNSCC include surgery, chemotherapy, radiotherapy, and targeted agents, including small molecular inhibitors or antibodies. Despite advances in treatment, the estimated 5-year overall survival rate of HNSCC has not significantly improved. Recently, there have been several studies that show immune checkpoint blockade appears to provide a promising new avenue for treatment in HNSCC.
Programmed death-1 (PD-1), a member of the immunoglobulin superfamily associated with CD28 and CTLA-4, may be expressed on the surface of activated T cells, B cells, and monocytes. Programmed death-ligand 1 (PD-L1) binding to PD-1 on T cells results in suppression of the T cell immune response. Cancer cells may develop several mechanisms of escaping immune-mediated surveillance and death, including surface expression of PD-L1. The interruption of PD-1 engagement by its ligand reinvigorates the immune system, allowing immune-mediated anti-cancer responses to resume, leading to marked clinical responses in some cancers. Anti-PD-1 and anti-PD-L1 antibodies such as nivolumab, pembrolizumab, cemiplimab, and atezolizumab, durvalumab, avelumab have shown promising results in several cancer types. Nivolumab and pembrolizumab have been approved as first-line agents in recurrent/metastatic HNSCC patients by the United States Food and Drug Administration (FDA).
Despite a declining trend in smoking and drinking rates in the United States, the incidence of a proportion of HNSCC related to human papillomavirus (HPV) infection has been increasing. HPV positive and negative HNSCC are considered two entirely different types of cancer, in part due to their unique molecular landscapes. HPV associated oncogenes E6 and E7 drive oncogenesis in HNSCC by inactivating tumor suppressors TP53 and Rb and activating oncogenic signaling pathways including EGFR and PI3K etc. Nevertheless, numerous clinical studies have demonstrated that HPV positivity in HNSCC confers a clear survival benefit as compared to HPV negative HNSCC patients after surgery with or without chemoradiotherapy. One possible factor contributing to this survival difference is that HPV may elicit inherent local or systemic immunity against tumor cells in HNSCC patients, even in the absence of therapy, leading to the hypothesis that HPV positive HNSCC patients may show increased benefit from immune checkpoint blockade.
There have been conflicting results from published HNSCC clinical trials involving either anti-PD-1 or anti-PD-L1 therapy. The HAWK (20) study concluded that HPV positive patients had a higher objective response rate and survival rate than HPV negative patients. However, Keynote012 (21), NCT01375842, and Keynote055 (23) trials reported that HNSCC patients' tumor response did not correlate with HPV status. Data from two recent meta-analysessuggest there is a trend towards significance favoring higher response rates in HPV positive vs. HPV negative tumors in patients receiving anti-PD-1/PD-L1 therapy. One study by used odds ratio (OR) in the analysis of overall survival, however this calculation does not take into account the effect of time. Furthermore, key limitations in these studies include a lack of stratification by anti-PD-1 or anti-PD-L1 therapy separately and inadequate selection of trials based on what is publicly available. Based on these inconsistent findings, we posited that there might be a difference in outcomes in HPV positive patients treated with immunotherapy depending on the use of either PD-1 or PD-L1 agents disrupting the PD-1/PD-L1 axis.
To further understand the importance of HPV status in HNSCC patients treated with anti-PD-1 or anti-PD-L1 agents, we systematically pooled the results from available trials together and conducted the present meta-analysis, which ultimately may help inform further investigation and ultimately clinical decision making.
# Materials and methods
## Search strategy and eligibility criteria
This systematic review and meta-analysis was conducted according to the Cochrane Handbook for Systematic Reviews of Interventionsand reported by adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement. Our protocol has been registered in the PROSPERO platform (ID: CRD42020175779).
Two independent authors systematically searched PubMed, EMBASE, Cochrane Library, and Web of Science for relevant articles published in English until February 28, 2021. Our search strategies included the following terms: "HPV or Human papillomavirus", "Immunotherapy or Cemiplimab or Atezolizumab or Nivolumab or Pembrolizumab or Durvalumab or Avelumab or PD-1 or PD-L1 or PD1 or PDL1 or checkpoint" and "head and neck or head and neck cancer or head and neck neoplasm or head and neck tumor or head and neck carcinoma or HNC or HNSCC or SCCHN". The complete search strategies used are found in Supplementary Data. We also manually checked the reference lists of identified studies and reviews to include more eligible trials. The search results were imported into Endnote (version 9.2).
Studies were included if they satisfied the following criteria: clinical trials of HNSCC patients treated with only a PD-1 or PD-L1 inhibitor agent, regardless of region, race, age, and gender; studies with a primary or secondary outcome that included objective response rate (ORR); reporting of ORR stratified by HPV status; studies reported in English. Clinical trials allowing participants with prior exposure to any immune-checkpoint blockade were excluded. If the same clinical study was reported in more than one publication, only the one with the most recent or complete data was analyzed. The methodological quality of randomized controlled trials (RCT) was evaluated by the recommendations in the Cochrane Collaboration handbookto assess the risk of bias. The quality of non-RCTs was judged by the Newcastle-Ottawa Scale (28) by two of the authors independently.
## Data extraction
We included the following data extracted from the eligible studies: trial name, publication year, study design, drug and dose, number of participants, age, gender, HPV status, anatomical subsite, ORR, overall survival (OS), progressionfree survival (PFS), median time of follow-up, median OS, median PFS, median duration of response and the median time to response. The disease control rate (DCR) was extracted as the percentage of patients with complete response, partial response, or stable disease in the trial according to the guideline of response evaluation criteria in solid tumors (RECIST version 1.1) (29).
# Statistical analysis
Continuous variables were reported as mean ± standard deviation (or median and range). Categorical variables were expressed as count and percentage. Measures of ORR and DCR stratified by HPV status were assessed by odds ratio (OR) and 95% confidence interval (CI). Subgroup analysis of ORR was performed according to the treatment agent used. OS and PFS data were evaluated by hazard ratio (HR) and 95% CI, and Tierney methodology was used for calculation if the data were not directly available in the original report. Statistical heterogeneity was detected using the Cochran Q chi-square test and inconsistency index (I 2 ). If the studies were low heterogeneity (P>0.1, I 2 < 50%), a fixed-effects model was used. Otherwise, a random-effects model was applied. We did not assess publication bias because only a small number of studies were included in the meta-analysis (n max = 7). All statistical analyses were conducted with Review Manager version 5.3 and STATA version 16.
# Results
## Study search, selection, and characteristics
A literature search identified 829 records after removing duplicates, and seven studies (20-23, 31-33) met the inclusion criteria after screening by title, abstract, and full text. The seven clinical trials included patients treated with anti-PD-1/ PD-L1 agents and standard treatment and were comprised of two randomized controlled trials (RCT) and five singlearm trials.
The summary of the risk of bias for the two RCTs was shown in. The Newcastle-Ottowa Scalescore of the five single-arm studies was 5 (Supplementary.
## Patients characteristics
A total of 814 patients were included, 671 (82.4%) of which had HPV status reported. Most of the patients were male (80.5%); the mean of the median age was 60.2 years (range 20-90) across the included trials. There were 217 (32.3%) HPV positive patients and 454 (67.7%) HPV negative patients. A summary of the anatomical subsites included was reported in. The most common subsite in included trials was the oropharynx (n = 259, 31.8%). As shown in, the median OS and duration of response across the included studies were longer than the standard therapy arm in the Checkmate141 study (6.0-13.0 months vs. 5.1 months, 7.4-12.4 months vs. 4.0 months).
## Higher objective response rate in hpv positive hnscc patients
We conducted a pooled analysis to assess the clinical efficacy of anti-PD-1/PD-L1 agents in HNSCC patients grouped by agents and HPV status.
A total of 665 patients from seven studies with a reported ORR were included in this analysis. As shown in, the results revealed that HPV positive patients had a higher ORR than HPV negative patients, regardless of anti-PD-1 or anti-PD-L1 treatment (ORR: 21.5% vs 13.7%, odds ratio (OR) = 1.77, 95% confidence interval (95%CI) = 1.14-2.74; P = 0.01). Subgroup analysis demonstrated that the pooled OR with use of anti-PD-1 agents was 1.51 (95%CI = 0.90-2.54; P = 0.12). In comparison, the pooled OR with use of anti-PD-L1 agents was 2.66 (95%CI = 1.16-6.11; P = 0.02).
Favorable Overall Survival in HPV Positive HNSCC Patients 447 patients available from four studies showed that HPV positive patients had significantly better overall survival than
# Discussion
The impact of immunotherapy in the treatment of head and neck squamous cell carcinoma (HNSCC) has been rapidly progressing, with an associated survival benefit in approximately 20-30% of patients. We hypothesized that the distinct immunological tumor landscapes of HPV positive and negative HNSCC patients might confer a difference in survival rates and tumor response after anti-PD-1/ PD-L1 therapy. Previous evidence suggests that HPV may promote the expression of PD-L1 and PD-1 mediated through an IFN-g related response. Consequently, this may support the hypothesis that HPV is a favorable factor in both anti-PD-1 and anti-PD-L1 treated cancer patients. Through analysis of seven studies, including 814 patients, we demonstrated that HPV positive HNSCC patients treated with anti-PD-1 or anti-PD-L1 agents displayed significantly longer OS than HPV negative HNSCC patients, which is in concordance with a similar association observed in HPV positive vs. negative patients undergoing surgery or chemoradiotherapy. While the difference in PFS and DCR was not significant between HPV positive and negative patients, the limited number of studies here may be a potential factor influencing this result. Specifically, there were only four studies that reported DCR and two studies that reported PFS. As more trial data emerge with longer followup time, the true association of HPV status with DCR and PFS will be more definitively ascertained.
To our knowledge, this is the first study to report an association with improved outcomes using anti-PD-L1 agents in HPV positive HNSCC patients. The explanation for this observed association is likely complicated and multifactorial. In addition to binding to PD-1, PD-L1 may inhibit T cell proliferation and induce immune tolerance in vivo and in vitro via the interaction with other receptors such as CD80. Blocking PD-L1 on dendritic cells (DC) relieves cis sequestration of CD80, which allows CD80/CD28 interaction to enhance T cell priming. We have previously shown that HPV positive HNSCC patients might have a higher proportion of DCs than HPV negative patients. Furthermore, it has been shown that the HPV16 E7 oncoprotein may promote increased CD80 expression on DCs. Therefore, combined blockade of PD-1/PD-L1 and CD80/PD-L1 interactions by anti-PD-L1 agents in HPV positive HNSCC patients may represent a possible mechanism for increased benefit in these patients. Additionally, HPV16 E6/E7 oncoprotein may promote Akt activityand glucose consumption. In vitro culture of tumor cell lines with anti-PD-L1 directly might decrease AKT phosphorylation and glucose uptake in the absence of PD-1expressing T cells, which may directly restrain tumor cell growth in turn. This could be another potential mechanism of increased benefit from anti-PD-L1 agents in HPV positive HNSCC patients. While these pathways provide a rational hypothesis towards explaining the difference in outcomes seen in our analysis, this does not imply that the mode of action of anti-PD-1/anti-PD-L1 inhibitors is different in HPV positive HNSCC patients compared with HPV negative. Rather, this demonstration in outcome difference suggests a need for translational research to better elucidate the underlying mechanism.
We noticed that there was a higher ORR in HPV positive over HPV negative HNSCC patients undergoing anti-PD-1 therapy in our analysis; however, this difference failed to reach significance (P = 0.12). As more and higher-quality trial data emerges, this difference may also trend toward significance. Importantly, the number of patients in our anti-PD-1 treated studies (476 patients) outnumber those of the anti-PD-L1 studies (189 patients) in our analysis, suggesting that the preferential effect of anti-PD-L1 therapy cannot merely be explained secondary to low numbers in the anti-PD-1 treated studies. Furthermore, the proportion of HPV positive patients in the two subgroups of anti-PD-1 and anti-PD-L1 studies is similar (31.3% vs. 34.4%,, underlining the evenness of HPV positive patients across the two different treatment-subgroups. From an immune perspective, it has been theorized in previous studies that anti-PD-1 agents would have a more extensive effect beyond the PD-L1 pathway, with the notion that blocking the PD-1 receptor would interfere with interactions with multiple ligands, including PD-L1 and PD-L2. Here, it is important to note that PD-L2 expression is variable, and it is not as highly expressed as PD-L1 on tumor and immune cells, making its role in anti-cancer immune suppression unclear. Differences in the expression of PD-L2 between HPV-positive and HPV-negative tumors may offer a potential explanation for the differences observed in our study.
Previous work has been published that investigates the relationship between HPV status and response to anti-PD-1/ PD-L1 therapy. Patel et al.and Wang et al.both showed a trend towards significance for higher response rates in HPV positive vs. HPV negative tumors in patients receiving anti-PD-1/PD-L1 therapy. Additionally, a recent report indicated that immune checkpoint blockade immunotherapy enhances ORR in HPV positive HNSCC patients compared with HPV negative patients. However, a major limitation of this report is that it only included four clinical trials, which does not encompass the full scope of the present literature on the topic. Furthermore, the key limitation in these previously published studies is the lack of stratification by anti-PD-1 or anti-PD-L1 therapy separately. Our study is the first to do so, and this has demonstrated the possibility of a meaningful difference in outcome for HPV positive HNSCC patients treated with either anti-PD-1 or anti-PD-L1 blockers. In our study, we used restricted inclusion criteria to ensure the quality of all the eligible studies (Keynote-040 (34) and Keynote-048were excluded due to no published ORR for HPV positive patients, and other data from trials like EAGLE (NCT02369874), NCT02684253were not available yet), but were still able to include a larger number of patients with HPV information.
# Limitations
Our study has several limitations. Firstly, some information was not available in all included studies, which prevented us from performing other informative sub-analyses such as stratification by anatomic subsite, the combination of PD-L1 expression and HPV subgroups, and drug dosage. In further studies, anatomic subsite in the head and neck is an essential factor to consider as it is known that the prognostic value of HPV status, based on available data, is thus far limited to the oropharynx. Particularly, as PFS is really the gold standard for the effect of therapy, the limited data of PFS restrict the value of our conclusion. Additionally, as there are no HPV diagnostic tests with FDA regulatory approval for head and neck cancers, the methodology to determine HPV status across the included trials differed based on the local institution or licensed lab. The most c o m m o n m e t h o d i n t h e i n c l u d e d t r i a l s w a s p 1 6 immunohistological staining applied to oropharyngeal cancer as well as non-oropharyngeal cancer, which may be imperfect and would therefore bring bias, albeit minor, to the analysis. Furthermore, a vast majority of HPV positive tumors are located within the oropharynx. The overall PD-L1 expression information is summarized in . Nevertheless, the interaction between PD-L1 and HPV is difficult to estimate due to the currently available data. It is possible that elements of our analysis are confounded by differences in response and survival related to the subsite and PD-L1 expression itself. These considerations should be addressed carefully as more randomized controlled trial data matures in order to confirm our findings and to explore other possible factors related to response to immunotherapy in HNSCC.
# Conclusion
HPV positive HNSCC patients display improved outcomes with PD-1/PD-L1 axis blockade as compared to HPV negative HNSCC patients. These improved outcomes are likely driven to a greater extent by anti-PD-L1 inhibitors. However, randomized controlled trials with greater numbers of patients are needed for validation of these early findings.
# 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.
# Author contributions
GZ, YL, RM, and XZ conceived and designed the study. YX, DH, and IW extracted, analyzed, and interpreted the data. YX and GZ drafted the article. TS, CM, YL, RM, and XZ did the critical revision of the article for important intellectual content. All authors contributed to the article and approved the submitted version. |
Bayesian modelling of population trends in alcohol consumption provides empirically based country estimates for South Africa
# Gather statement
This study complies with the Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER) recommendations. [bib_ref] Guidelines for Accurate and Transparent Health Estimates Reporting: the GATHER statement, Stevens [/bib_ref] The step involved in the analytical procedures and the data sources upon which the estimates reported in this article are based are described as detailed in [fig_ref] Table A1: GATHER checklist [/fig_ref] below.
## Data inputs
for all data inputs from multiple sources that are synthesized as part of the study:
3
Describe how the data were identified and how the data were accessed.
Main text (Methods) and Additional File 1 (Data Sources).Specify the inclusion and exclusion criteria. Identify all ad-hoc exclusions.
Additional File 1 (Data Sources).Provide information on all included data sources and their main characteristics. For each data source used, report reference information or contact name/institution, population represented, data collection method, year(s) of data collection, sex and age range, diagnostic criteria or measurement method, and sample size, as relevant.
Additional File 1 (Data Sources).Identify and describe any categories of input data that have potentially important biases (e.g., based on characteristics listed in item 5).
## Main text (introduction) and additional file 1 (data sources)
For data inputs that contribute to the analysis but were not synthesized as part of the study:Describe and give sources for any other data inputs.
## Additional file 1 (data sources)
For all data inputs:Provide all data inputs in a file format from which data can be efficiently extracted (e.g., a spreadsheet rather than a PDF), including all relevant meta-data listed in item 5. For any data inputs that cannot be shared because of ethical or legal reasons, such as third-party ownership, provide a contact name or the name of the institution that retains the right to the data.
Additional File 1 (Data Sources) and Additional File 2 (Dataset 1).
# Data analysis
9
Provide a conceptual overview of the data analysis method. A diagram may be helpful.
Main text 10 Provide a detailed description of all steps of the analysis, including mathematical formulae. This description should cover, as relevant, data cleaning, data pre-processing, data adjustments and weighting of data sources, and mathematical or statistical model(s).
Main Text (Statistical modelling) and Additional File 1 (Additional methods)Describe how candidate models were evaluated and how the final model(s) were selected.
Main Text (Statistical modelling) and Additional File 1 (Additional methods)Provide the results of an evaluation of model performance, if done, as well as the results of any relevant sensitivity analysis.
Additional File 1 (Model checking, Sensitivity analysis)Describe methods for calculating uncertainty of the estimates. State which sources of uncertainty were, and were not, accounted for in the uncertainty analysis.
Additional File 1 (Additional Methods) and Main Text (Methdos, Discussion)State how analytic or statistical source code used to generate estimates can be accessed.
## Additional file 1 (stan model and r code)
# Results and discussion
## 15
Provide published estimates in a file format from which data can be efficiently extracted. 3. Sampling design and realisation and/or data collection methods not described with sufficient detail to evaluate the level of representativeness and quality.
The search with the specified inclusion and exclusion criteria retrieved the 17 surveys listed in [fig_ref] Table A2: Data SourcesFurther 15 surveys -subsequent iterations of the All Media Products Survey [/fig_ref] , with data collection spanning between 1998 and 2016. In all surveys, the target population was the South African population resident in private households, workers' hostels, convents and monasteries. The data frame excluded other collective living quarters, such as student hostels, old age homes, hospitals, prisons and military barracks. In all cases the target population included subjects 15 years old and above, and in some cases also younger individuals (of no interest for this study and excluded from the datasets). 2. the reference period to distinguish between former and current drinkers and to recall alcohol consumption varied across surveys, as per [fig_ref] Table A3: Recall period ‡= recall period to calculate the frequency of drinking/average consumption [/fig_ref] below: For all sources, details on the sampling strategy and realisation (including response rates at the different levels of clustering), survey questionnaire and other metadata are publicly available from the repositories above and/or the references in [fig_ref] Table A2: Data SourcesFurther 15 surveys -subsequent iterations of the All Media Products Survey [/fig_ref]. [fig_ref] Table A4: Estimated recorded, unrecorded and total alcohol consumption per capita in South Africa... [/fig_ref] shows yearly estimates of recorded, unrecorded and total alcohol consumption per capita (APC) between 1998 and 2016 obtained from the study by Manthey et Al. [bib_ref] Global alcohol exposure between 1990 and 2017 and forecasts until 2030: a..., Manthey [/bib_ref] * and converted from litres per year to grams per day by using the conversion equation:1 l pure alcohol ≡ 793 g (A1)
## Alcohol per capita
The 95% confidence intervals for total APC in the table are obtained by approximating the standard error (se) with the relative uncertainty related to recorded/unrecorded APC: ]
[formula] se AP C = 0.1 · AP C recorded + 0.5 · AP C unrecorded √ 100 (A2) [/formula]
and then calculating the bounds of the intervals with the normal approximation.
## Population structure
Estimates on the distribution of the South African adult population (15+) across sex and 10years age groups for each year between 1998 and 2013 were obtained from the Centre for Actuarial Research (CARe, http://www.care.uct.ac.za/) at the University of Cape Town. Distributions for 2014, 2015 and 2016 were approximated by linear extrapolation of the last two years (see Dataset 1, Additional File 2).
## Potential sources of bias
The general limitations of survey data highlighted in the main text (likely underreporting of consumption, uncertainty of alcohol content of the various types of drinks, categorisation of responses, incoherence or lack of precise definition of reporting periods) apply to the data sources considered for this study. Of particular relevance:
1. Current use and quantity consumed are all self-reported and thus subject to a potentially large degree of bias. Probst and colleagues [bib_ref] Coverage of Alcohol Consumption by National Surveys in South Africa, Probst [/bib_ref] investigated the level of underreporting for some of these surveys and 2012, NIDS 2012) and found that it varies between -84% to -72% (with reference to recorded alcohol consumption);
2. as shown in [fig_ref] Table A3: Recall period ‡= recall period to calculate the frequency of drinking/average consumption [/fig_ref] , the reference period for distinguish former from current drinkers is not completely consistent across surveys. While the relationship between recall period and bias is a complex one and moderated by drinking patterns, longer recall periods are generally associated with higher levels of underreporting. [bib_ref] Influence of the recall period on self-reported alcohol intake, Ekholm [/bib_ref] [bib_ref] Under-reporting of alcohol consumption in household surveys: a comparison of quantity-frequency, graduated-frequency..., Stockwell [/bib_ref] 3. the categories used to quantify drinking frequency and typical quantity per drinking occasion are not identical across surveys. A further source of bias in survey data relates to the use of sampling weights. All surveys considered in this study collected data using a multi-stage clustered sampling design and included stratification and oversampling of sub-populations of specific interest. Each dataset included, as per common practice, a set of sampling weights calculated in order to inflate the sample and represent the target population, taking into account not only the survey design (i.e. the inverse of the probability of being included in the sample, given the survey design) but also (as far as possible) non-response and coverage errors. To obtain the latter result, the weights provided with the surveys are calibrated, i.e. adjusted so that their sum across specific population strata (defined by sex, age category, race and province in our datasets) matched externally supplied population totals. Unfortunately, it is known that the population totals used for the calibration of weight in the various surveys do not represent a consistent temporal series, because the different surveys used the population totals available at that point in time from the national statistics agency (Statistics South Africa, http://www.statssa.gov.za) which are periodically adjusted when more precise data become available from a census or other sources.This might introduce bias when, as in this case, the interest lies in the joint analysis of multiple datasets.
# Additional methods
## Recalibration of sampling weights
We used the population totals described in Section 2.3 to recalibrate the sampling weights in each survey in order to match as closely as possible the gender and 5-years age group distribution in each of the 9 Provinces (first level of administrative subdivision) of South Africa in the corresponding year. We used the Deville and Sarndal calibration method described by Pacificoand implemented in the Stata Rcommand reweight and we bound the resulting weights to the interval [0.2;-5].
The iterative calibration algorithm converged in all cases with a number of iterations between 25 and 345 and tolerance < 10E-7. [fig_ref] Figure A1: Original vs [/fig_ref] illustrates the differences between the original and recalibrated population totals per sex and age group. [fig_ref] Table A6: Definition of 'current drinking status' in each survey [/fig_ref] summarises the strategy used to recover a binary indicator of 'current drinking' at individual level from the response to the relevant items in each survey dataset.
## Extracting drinking status form survey items
## Calculating consumption intervals
For surveys that collected data with frequency/quantity questionnaires, we calculate individual consumption intervals by multiplying the bounds of the frequency interval by the bounds of the typical quantity interval and converting the result in g/day.
For example, if an individual indicated that he/she drunk between 2 and 3 times a week, and the typical quantity consumed in a drinking occasion was between 1 and 2 standard drinks, the resulting consumption interval ranged between:
## Quality effect weighting and effective sample size
For the preprocessed datasets including the estimated proportion of individuals in each consumption class, we calculated quality effect weights with the procedure described by Doi et al. [bib_ref] Advances in the Meta-Analysis of Heterogeneous Clinical Trials II: The Quality Effects..., Doi [/bib_ref] We used the variance of the estimated proportion in each class as a measure of random variability (the ν j in Doi's formulae) and the risk of bias scores from [fig_ref] Table A5: provides a summary measure of the risk of bias associated with alcohol... [/fig_ref] rescaled to the 0-1 interval as a measure of the variability due to study bias (the Q j in Doi's formulae).
We then calculated the effective sample size of each estimate (the ne s,g,a in the expression of the model likelihood function) by redistributing the total sample size across all surveys proportionally to the quality effect weights (the w " j in Doi's formulae).
## Parametrization of the gam and choice of the number of bases
In both the consumption and prevalence splines, we impose relatively low 'flexibility' (i.e. less degrees of freedom) along the temporal axis, deeming implausible large variations year-by-year. Conversely, we allowed higher flexibility (more degrees of freedom) on the age axis, because of the evidence from various populations that relatively sharp variations in alcohol consumption patterns with age are common, especially at younger ages. [bib_ref] Life Course Trajectories of Alcohol Consumption in the United Kingdom Using Longitudinal..., Britton [/bib_ref] This translated in the following choices for the number of bases: dc 1 = 3 ; dc 2 = 5 ; dp 1 = 3 ; dp 2 = 5 (A3)
As a sensitivity analysis, we re-fitted the model with increased dimension for the time bases (dc 1 = dp 1 = 5), and the results shown in Section 4.2.1 did not alter the substantive conclusions regarding time and age patterns in the population.
We implemented the GAMs described in Eq. 7 and 8 in the main text using the mixed effects parametrization from the R package mgcv.In this representation, the wiggly parts of the spline basis are treated as a random effect and their associated variance parameter controls the degree of wiggliness of the fitted spline. The perfectly smooth parts of the basis are treated as a fixed effect. With this parametrization, Eq. 7 becomes (vectors and matrices in bold): With the choice of number of bases indicated above, the model A4 has a total of 15 coefficients to be estimated.
[formula] µ i = exp(b i + [/formula]
The model in Eq. 8 of the Article is parametrized in the same way.
## Soft-capping the distribution
To formally translate the assumption that average consumptions of more than 150 g/day of pure alcohol are extremely unlikely, we impose the prior shown is [fig_ref] Figure A2: Custom prior distribution for the 97 [/fig_ref] to the 97.5 th percentile of the population distribution. The prior is uniformly distributed in the interval (0; 150] g/day with 97.5% of probability and decays exponentially for higher values. It is formally defined by Equation .
[formula] f (x) = 0.975 150 x < 150 0.0250e −0.26(x−150) x >= 150 (A5) [/formula]
Because no closed form exists that expresses the quantiles of the Gamma distribution as a function of its mean µ and shape α, imposing exactly this constraint would have required solving numerically an equation at each iteration during the sampling process. To reduce the computational burden, and given the limited range of µ and α of interest in our study, we considered the following linear function to approximate the relationship in that range:
[formula] 97.5 th percentile = β 0 + β 1 µ + β 2 α + β 3 µα (A6) [/formula]
and we recovered the best-fitting parameters (for α ∈ [0.5; 0.9] and µ ∈ [10; 350]) with ordinary least square:
[formula] β 0 = 3.259e −12 β 0 = 6.397 β 0 = −1.887e −12 β 0 = −2.884 (A7) [/formula]
We deemed the result (see [fig_ref] Figure A3: Approximate linear relationship between mean and 97 [/fig_ref] adequate for the purpose. [fig_ref] Table A7: Prior distribution of model parameters [/fig_ref] lists the prior distributions assigned to the 324 model parameters.
## Priors
## Stan model and r code
The Stan model and the associated R code used to produce the results presented in this article are available on request from the corresponding author ([email protected]). [fig_ref] Figure A4: Posterior log-likelihood * [/fig_ref] shows the trace plot for the log-likelihood, its posterior distribution and the autocorrelation between draws. The potential scale reduction statisticR varied between 1.000 and 1.023 across the 856 parameters and the effective sample size ECC between 539.7 and 13613.7. Dataset 4 in Additional File 2 shows summary statistics for the distribution of each parameter. shows the quantile-quantile plots for the residuals of the prevalence estimates from each survey. shows the QQ plots for the randomised quantile residuals of the consumption estimates. [bib_ref] Randomized quantile residuals, Dunn [/bib_ref] Randomised residuals are reported in the latter case because the interval-censored nature of the input data makes the calculation of ordinary probability residuals not meaningful (see: https://mjskay.github.io/tidybayes/articles/tidybayes-residuals.html qqq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qq q qq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qq qq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qq qq q q q q q q qq qq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qqq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qq qq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qq qqq q q q q qqq qq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q qqqq q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q shows the predicted trends in prevalence of drinking and average consumption among drinkers when the dimension of the spline bases is increased. In both cases the results are substantively similar to the results of the main analysis. : Trends in prevalence of drinkers. South Africa, population 15+, 1998-2016. Per sex and age category. Sensitivity analysis for increased spline base dimension (dc 1 = 5 ; dc 2 = 5 ; dp 1 = 5 ; dp 2 = 5) [fig_ref] Figure A9: Trends in prevalence of drinkers and average consumption among drinkers [/fig_ref] shows how trends estimates changed when we dropped the assumption of a 20% wastage of alcohol, and we assumed than all APC was actually used for drinking. The new estimates show limited (if any) change in the prevalence across all age classes, and, as expected, a marked overall increase in the average consumption. Interestingly, the increase is not distributed uniformly across age categories and sexes. On the contrary, it appears mostly concentrated in the highest consumption categories, i.e. men 25-44 years. [fig_ref] Table A8: Differences in the estimated prevalence of drinkers and average alcohol consumption among... [/fig_ref] shows the differences in prevalence of drinkers and average consumption among drinkers for selected years estimated by the model in the the two hypotheses of 20% wastage and no wastage. [fig_ref] Table A9: Average alcohol consumption and proportion of total volume consumed at country level... [/fig_ref] shows the average alcohol consumption by light, intermediate and heavy drinkers, and the proportion of the total consumption at country level accounted for by each category. Light = Light drinkers: average daily consumption < 12/24 g; Inter = Intermediate drinkers: average daily consumption ≥ 12/24 g and < 40/60 g; Heavy Heavy drinkers: average daily consumption > 40/60 g. The first figure refers to females, the second to males.
# Additional results
## Model checking
## Convergence statistics and posterior distribution of parameters
## Prevalence/consumption estimates: residuals and posterior predictive check
# Sensitivity analysis
## Increasing spline bases dimension
## Prevalence and consumption assuming no wasted alcohol
## Consumption by drinking category
## References
[fig] Figure A1: Original vs. recalibrated population totals. marks = original; red dots: recalibrated. [/fig]
[fig] Figure A2: Custom prior distribution for the 97.5 th percentile of the distributions of average alcohol consumption among drinkers. [/fig]
[fig] Figure A3: Approximate linear relationship between mean and 97.5 th percentile of the distribution of average alcohol consumption among drinkers for different values of the shape parameter α. Solid lines: exact percentile (by numerical solution of the equation); + = model-predicted percentile [/fig]
[fig] Figure A4: Posterior log-likelihood * : trace , density, autocorrelation plot.* Un-normalised. Plots exclude warm-up samples. [/fig]
[fig] Figure A5, Figure A6: Standardised residuals for the prevalence of drinkers: QQ plots. Per survey. Randomised Quantile Residual for average consumption by drinkers: QQ plots. [/fig]
[fig] Figure: A7 compares the observed cumulative distribution of average consumption of alcohol among drinkers from each survey (recovered non-parametrically for the surveys with censored consumption data) with the distribution predicted by the model. [/fig]
[table] Table A1: GATHER checklist [/table]
[table] Table A2: Data SourcesFurther 15 surveys -subsequent iterations of the All Media Products Survey (AMPS) carried out annually by the South African Audience Research Foundation (SAARF) to collect data on ownership or usage of certain products and services -were also identified in the repositories, but excluded from the analysis for lack of information on sampling design and realisation. A further nationally representative survey -the South African Stress and Health (SASH) Study[19] -was suggested for inclusion by a member of the expert group which consult the Burden of Disease Research Unit on substance abuse related matters. We were, however, unable to access the microdata, which are not in the public domain.Of the 17 surveys inTable A2, the WHS 2003 and NIDS datasets are publicly available from DataFirst at the University of Cape Town (https://www.datafirst.uct.ac.za/dataportal/). The DHS 1998 and DHS 2016 datasets are also in the public domain and available for download from the DHS program data repository (https://dhsprogram.com/Data/). The SABSSM, SASAS and SANHANES dataset are available for download from the Human Sciences Research Council data repository (http://datacuration.hsrc.ac.za/), subject to approval by the curator. The DHS 2003 dataset is not on the public domain, but a copy can be requested to the South African National Department of Health through the National Health Research Database (https://nhrd.hst.org.za/). [/table]
[table] Table A3: Recall period ‡= recall period to calculate the frequency of drinking/average consumption. [/table]
[table] Table A4: Estimated recorded, unrecorded and total alcohol consumption per capita in South Africa between 1998 and 2016 [g/day per population 15+] [/table]
[table] Table A5: provides a summary measure of the risk of bias associated with alcohol related questions in the 17 data sources, as quantified by the Burden of Disease Review Manager BODRevMAN [/table]
[table] Table A6: Definition of 'current drinking status' in each survey [/table]
[table] Table A7: Prior distribution of model parameters. [/table]
[table] Table A8: Differences in the estimated prevalence of drinkers and average alcohol consumption among drinkers in the hypothesis of 20% vs. 0% wastage. Year 1998, 2008,2016. By age and sex. [/table]
[table] Table A9: Average alcohol consumption and proportion of total volume consumed at country level per drinking category. By year and sex. [/table]
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Real-time monitoring and just-in-time intervention for adherence to pre-exposure prophylaxis among men who have sex with men in China: a multicentre RCT study protocol
Background: Pre-exposure prophylaxis (PrEP) is an effective biomedical strategy to prevent transmission of HIV infection, although medication adherence remains a challenge. We present the protocol for a multicentre randomised controlled trial to measure the effectiveness of a real-time monitoring and just-in-time intervention on medication adherence among PrEP users in China.Methods: Study participants will include 1000 men who have sex with men (MSM) from four cites in China (Shenyang, Beijing, Chongqing and Shenzhen) attending a tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) PrEP project as part of a real-world, prospective multicentre cohort study (CROPrEP). Participants will be randomised into the intervention and control arms in a 1:1 ratio. Participants in the intervention arm will be provided with remote real-time monitoring equipment that triggers twice just-in-time SMS (Short Messaging Service) medication reminders to PrEP users every half an hour when a scheduled dosage is missed, and followed with just-in-time SMS medication reminders to clinicians half an hour when there is no supplement after the second just-in-time SMS reminder to PrEP users. Clinicians will initiate individualised telephone intervention as soon as possible upon receipt of the just-in-time SMS missed dose alert. Those in the control arm will only receive generic weekly SMS reminders. The study will last 6 months. Participants will be seen at follow-up visits at three and 6 months. Trial outcomes to be measured include self-reported adherence assessed via questionnaire and pill counts, as well as drug concentration test results.(Continued on next page)
# Background
Pre-exposure prophylaxis (PrEP) is recognized as highly effective in preventing new HIV infections in high-risk populations. However, multiple clinical trials worldwide have shown that medication adherence to PrEP is suboptimal, which reduces its effectiveness. The literature on medication adherence interventions provides several examples in the field of antiretroviral therapy (ART) and other clinical areas. However, while scientific consensus on the efficacy of PrEP has been attained, there has been little work in development and evaluation of interventions to improve PrEP medication adherence.
Measuring adherence to PrEP is challenging and has traditionally been done using self-report questionnaires and by measuring pill counts and blood drug concentrations. All of these methods are subject to limitations posed by the intermittent nature of data collection. For example, missed doses may be detected several weeks to months after they occur. In addition, interventions can only be conducted after poor medication adherence is detected by clinicians through follow-up interviews, pill counts, drug concentration, and adherence scales. Therefore, it is often impossible to initiate medication adherence interventions before viral rebound that would lead to treatment failure and drug resistance.
Electronic drug monitors (EDM) constitute a novel technology that has been applied in clinical practice for real-time adherence detection, reminder of adherence lapses, and interventions to resume treatment before treatment failure and onset of drug resistance.
The EDM system involves the use of a microchip embedded inside an intelligent pill container that records when the container is opened as an indication that the medication has been taken. A key innovation of advanced EDM technologies is to combine the Internet of Things with smartphone technology to monitor adherence in real time and provide personalised intervention. Patients can set their medication reminders (pillbox ringing and mobile phone reminders) to notify them when a scheduled dose is missed. The EDM system stores and uploads medication use data to support joint management by clinicians, the patient, and family members to improve medication adherence. Graphical adherence curves and scoring information can be generated on linked mobile phones, supporting real-time intervention from professionals when needed to avoid missed doses and overdoses.
This technology has been used to improve medication adherence of HIV treatment and other chronic diseases. However, the device has not yet been tested for PrEP. We now present a study protocol examining the use of EDM technology to monitor PrEP adherence in real time, along with personalised intervention to users to assess improvements in medication adherence with this system. This randomised controlled trial will prospectively assess the efficacy of this approach, providing scientific evidence to guide development of future PrEP intervention strategies.
# Methods
## Objectives
The objective of the study is to estimate the effectiveness of real-time monitoring and just-in-time intervention technology for improving PrEP medication adherence among Chinese MSM.
## Study setting
The study includes four study centres (First Affiliated Hospital of China Medical University in Shenyang, Beijing You'An Hospital of Capital Medical University in Beijing, Shenzhen Third People's Hospital in Shenzhen, and Chongqing Public Health Medical Treatment Centre in Chongqing).
## Trial design
This study is organized within the ongoing CROPrEP project, a multicentre, real-world prospective cohort study of daily or event-driven PrEP (emtricitabine/tenofovir disoproxil fumarate, TDF/FTC) among 1000 HIVnegative MSM most-at-risk of HIV infection in China. For the present study protocol, we randomly divided the 1000 CROPrEP study participants (500 from daily TDF/ FTC group and 500 from event-driven TDF/FTC group in the CROPrEP study) into intervention and control arms in a 1:1 ratio (250 in intervention arm and 250 in control arm for each of the original CROPrEP study groups).
## Eligibility criteria
The current RCT will be conducted with participants in the ongoing CROPrEP study. The inclusion criteria for the CROPrEP study include: (1) HIV-negative MSM; (2) aged 18-65 years; (3) report no severe damage to liver or kidney function; (4) self-report at least one of the following behaviours during the last 6 months: engaging in unprotected receptive anal sex with men, multiple male sexual partners, current or previous STIs (syphilis, gonorrhoea, chlamydia, chancroid, venereal lymphogranuloma, or other STI), had a history of post-exposure prophylaxis (PEP) but did not receive PEP in the last month; (5) not planning to leave local area of residence within the next 6 months (to ensure cohort retention); and (6) being able and agreeing to provide written informed consent. Individuals who maintained a monogamous sexual relationship with only one HIV-1 negative partner or one virologically suppressed HIV-1 positive partner for more than 1 year will be excluded.
## Interventions
Participants in the intervention arm will be provided with a real-time monitoring device administering twice just-in-time SMS medication reminders to PrEP users every half an hour when a scheduled dosage is missed and followed with just-in-time SMS medication reminders to clinicians half an hour when there is no supplement after the second just-in-time SMS reminder to PrEP users. Clinicians will initiate individualised telephone intervention as soon as possible when they receive the just-in-time SMS medication reminder. Telephone interventions will be carried out at the same day for reminders received before 4:00 p.m. and 8:00 a.m. the next day for reminders received after 4:00 p.m., whereas control arm participants will receive general weekly SMS medication reminders. Participants in both groups will be invited to attend follow-up clinic visits at three and 6 months. The flow chart depicting the study design is shown in.
## Sample size
We have powered the RCT to be able to detect meaningful changes in optimal adherence. Our estimates of outcome differences are based on our basic findings from our earlier pilot study with Chinese PrEP users, in which we found that proportions of adherence were 94% versus 86%, in intervention versus control patients after 6 months of the intervention. We will recruit 500 PrEP users per study arm. With the consideration of a twosided alpha of 0.05 and 40% loss to follow-up of our study, we will have a 90.6% power at to detect specified differences in adherence between the two arms after intervention.
## Real-time monitoring equipment
The medication adherence monitor (Msense, Icompanion Health Technologies, Guangzhou, China) records dosing behaviour in real time using microchips embedded in the container vial cap to detect when the container is opened. This approach assumes that the participant takes the medication every time the monitored equipment is opened.
The real-time monitoring device can set the medication time, issue reminders for medicine to be taken, and trigger voice alerts for repeat doses. Data are automatically uploaded to a cloud-based server and can then be accessed by clinicians, PrEP users and family members via WeChat platform. Clinicians can monitor real-time data on PrEP use in the intervention arm from a backend platform and can provide just-in-time intervention to participants who did not take medicine according to prescription. PrEP users can also check their records on medication taken and not taken, to monitor their own medication usage and for reference during clinic visits. Graphical medication adherence curves and scoring data can be generated on mobile phones to support selfmanagement and personalised reminders. If participants in the intervention arm do not take medication on time according to prescription, clinicians and family members can send a message or initiate voice calls directly to the patient. Details on the intervention procedure are illustrated in.
## Randomisation
Following the baseline study visit, all participants are randomised to one of the two study arms using 1:1 randomisation. The PASS 11.0 randomisation module will be used to ensure equal sizes of the two study groups. Each group will have 500 participants, and two groups of 500 random numbers will be generated for randomised grouping of the daily dose group and the eventdriven group to ensure equality of the two medication dosing strategies within the intervention arm.
Accordingly, both the PrEP daily FTC/TDF group and the event-driven FTC/TDF group will have 250 PrEP users randomly assigned to the intervention arm and 250 assigned to the control arm. Recruitment targets of four study sites were determined based on patient volume and are shown in.
All study participants will be seen for a baseline study visit, followed up at 3 and 6 months, and fill an online self-administered questionnaire assessing sexual behaviours, psychological status, self-reported medication adherence, attitude and side effects towards PrEP use.
Blood samples and dry blood spots will be collected and tested as previously described for the CROPrEP project. Pills remaining will be counted at the 3-and 6month follow-up visits, and refills will be provided to last until the next study or clinic visit. Participants will be required to return the real-time monitoring device provided by the program when the study is completed.
The program will incorporate locally trained community-based organizations (CBOs) serving MSM to recruit eligible participants for the study. CBO peer educators will be responsible for guiding the correct use of the monitoring device and will provide support services through social media platforms (WeChat, QQ). The study also offers incentives of US $10 upon completion of the enrolment visit and US $10 each at the 3-and 6-month visits (US $30 total) to compensate the time contributed by participants.
## Laboratory procedure
The laboratory testing procedures and parameters are described in the published protocol of the CROPrEP project. MSM who are enrolled in the study and later screen as HIV positive will be instructed to discontinue PrEP and visit the local study hospital for HIV confirmation.
## Data collection and management
The study questionnaire will be administered via a specialised online platform (Gold Data Technologies, Sichuan, China). Trained staff will collect data on remaining pill counts and verify test results.
CBO peer educators will instruct study participants on use of the real-time monitoring device at the baseline study visit and throughout follow-up using related images and videos in order to avoid overestimates (opening the container without removing pills) or underestimates (multiple pills removed during one opening of the monitoring device) of actual medication adherence. The monitoring device battery is sufficient to run in standby mode for 45 days after a 2-h charge.
## Study endpoints
Medication adherence refers to the degree to which a patient complies with the doctor's prescription to complete the treatment plan. It involves taking regularly, not forgetting to take medication, and taking medication at the correct time, i.e., taking medicine according to the prescribed timing, dosage, and administration instructions. PrEP medication adherence will be measured via self-report questionnaires and pill counts as well as drug concentration test results. Details on the study endpoints are shown in .
# Statistical methods
Normal distribution continuous variables will be presented by means and standard deviations while nonnormal distribution continuous variables will be presented by medians and interquartile ranges. For categorical variables, composition ratios and 95% confidence intervals will be used to describe the variables. Chi-square analysis will be used to assess differences between the study arms in HIV testing history, HIV self-testing (HIVST) characteristics, and high-risk behaviours. Multinomial logistic regression will be used to measure the outcome of real-time monitoring equipment on medication adherence.
## Ethics and dissemination
Written informed consent will be provided to all study participants before take part in this study.
# Discussion
Traditional methods of measuring medication adherence such as self-report and pill counting are executed intermittently or at the end of the study period, often several months after the dosage has been missed. Accordingly, these methods often cannot guide intervention before the disease recurs or worsens. Hence, the ability to change medication adherence and prevent treatment failure or drug resistance with these methods is limited. In contrast, EDM is characterised by real-time monitoring and data processing, which allow healthcare teams and family members to monitor medication adherence and send reminders just-in-time to patients who have missed doses, before the disease may worsen. EDM is therefore a more efficient strategy to improve medication adherence.
Medication adherence is crucial for achieving optimal benefits from PrEP. However, many participants overreport PrEP medication adherence by traditional approaches such as self-reporting and pill counting, which adds to the challenge of accurately measuring medication adherence. With traditional approaches, measurement of adherence after doses have been missed leads to an inability to provide timely intervention and thus to worse adherence. Real-time monitoring technology creates the ability to monitor medication adherence between routine visits and can trigger just-in-time intervention before adherence has lapsed for a substantial amount of time. Our study will be the first to directly compare traditional measurement of adherence versus real-time monitoring with just-in-time individualised intervention in PrEP users. This study is focused on a novel, individualised behaviour intervention to reduce the risk of decreased medication adherence among PrEP users in China. Ours will be the first study of this type of intervention for improvement of PrEP medication adherence. Just-in-time telephone intervention requires investment of more time and energy. Our intervention employs novel technology combined with just-in-time SMS medication reminders and individualised telephone intervention to increase the intensity and efficiency of improving PrEP medication adherence in real time. Furthermore, telephone intervention only focuses on those with a missed dosage after just-in-time SMS reminders which can save time and energy. The control group will receive weekly universal undifferentiated SMS medication reminders since the real-time medication condition is unknown. Clinicians can provide just-in-time professional intervention based on information from the back-end real-time monitoring platform to improve the efficacy of PrEP in preventing the transmission of HIV infection. Dose-to-dose medication adherence data and curves generated on adherence patterns are informative in promoting selfmanagement and intervention from others who are engaged with participants. The study has several important strengths, including capitalizing on the infrastructure of a large-scale, real-world multicentre study in China, use of real-time monitoring devices with just-in-time intervention to advance medication adherence, and measure the efficacy of such devices compared to traditional approaches.
Our study tests a real-time monitoring device with just-in-time intervention against other traditional approaches to address medication adherence in PrEP users. We hypothesize that by targeting real-time monitoring and intervention to reduce the risk of viral rebound, our strategy will improve TDF/FTC medication adherence, allowing clinicians and family members to maintain confidence regarding adherence.
Our study should be evaluated considering several limitations. First, the CROPrEP project is focused on the MSM population, in which medication adherence characteristics may be different from other patient populations. It has been shown that research study participants generally exhibit higher medication adherence levels than other clinical patients. Accordingly, we recommend that future studies be conducted in other key populations to validate the effects of real-time monitoring and just-in-time intervention on PrEP medication adherence. In addition, the cost of real-time monitoring devices has limited their large-scale uptake in research and general clinical care. Thus, development of low-cost versions of these devices will be critically important for their future application.
# Acknowledgments
We would like to thank Yao Li, Fang Zhao for their efforts in the implementation of the project. We would also like to express our sincere appreciation to the study participants and community partners for their participation and support with recruitment for the study. Authors' contributions HS and JX is responsible for all aspects of the study. XJ, HW, ZC and HL contributed to participant selection and IRB application. XH, YC, HW, XH, LZ, JZ, QH, ZH, RB, HD and LS performed the study. XJ drafted the manuscript. JX, HW, YJ, WG and HS reviewed the manuscript and provided edits and comments. WL and XH provided input into the study design. All authors have read and approve the final submission manuscript. Study endpoints
Medication adherence scores -The proportion of self-reported number of pills taken among the number of pills prescribed by the doctor.
-The proportion of the counted number of pills taken among the number of pills prescribed by the doctor.
-The proportion of pills taken as calculated by remote real-time monitoring equipment among the number of prescribed pills by the doctor. Scores will be classified as high medication adherence (≥90%), intermediate medication adherence (≥60 and < 90%), or low medication adherence (< 60%).
Blood medicine concentration monitoring (TDF/FTC) Blood TDF/FTC concentration will be classified as high medication adherence (≥90% concentration), intermediate medication adherence (≥60 and < 90%), or low medication adherence (< 60%). |
Prevalence of dysmenorrhea among reproductive age group in Saudi Women
Background: The condition of recurrent, crampy, lower abdominal pain during menses is defined as dysmenorrhea. The study aims to assess the factors affecting the prevalence of primary and secondary dysmenorrhea among Saudi women from the reproductive age group.Methods:A cross-sectional survey-based study recruited 1199 participants through a systematic random sampling technique. The study was carried out among the reproductive age group in Saudi women (total number of 1199) who are more than 18-year-old and less than 45-year-old in Riyadh, King Dom of Saudi Arabia, using an electronic questionnaire.Results:The observed dysmenorrhea in the study; 1107 (92.3%) women had non-pathological dysmenorrhea (primary) while 92 (7.7%) women had pathological dysmenorrhea (secondary) respectively.Conclusion:In the present study, the prevalence of dysmenorrhea was high among the recruited Saudi women. The study suggests the inclusion of health education programs for students at the school and university level to deal with problems associated with dysmenorrhea that limit their interference with the student's life.
# Background
The crampy, recurrent pain in the lower abdomen during menses is defined as dysmenorrhea [bib_ref] Diagnosis and management of primary dysmenorrhea, Kho [/bib_ref]. Dysmenorrhea is divided into two broad categories, i.e., primary and secondary dysmenorrhea. The presence of crampy, recurrent pain in the lower abdomen during menses in the absence of demonstrable disease is primary dysmenorrhea. Adolescents and young women are more likely to be diagnosed with primary dysmenorrhea, an exclusionary diagnosis. Women suffer from pain related symptoms in secondary dysmenorrhea, with a disorder accounting for symptoms like endometriosis, uterine fibroids, or adenomyosis. The significant clinical features experienced by women suffering from secondary dysmenorrhea include pain during intercourse, resistance to effective treatment, and enlarged uterus [bib_ref] • fast, convenient online submission • thorough peer review by experienced researchers..., Iacovides [/bib_ref].
Prostaglandins play a significant role in inducing uterine contractions released from endometrial sloughing at the start of menses [bib_ref] Prostaglandin level of primary dysmenorrhea pain sufferers, Fajrin [/bib_ref] [bib_ref] The role of prostaglandins in dysmenorrhea and menorrhagia, Smith [/bib_ref]. The contractions occur at a frequency of > 4-5 per minute (high frequency) and are incoordinate and nonrhythmic. These contractions result in increased intrauterine pressures, which may even exceed 400 mmHg (ranging between 150 and 180 mmHg) [bib_ref] Reappraisal on the Management of Primary Dysmenorrhoea in Adolescents, Najimudeen [/bib_ref]. There is the development of uterine ischemia and accumulation of anaerobic metabolites as uterine pressure exceeds the arterial pressure stimulating type C pain neurons that cause dysmenorrhea. The pain perception can also be determined through stretch receptors' activation.
Dysmenorrhea is a common problem, and it is experienced by 50-90% of women in their reproductive years worldwide, describing having painful menstruation [bib_ref] Continuous norethisterone acetate versus cyclical drospirenone 3 mg/Ethinyl estradiol 20 μg for..., Nawaiseh [/bib_ref]. Young women with primary dysmenorrhea make the Open Access *Correspondence: [email protected] [bib_ref] Diagnosis and management of primary dysmenorrhea, Kho [/bib_ref] Clinical Sciences Department, College of Medicine, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia Full list of author information is available at the end of the article majority group of these women. Primary dysmenorrhea tends to decrease with advancing age. However, secondary dysmenorrhea develops later in life. The dysmenorrhea prevalence among Saudi young women ranges from 60.9 to 89.7% [bib_ref] Dysmenorrhea among female medical students in King Abdulaziz University Prevalence, Predictors and..., Ibrahim [/bib_ref] [bib_ref] Prevalence of menstrual problems and their association with psychological stress in young..., Rafique [/bib_ref] [bib_ref] Prevalence of menstrual problems and their association with psychological stress in young..., Rafique [/bib_ref] [bib_ref] Dysmenorrhea, associated symptoms, and management among students at King Khalid University, Saudi..., Saleem [/bib_ref] [bib_ref] Prevalence of primary dysmenorrhea and its effect on the quality of life..., Hashim [/bib_ref]. These studies were performed in different cities in Saudi Arabia [bib_ref] Dysmenorrhea among female medical students in King Abdulaziz University Prevalence, Predictors and..., Ibrahim [/bib_ref] [bib_ref] Prevalence of menstrual problems and their association with psychological stress in young..., Rafique [/bib_ref] [bib_ref] Prevalence of menstrual problems and their association with psychological stress in young..., Rafique [/bib_ref] [bib_ref] Dysmenorrhea, associated symptoms, and management among students at King Khalid University, Saudi..., Saleem [/bib_ref] [bib_ref] Prevalence of primary dysmenorrhea and its effect on the quality of life..., Hashim [/bib_ref]. Dysmenorrhea-associated risk factors include younger age (adolescents in particular), smoking, and stress [bib_ref] The prevalence and risk factors of dysmenorrhea, Ju [/bib_ref] [bib_ref] 345-Primary Dysmenorrhea Consensus Guideline, Burnett [/bib_ref]. Risk reduction is accompanied by hormonal contraceptives, higher parity, and having the first childbirth at a younger age [bib_ref] An epidemiologic study of young women with dysmenorrhea, Andersch [/bib_ref] [bib_ref] Use of medication by adolescents for the management of menstrual discomfort, Campbell [/bib_ref]. The severity of dysmenorrhea ranges from mild to severe [bib_ref] Diagnosis and management of primary dysmenorrhea, Kho [/bib_ref]. Patients with dysmenorrhea often report depressed mood, anger, eating more than usual, nausea, dizziness, headache, fatigue, diarrhoea, or constipation associated with dysmenorrhea [bib_ref] Dysmenorrhea, associated symptoms, and management among students at King Khalid University, Saudi..., Saleem [/bib_ref].
To attain targeted intervention and timely prevalence, it is significant to understand the disease burden among the population. In a similar context, the present study aims to determine factors affecting dysmenorrhea prevalence (primary and secondary) among Saudi women from the reproductive age group. The secondary objectives of the study are as follows;
- To determine the relationship between dysmenorrhea and intake of dairy products. - To determine the relationship between dysmenorrhea and exercise - To evaluate the effects of dysmenorrhea on the quality of life
# Methods
## Study design and sampling
A cross-sectional survey-based study was conducted, and the participants were recruited through a systematic random sampling technique. The study was carried out among 1199 Saudi women. Epi Info was used to calculate the sample size at a 95% confidence level.
## Inclusion criteria
The inclusion criteria for the study was Saudi women aged between 18 and 45 years and visiting the private clinic to undergo gynaecological examination for dysmenorrhea problems in Riyadh, King Dom of Saudi Arabia.
## Study tool
The self-administered online questionnaire was used as a study tool on Google Forms. This type of survey was easy to administer and could include many participants.
## Data collection
The study took place between July and September 2020. The questionnaire was translated into validated Arabic language and back-translated into English. The questionnaire contained many parts, including sociodemographic questions such as (age, marital status, weight, height), menses related information such as (age at menarche, duration of menstrual cycle in last 12 months, regularity of menstrual cycle, duration of the menstrual flow, number of used pads, type of dysmenorrhea) and any medical or psychological illness. Other questions included the severity of menstrual cramps and treatment, possible symptoms associated with dysmenorrhea, general assessment of dairy products intake, and the effects of dysmenorrhea on activities of daily living.
# Data analysis
The data gathered from the survey were entered on a Microsoft excel sheet and then analysed using Statistical Package of Social Sciences (SPSS) version 25 (SPSS Inc., Chicago, IL, USA). Frequencies and percentages were used to represent categorical variables; while, means and standard deviations represented continuous variables. Kolmogorov-Smirnov test was used to determine the normality of the tested data. However, groups on normally distributed variables were compared through parametric tests; while, skewed data was represented through non-parametric tests. The significant association between variables was determined using the chi-square / Fisher's exact test, considering that cell expected frequency is < 5. The mean significant differences between the patient's age and the dysmenorrhea group were determined using an independent sample t-test. The results were considered statistically significant with a P value < 0.05. [fig_ref] Table 1: Demographic profile of patients [/fig_ref] shows the demographic characteristics of 1199 women recruited in the study according to inclusion criteria. The mean age of women observed was 27.49 years, [fig_ref] Table 2: Past medical history and psychological illness of patients [/fig_ref]. [fig_ref] Table 3: Descriptive analysis of menstrual cycle, regularity, and type of dysmenorrhea [/fig_ref] shows a descriptive analysis of the menstrual cycle, regularity, and type of dysmenorrhea. According to it, 296 (24.7%) women had irregular menstrual cycles, and 103 (8.6%) had less than 21 days of the menstrual cycle, and 76 (6.3%) had irregular bleeding. The majority of them, 979 (81.7%), had the duration of the menstrual flow between 3 and 7 days. Around 92 (7.7%) women had pathological dysmenorrhea. [fig_ref] Table 4: Descriptive analysis of classification of dysmenorrhea pain, therapy and associated symptoms [/fig_ref] depicts the analysis of the classification of dysmenorrhea pain, therapy and associated symptoms. Around 170 (14.2%) patients had severe pain. The majority of women, 1086 (88.4%), experienced pain in the lower abdomen, and the majority, 808 (67.4%), had pain for more than 3 days. For relieving pain, only 55 (4.6%) patients used NSAIDs only, and 747 (62.3%) patients used more than one analgesic or other alternative therapies used for relieving menstrual cramps. Considering the possible symptoms associated with dysmenorrhea, abdominal bloating is the most common single symptom that was observed among 35 (2.9%) women with dysmenorrhea, whereas, majority of them, 926 (77.2%), had more than one possible symptom associated with dysmenorrhea [fig_ref] Table 4: Descriptive analysis of classification of dysmenorrhea pain, therapy and associated symptoms [/fig_ref]. [fig_ref] Table 5: Distribution of dysmenorrhea limitation, academic performance, exercise and diet during menstruation [/fig_ref] displays the distribution of dysmenorrhea limitation, academic performance, exercise and diet during menstruation. The different stress level was found during menstruation, where the majority, 614 (51.2%) of the women, usually had stress. Regarding limitation during menstruation, 161 (13.4%) women with dysmenorrhea reported to have a physical limitation, and 543 (45.3%) reported to have more than one limitation. Likewise, 122 (10.2%) reported dysmenorrhea affecting their concentration aspect of academic performance, and 462 (38.5%) patients had more than one factor affecting their academic performance. Around more than 512 (42.7%) women reported changes in sleeping routine. Furthermore, 268 (22.4%) performed more than one exercise during one exercise, and 260 (21.7%) reported exercise to reduce period pain. In connection with diet, 745 (62.1%) had all types of diet during the period. [fig_ref] Table 6: Impact and Association between dysmenorrhea and clinical and demographic characteristics of patients [/fig_ref] shows no statistically significant association among age, marital status, diabetes mellitus, IBS, Schizophrenia, and OCD with primary and secondary dysmenorrhea. However, most 775 (70%) women with age less than 30 years and the majority of the single women, 729 (65.9%), had primary dysmenorrhea. Around 55 (59.8%) women who had irregular menstrual cycles had significantly secondary dysmenorrhea (P < 0.001). Similarly, there was a significant association between the menstrual cycle duration in the last 12 months and primary and secondary dysmenorrhea (P < 0.001). Here, 11 (12.0%) women with less than 21 days cycle had secondary dysmenorrhea compared to 92 (8.3%) women with less than 21 days cycle with primary dysmenorrhea. Likewise, duration of the menstruation flow was significantly associated with the type of dysmenorrhea, were among women with more than 7 days cycle, 103 (9.3%) had primary and 19 (20.7%) had secondary dysmenorrhea (P = 0.002). Moreover, around 801 (72.4%) women with primary and 52 (56.5%) with secondary dysmenorrhea significantly used 3-5 pads per day (P = 0.004). [fig_ref] Table 7: Impact and Association between dysmenorrhea and intensity of pain and its management [/fig_ref] shows a statistically significant association between the intensity of pain and primary and secondary dysmenorrhea (P = 0.006). Where majority of 539 (48.7%) women with primary dysmenorrhea had mild and 32 (34.8%) with secondary dysmenorrhea had moderate pain. Moreover, no significant association was demonstrated between the type of dysmenorrhea and pain localisation, duration and period of pain, and use of NSAIDs or paracetamols. Whereas, majority of the women with both types significantly did not use alternative therapies (hot pack) to relieve menstrual cramps (P = 0.013). There was no statistically significant association between dysmenorrhea and associated symptoms, limitation and affected academic performance [fig_ref] Table 8: Impact and Association between dysmenorrhea and associated symptoms, limitation and effected academic... [/fig_ref].
# Results
There was a statistically significant association between dysmenorrhea type and dietary habits, exercise and quality of life, as shown in [fig_ref] Table 9: Impact and association between dysmenorrhea and dietary habits, exercise and quality of... [/fig_ref]. There was a statistically significant association between exercise during menstruation and primary and secondary dysmenorrhea (P = 0.001). It is shown that 1105 (99.8%) patients from primary and 90 (97.8%) secondary dysmenorrhea did not use bikes, respectively.
# Discussion
The study aims to determine the factors affecting dysmenorrhea (primary and secondary) prevalence among Saudi women from the reproductive age group. Dysmenorrhea is an important symptom among many women of reproductive age. Dysmenorrhea has a significant impact on the health-related quality of life, work productivity, and health-care utilisation. The dysmenorrhea prevalence came out to be 95.3% in the present study. This prevalence was higher than that observed in the other studies in Saudi Arabia and worldwide [bib_ref] Dysmenorrhea among female medical students in King Abdulaziz University Prevalence, Predictors and..., Ibrahim [/bib_ref] [bib_ref] Prevalence of menstrual problems and their association with psychological stress in young..., Rafique [/bib_ref] [bib_ref] Prevalence of menstrual problems and their association with psychological stress in young..., Rafique [/bib_ref] [bib_ref] Dysmenorrhea, associated symptoms, and management among students at King Khalid University, Saudi..., Saleem [/bib_ref] [bib_ref] Prevalence of primary dysmenorrhea and its effect on the quality of life..., Hashim [/bib_ref]. The worldwide prevalence ranged from 50 to 90% [bib_ref] Primary dysmenorrhea magnitude, associated risk factors, and its effect on academic performance:..., Hailemeskel [/bib_ref] [bib_ref] Prevalence and severity of dysmenorrhoea, and management options reported by young Australian..., Subasinghe [/bib_ref] [bib_ref] Prevalence of primary dysmenorrhea and factors associated with its intensity among undergraduate..., Habibi [/bib_ref] [bib_ref] Primary dysmenorrhea among Mexican university students: prevalence, impact and treatment, Ortiz [/bib_ref]. The high majority observed in this study could be due to the different population age groups included in it or that the women who had the symptoms were more interested in taking part in it.
The study also determined the relationship between dysmenorrhea and intake of dairy products, which showed no significant association. A systematic review conducted by Zahra et al. [bib_ref] Nutrition as a potential factor of primary dysmenorrhea: a systematic review of..., Bajalan [/bib_ref] found that fruits, vegetables, milk, fish and dairy products had positive associations with decreased menstrual pain in primary dysmenorrhea. However, the majority of the participants did not have fruits, vegetables, and dairy products as part of their primary diet, which could be why there was a high prevalence of dysmenorrhea in the studied population.
Also, this study had the objective to elucidate the possible relationship between exercise and dysmenorrhea and to evaluate the impact of dysmenorrhea on the quality of life. Varied researches previously have shown dysmenorrhea to be impacted by multiple factors, of these were physical and daily activities, emotional health, social activities, family and friends' relationships, in addition to the academic performance regarding concentration, attendance, class participation, and study time [bib_ref] Dysmenorrhea: prevalence and impact on quality of life among young adult Jordanian..., Seham [/bib_ref] [bib_ref] Primary dysmenorrhea magnitude, associated risk factors, and its effect on academic performance:..., Hailemeskel [/bib_ref]. In this study, the impact and association between dysmenorrhea and associated symptoms, limitation and affected academic performance were not statistically significant. As most of the women included in the study reported not to have done an adequate amount of exercise, this could be another factor that the prevalence among participants was high.
Another notable finding of this study was the significant association between dysmenorrhea and irregularity of the menstrual cycle. Similarly, a survey by Ameade et al. [bib_ref] Prevalence of dysmenorrhea among University students in Northern Ghana; its impact and..., Ameade [/bib_ref] showed a statistically significant association between the severity of dysmenorrhea and irregularity of menstruation. Also, the menstrual flow and dysmenorrhea were necessary, similar to the results of previous studies [bib_ref] Dysmenorrhea among high-school students and its associated factors in Kuwait, Al-Matouq [/bib_ref] [bib_ref] Factors associated with increased pain in primary dysmenorrhea: analysis using a multivariate..., Tomás-Rodríguez [/bib_ref]. There was a statistically significant association between the type of dysmenorrhea and pain intensity (P = 0.006). The severity of pain was high among women with secondary dysmenorrhea compared to women with primary dysmenorrhea.
Menstrual abnormalities, dysmenorrhea, infertility, chronic pelvic pain (CPP), and dyspareunia are endometriosis's most prevalent clinical indications. Endometriosis symptoms frequently impact patients' social and psychological functioning. As a result, endometriosis is considered a debilitating disease that can jeopardise social interactions and mental health [bib_ref] Anxiety and depression in patients with endometriosis: impact and management challenges, Laganà [/bib_ref]. Endometriosis can be effectively treated with progestins. The effects of the etonogestrel implant on pelvic discomfort, sexual function, and quality of life in women needing long-term reversible contraception and having ovarian cysts of possible endometriotic origin are investigated in the study by Sansone et al. [bib_ref] Effects of etonogestrel implant on quality of life, sexual function, and pelvic..., Sansone [/bib_ref]. In patients with ovarian cysts suspected of being caused by endometriosis, etonogestrel implants appear to relieve pelvic discomfort, enhance sexual function, and improve quality of life. Endometriosis is characterised by endometrial-like tissue outside the uterus, which is accompanied by a persistent and inflammatory response. Brasil et al. [bib_ref] Psychological stress levels in women with endometriosis: systematic review and meta-analysis of..., Brasil [/bib_ref] determined the prevalence and degrees of psychological stress among endometriosis patients. The study showed that multidisciplinary illness management should include mental health assistance inpatient care beyond pain treatment. Moreover, the medical team's attitude toward the patients' psychological stress may positively impact their therapy.
It is essential to encourage modifications in the diet and lifestyle of individuals like restricted intake of salt and excessive caffeinated drinks with effective exercising for reducing the severity of dysmenorrhea symptoms. The possible side effects of using analgesics also need to be informed to the women, and they need to be encouraged for other management techniques like the use of hot pads [bib_ref] Primary dysmenorrhea and herbals, Aksu [/bib_ref] [bib_ref] A study to assess the effectiveness of structured teaching programme on knowledge..., Savitha [/bib_ref]. Measures to deal with dysmenorrhea need to be focused at the school and university level for limiting its interference with the student's life. Apart from these implications, there were some limitations of this study. For instance, the data was collected using self-administered questionnaires (electronic questionnaires), which decreased the reliability of the results. Moreover, the study only included females from a specific region. The study also fails to compare the sample to the number of Saudi women of reproductive age. As a result, the results just reflect a small portion of the sample. Future studies need to include females from other regions of Saudi Arabia to generalise the results to all Saudi females.
[table] Table 1: Demographic profile of patients (n = 1199) [/table]
[table] Table 2: Past medical history and psychological illness of patients [/table]
[table] Table 3: Descriptive analysis of menstrual cycle, regularity, and type of dysmenorrhea [/table]
[table] Table 4: Descriptive analysis of classification of dysmenorrhea pain, therapy and associated symptoms [/table]
[table] Table 5: Distribution of dysmenorrhea limitation, academic performance, exercise and diet during menstruation [/table]
[table] Table 6: Impact and Association between dysmenorrhea and clinical and demographic characteristics of patients [/table]
[table] Table 7: Impact and Association between dysmenorrhea and intensity of pain and its management [/table]
[table] Table 8: Impact and Association between dysmenorrhea and associated symptoms, limitation and effected academic performance [/table]
[table] Table 9: Impact and association between dysmenorrhea and dietary habits, exercise and quality of life Which type of exercise you did it during menstruation (Stretching various parts of the body)The major part of your diet during the menstruation is MeatThe major part of your diet during the menstruation is FruitThe major part of your diet during the menstruation is Eggs How many time eats dairy products per day (milk, yogurt, cheese and labanah) [/table]
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Isolation, Screening, and Identification of Cellulolytic Bacteria from Natural Reserves in the Subtropical Region of China and Optimization of Cellulase Production by Paenibacillus terrae ME27-1
From different natural reserves in the subtropical region of China, a total of 245 aerobic bacterial strains were isolated on agar plates containing sugarcane bagasse pulp as the sole carbon source. Of the 245 strains, 22 showed hydrolyzing zones on agar plates containing carboxymethyl cellulose after Congo-red staining. Molecular identification showed that the 22 strains belonged to 10 different genera, with the Burkholderia genus exhibiting the highest strain diversity and accounting for 36.36% of all the 22 strains. Three isolates among the 22 strains showed higher carboxymethyl cellulase (CMCase) activity, and isolate ME27-1 exhibited the highest CMCase activity in liquid culture. The strain ME27-1 was identified as Paenibacillus terrae on the basis of 16S rRNA gene sequence analysis as well as physiological and biochemical properties. The optimum pH and temperature for CMCase activity produced by the strain ME27-1 were 5.5 and 50 ∘ C, respectively, and the enzyme was stable at a wide pH range of 5.0-9.5. A 12-fold improvement in the CMCase activity (2.08 U/mL) of ME27-1 was obtained under optimal conditions for CMCase production. Thus, this study provided further information about the diversity of cellulose-degrading bacteria in the subtropical region of China and found P. terrae ME27-1 to be highly cellulolytic.
# Introduction
With decades of studies on cellulose bioconversion, cellulases have been playing an important role in producing fermentable sugars from lignocellulosic biomass. Usually, cellulases are mainly composed of three types of synergistic enzymes: endoglucanases (EC 3.2.1.4) that hydrolyze the exposed cellulose chains of the cellulose polymer, exoglucanases (cellobiohydrolases, EC 3.2.1.91) that act to release cellobiose from the reducing and nonreducing ends, andglucosidases (EC 3.2.1.21) that help to cleave the cellobiose and short-chain cello-oligosaccharide into glucose [bib_ref] Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis, Brás [/bib_ref].
Numerous microorganisms that are able to degrade cellulose have been isolated and identified. However, many studies have put more emphasis on fungi because the cellulases that they produce are abundant and easy to extract, and some of the fungal cellulases have been used as commercial cellulase [bib_ref] Cellulases from Penicillium species for producing fuels from biomass, Gusakov [/bib_ref]. Although fungi such as Trichoderma, Aspergillus, Penicillium, Phanerochaete, and Fomitopsis have been widely studied in recent years, researchers have also been paying attention to various bacteria that produce cellulases because of their fast growth, expression of multienzyme complexes, and resistance to extreme environments [bib_ref] Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their..., Martins [/bib_ref] [bib_ref] The prospects of cellulaseproducing bacteria for the bioconversion of lignocellulosic biomass, Maki [/bib_ref] [bib_ref] Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus..., Waeonukul [/bib_ref] [bib_ref] Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010..., Deswal [/bib_ref] [bib_ref] Cellulolytic bacteria from soils in harsh environments, Soares [/bib_ref] [bib_ref] Novel Penicillium cellulases for total hydrolysis of lignocellulosics, Marjamaa [/bib_ref]. Bacteria belonging to the genera Clostridium, Cellulomonas, Cellulosimicrobium, Thermomonospora, Bacillus, Ruminococcus, Erwinia, Bacteriodes, Acetovibrio, Streptomyces, Microbispora, Fibrobacter, and Paenibacillus have been observed to produce different kinds of cellulase when incubated under anaerobic or aerobic conditions [bib_ref] The prospects of cellulaseproducing bacteria for the bioconversion of lignocellulosic biomass, Maki [/bib_ref] [bib_ref] Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic..., Lo [/bib_ref] [bib_ref] Microbial diversity of cellulose hydrolysis, Wilson [/bib_ref].
Several studies have been carried out to investigate the carboxymethyl cellulase (CMCase) activity of aerobic bacteria. For instance, a maximum CMCase activity (0.48 U/mL) of Acinetobacter anitratus was observed in the late logarithm phase [bib_ref] Preliminary studies of cellulase production by Acinetobacter anitratus and Branhamella sp, Ekperigin [/bib_ref]. reported that a maximum CMCase activity of 0.02 and 0.058 U/mL was exhibited by Brevibacillus sp. DUSELG12 and Geobacillus sp. DUSELR7 on days 10 and 7, respectively [bib_ref] Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the..., Rastogi [/bib_ref]. Furthermore, Gupta et al. isolated several cellulose-degrading bacteria exhibiting CMCase activities in the range of 0.162-0.400 U/mL [bib_ref] Isolation of cellulosedegrading bacteria and determination of their cellulolytic potential, Gupta [/bib_ref].
With regard to studies on optimization of cellulase production by aerobic bacteria, Deka et al. used response surface methodology and found that Bacillus subtilis AS3 exhibited a maximum CMCase activity of 0.43 U/mL [bib_ref] Enhancement of cellulase activity from a new strain of Bacillus subtilis by..., Deka [/bib_ref]. Furthermore, using response surface methodology and orthogonal experiment design for medium optimization, Da Vinha et al. and Sheng et al. observed a maximum CMCase activity of 2.0 and 1.432 U/mL by Streptomyces viridobrunneus SCPE-09 and Pseudomonas sp. HP207, respectively [bib_ref] Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate, Da [/bib_ref] [bib_ref] Isolation, screening, and optimization of the fermentation conditions of highly cellulolytic bacteria..., Sheng [/bib_ref]. Thus, isolation of aerobic bacterial strains producing higher cellulase activity is gaining increasing interest.
In this study, diverse aerobic bacteria capable of hydrolyzing cellulose were isolated from the subtropical region of China, with Burkholderia sp. being the most ubiquitous. Furthermore, a bacterial strain ME27-1, producing CMCase at 2.08 U/mL after optimization of culture conditions, was isolated and identified.
# Materials and methods
## Strain isolation and
Screening. The soil sample suspensions were inoculated on Czapek's medium [bib_ref] The production of indole acetic acid by Ustilago zeae, and its possible..., Wolf [/bib_ref] containing sugarcane bagasse pulp (in g/L: NaNO 3 , 2; MgSO 4 ⋅7H 2 O, 0.5; NaCl, 0.5; FeSO 4 ⋅7H 2 O, 0.01; KH 2 PO 4 , 1.0; yeast extract, 0.4; pulp, 5 (containing 80% water); and agar, 15.0; pH 5.0) and incubated at 28 ∘ C. Subsequently, single colonies were picked , 0.002; pH 7.0). The bacterial isolates were precultured overnight in general bacteria medium (in g/L: beef extract, 2; yeast extract, 2; sucrose, 6; and peptone, 5) at 28 ∘ C and 180 rpm. Subsequently, 2 mL of the culture was inoculated into 250 mL conical flask containing 50 mL of basal medium with 10 g/L of CMC-Na as the sole carbon source and incubated at 28 ∘ C and 180 rpm for 60 h.
## Enzyme assay.
Enzyme production during cultivation was assayed at 12 h intervals up to 3 days. The cultures were centrifuged at 12,000 rpm for 10 min at 4 ∘ C. The supernatants were collected as crude enzyme for enzyme assay. CMCase, Avicel cellulase (Avicelase), and filter-paper cellulase (FPase) activities were determined using the 3,5-dinitrosalicylic acid (DNS) method [bib_ref] Use of dinitrosalicylic acid reagent for determination of reducing sugar, Miller [/bib_ref]. The reaction systems were prepared as follows: 250 L of crude enzyme (appropriately diluted) mixed with 250 L of 2% (w/v) CMC for determining the CMCase activity; 500 L of enzyme mixed with 1 mL of Avicel (1%, w/v) for determining the Avicelase activity; and 500 L of enzyme mixed with 50 mg of Whatman number 1 filter paper (1.0 × 6.0 cm) in 1 mL of buffer for determining the FPase activity. The buffer used for dissolving or resuspending the substrates was 100 mM sodium citrate buffer (pH 5.5). The mixtures were incubated at 50 ∘ C for 30 min for CMCase assay and for 1 h for Avicelase and FPase assay, respectively. Then, the reactions were stopped by adding 1 mL of DNS reagent for CMCase assay and 3 mL of DNS reagent for Avicelase and FPase assay, respectively. All the mixtures were heated in boiling water for 5 min for color development. Subsequently, 200 L of each sample was transferred to 96-well microplate
[formula] BM17-1 BM19-6 BM19-8 Burkholderia sp. bB24 (JF772524) Burkholderia cepacia G3 (KF493889) Burkholderia cepacia ATCC 21809 (AY741338) ME59-1 ME59-2 FCD2-1 Burkholderia cepacia ATCC 49709 (AY741349) FCD7-2 Burkholderia sp. B26 (KF788047) Burkholderia cenocepacia AU 1054 (CP000379) FCD6-1 Burkholderia sp. D414 (KF601211) Pandoraea sp. OXJ-11 (EF067851) HPA16-1 Pandoraea norimbergensis CCUG 39188 (AY268174) ME43-1 Dyella sp. BM6 (HM057825) Dyella marensis LNP9 (GQ181039) Dyella koreensis LNW11 (GQ181031) ME67-3 Pseudomonas sp. CK57 (EU686687) Pseudomonas poae BCHCNZ 253 (GU188947) Escherichia coli KCTC 2441 (EU014689) Citrobacter freundii KUDC1770 (KC355277) HPC15-3 HPA21-1 NG5-2 Citrobacter freundii AtetA (KF245926) Enterobacter aerogenes ATCC 13048 (KC429778) FCD2-2 Enterobacter aerogenes T2 (GU265554) FCD1-3 Arthrobacter sp. Am13 (KC853144) Arthrobacter woluwensis A12-1 (AB244301) FCD11-1 ME27-1 Paenibacillus terrae AM141 (AF391124) Paenibacillus brasilensis PB172 (AF273740) Paenibacillus polymyxa IAM 13419 (D16276) SK3-4 Bacillus subtilis IARI-NIAW1-13 (KF054916) DF2-1 Bacillus subtilis 0-2 (FJ959367) BS16-3+ Gelatin + Acid fermentation Glycerol − Ribose + -Methyl-D-xyloside − Mannose + Inositol − -Methyl-glucoside + Esculin + Lactose + Synanthrin − Glycogen + D-Lyxose − D(L)-Arabitol − 5-Keto-gluconate − Erythritol − D-Xylose + Galactose − Sorbose − Mannitol − N-Acetyl-glucosamine − Salicine + Melibiose + Melezitose − Xylitol − D-Tagatose − D-Arabinose − L-Xylose + Glucose − Rhamnose − Sorbitol − Amygdalin + Cellobiose + Sucrose + Raffinose + Gentiobiose + D-Fucose − Gluconate − L-Arabinose + Adonitol − Fructose + Dulcitol − -Methyl-D-xyloside − Arbutin +Maltose + Trehalose + Starch + D-Turanose − L-Fucose − 2-Keto-gluconate − [/formula]
"+": positive reaction; "−": negative reaction. and the absorbance was measured at 540 nm [bib_ref] Measurement of cellulase activity, Ghose [/bib_ref] [bib_ref] Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis..., Rawat [/bib_ref]. One unit (U) of the enzyme activity was defined as the amount of enzyme that released 1 mol of reducing sugars equivalent to glucose per minute during the reaction. The activity of -glucosidase was measured by using pnitrophenyl--D-glucopyranoside (p-NPG) as substrate. The enzyme activity was determined by detecting the amount of p-nitrophenol (p-NP) produced from p-NPG [bib_ref] Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit..., Feng [/bib_ref]. One unit (U) of -glucosidase activity was defined as the amount of enzyme liberating 1 mol of p-NP per minute.
## 16 rrna gene sequencing and phylogenetic analysis of the cmc-degrading
Isolates. The CMC-degrading isolates were cultivated in general bacteria medium at 28 ∘ C for 24 h. The culture was directly used for the amplification of bacterial 16S rRNA gene by PCR [bib_ref] One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Datsenko [/bib_ref]. Two universal 16S rRNA gene primers (F27: 5 -AGAGTTTGATCCTGGCTCAG-3 and R1492: 5 -TACGGTTACCTTGTTACGACTT-3 ) were used [bib_ref] 16S/23S rRNA sequencing, Lane [/bib_ref]. The 25 L mixtures were composed of 1 L of bacterial culture as template DNA, 12.5 L of 2 × Taq PCR Master Mix (containing 0.5 U Taq DNA polymerase/ L, 500 M of each dNTP, 20 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM MgCl 2 , and bromophenol blue, purchased from Tiangen Biotech, Beijing, China), 1 L of each primer (10 M), and 9.5 L of double-distilled H 2 O. The PCR procedure employed was as follows: primary denaturation for 5 min at 94 ∘ C; 30 cycles of denaturation at 94 ∘ C for 30 s; annealing at 55 ∘ C for 30 s, and extension at 72 ∘ C for 100 s; and an additional reaction for 10 min at 72 ∘ C. The PCR products were detected on 0.8% agarose gel to confirm its purity, quantity, and size. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd., China, for sequencing.
The 16S rRNA gene sequences were compared with other 16S rRNA gene sequences available in GenBank by using the BLASTN program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligned with similar sequences by using CLUSTX program. The phylogenetic tree was constructed by applying the neighbor-joining method using MAGA4.1 program based on Kimura-2 parameters with 1000 replicates of bootstrap values [bib_ref] MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences, Kumar [/bib_ref].
## Morphological, physiological, and biochemical
Identification of the Bacterial Strain ME27-1. The morphological properties of the strain ME27-1, including shape, size, colony characteristics (color, shape, surface, elevation, and edge), and Gram staining were evaluated [bib_ref] Paenibacillus kribbensis sp. nov. and Paenibacillus terrae sp. nov., bioflocculants for efficient..., Yoon [/bib_ref]. The physiological and biochemical characterization of the strain ME27-1 was carried out by using API 50CHB microtests (bioMérieux). The effect of carbon and nitrogen sources on cellulase production by the strain ME27-1 was determined by using 11 different carbon sources (fructose, glucose, glycerol, lactose, sucrose, maltose, CMC-Na, filter paper (chopped into 20 mesh size), Avicel, soluble starch, and wheat bran which was chopped into 80 mesh size) and 10 different nitrogen sources as below: (NH 4 ) 2 SO 4 , NH 4 NO 3 , NaNO 3 , KNO 3 , NH 4 Cl, urea, soybean, yeast extract, tryptone, and beef extract. The carbon sources were used at a concentration of 10 g/L, instead of the carbon source in the basal medium. Furthermore, different concentrations (10-100 g/L with an interval of 10 g/L) of optimal carbon source were examined. Similarly, the effect of nitrogen sources was also studied with an initial concentration of 1.5 g/L.
## Optimization of cultivation conditions for cmcase
The effect of different inoculum sizes (2%, 4%, 6%, 8%, and 10%) on enzyme production was tested. All media were in pH 8.0. All the flasks were incubated at 28 ∘ C. The CMCase activity was detected at an interval of 12 h.
## Properties of cmcase produced by the bacterial strain
ME27-1. To determine the optimal pH, 250 L of crude CMCase supernatant was incubated with 250 L of CMC-Na (2%, w/v) at 50 ∘ C and different pH (3.0-11.0 with an interval of 0.5), respectively. To observe the effect of temperature, CMCase was incubated with 2% CMC-Na at a pH of 5.5 and temperature ranging from 30 to 75 ∘ C with an interval of 5 ∘ C.
The maximum CMCase activity obtained at different pH and temperatures was considered to be 100%.
The effect of pH on the stability of CMCase was studied by mixing the crude enzyme with different buffers (1 : 9, v/v) with pH ranging from 3.0 to 10.0. The CMCase activity of the crude enzyme after incubating at 4 ∘ C for 24 h at different pH was detected. To study the thermostability of the CMCase produced by the strain ME27-1, the crude enzyme was preincubated at different temperatures (varying from 30 to 75 ∘ C with an interval of 5 ∘ C) for 1 h. The residual CMCase activity was detected. The maximum CMCase activity obtained at pH 3.0-10.0 or temperature 30-75 ∘ C was considered to be 100%. All the enzyme assays were carried out in triplicate.
## Nucleotide sequence accession numbers.
All the DNA sequences of the partial 16S rRNA genes of the 22 strains reported in this study have been deposited into the GenBank database under the accession numbers from KF536877 to KF536898.
# Results and discussion
## Isolation and screening of cellulose-degrading bacteria.
A total of 245 cellulose-degrading aerobic bacterial strains were isolated from different natural reserves in the subtropical region of China, which were cultured in agar medium containing sugarcane bagasse pulp as the sole carbon source. Out of these strains, 22 isolates showed hydrolyzing zones on agar plates containing CMC-Na after Congo-red staining [fig_ref] Figure 1: Hydrolyzing zones produced by bacterial strains on agar plates containing CMC after... [/fig_ref]. The hydrolyzing zone diameter and colony diameter are listed in [fig_ref] Table 1: Cellulose-degrading bacteria isolated from different natural reserves of subtropical region in China [/fig_ref].
Among the 22 isolates, only three isolates (ME27-1, FCD1-3, and SK3-4) were found to produce measurable CMCase after liquid cultivation, and isolate ME27-1 showed the highest CMCase activity (0.17 U/mL) after incubation for 60 h in basal liquid medium containing 10 g/L of CMC-Na [fig_ref] Table 1: Cellulose-degrading bacteria isolated from different natural reserves of subtropical region in China [/fig_ref] after cultivating in various liquid media for up to 6 days, and the Avicelase, FPase, and -glucosidase activities of all the 22 bacterial strains were also undetectable.
Congo-red staining has been widely used in many studies for screening cellulose-degrading microorganisms. Although Teather and Wood described the relationship between the diameter of hydrolyzing zone and log enzyme concentration, this correlation could not represent the enzyme-producing ability of the microorganisms [bib_ref] Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria..., Teather [/bib_ref]. In the present study, although some strains presented large and clear hydrolyzing zones, the activities of CMCase and other cellulases produced by them were undetectable in various liquid media containing CMC and other cellulosic materials, suggesting that either the concentration of the enzyme produced by these strains was very low to be detected after cultivation in liquid medium or the ability of the strains to secrete CMCase was weak. Sadhu and Maiti also reported that the diameter of the hydrolyzing zone may not accurately reflect the real cellulase activity [bib_ref] Cellulase production by bacteria: a review, Sadhu [/bib_ref].
In general, aerobic bacteria produce low levels of Avicelase, FPase, and -glucosidase. In a study carried out by Rastogi et al., Brevibacillus sp. DUSELG12 and Geobacillus sp. DUSELR7 were found to produce a maximum FPase activity of 0.027 and 0.043 U/mL on days 7 and 8, respectively [bib_ref] Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the..., Rastogi [/bib_ref]. Recently, Soares et al. found that only 9.1% of bacterial strains were able to degrade Avicel on agar plates [bib_ref] Cellulolytic bacteria from soils in harsh environments, Soares [/bib_ref].
## Identification of cellulose-degrading bacteria.
The DNA fragments containing partial 16S rRNA genes of the 22 isolates were amplified and sequenced. The sequences obtained were matched with those available in GenBank, which revealed maximum identity of these isolates and allowed identification of these cellulose-degrading bacterial strains [fig_ref] Table 1: Cellulose-degrading bacteria isolated from different natural reserves of subtropical region in China [/fig_ref]. It was found that the 22 aerobic bacterial strains that could hydrolyze cellulose belonged to 10 different genera: Burkholderia (36.36%), Bacillus (13.65%), Citrobacter (13.65%), Arthrobacter (9.10%), Enterobacter (4.54%), Chryseobacterium (4.54%), Pandoraea (4.54%), Paenibacillus (4.54%), Dyella (4.54%), and Pseudomonas (4.54%). The phylogenetic tree of the 22 strains was constructed by using MAGA4.1 program [fig_ref] Figure 2: Phylogenetic tree for the 22 strains and related bacterial strains [/fig_ref].
Various cellulose-degrading bacteria have been found in different environments. The genus Burkholderia was observed to be the main cellulose-hydrolyzing bacteria and was considered to play an important role in cellulose degradation in the subtropical region of China in this study. In addition, bacteria belonging to the genera Arthrobacter, Chryseobacterium, Pandoraea, and Dyella were also found to be cellulolytic in the present study, which have been rarely reported as cellulose-degrading bacteria. In a previous study, reported that the cellulase-producing bacterial strains isolated from a rice field in southern Taiwan mainly belonged to the genus Cellulomonas [bib_ref] Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic..., Lo [/bib_ref]. On the other hand, Bacillus was reported to be the dominant cellulose-degrading bacteria in samples collected from paper mill sludges and organic fertilizers from Red Rock, Canada, as well as in those from soil, compost, and animal waste slurry from Jeju Island [bib_ref] Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges..., Maki [/bib_ref] [bib_ref] Isolation of cellulolytic Bacillus subtilis strains from agricultural environments, Kim [/bib_ref]. Similarly, Burkholderia was found to be the main genus of cellulase-producing bacteria in the subtropical rainforest in Okinawa Island, Japan [bib_ref] Cellulolytic microbes in the Yanbaru, a subtropical rainforest with an endemic biota..., Fujii [/bib_ref].
The strain ME27-1, with higher CMCase activity, was thoroughly examined. The partial 16S rRNA gene (1309 bp) from the strain ME27-1 showed a maximum identity of 99% with that of Paenibacillus terrae AM141 T (T: type strain). Morphological tests revealed that the cells of the strain ME27-1 were rod-shaped, endospore-forming, Gram-positive, and 0.8 × 1.9-3.2 m in size. The appearance of the colony on the TSA medium was cream-colored, moist, irregular, swollen, and pigment-free. The biochemical properties of the strain ME27-1 are listed in [fig_ref] Table 2: Physiological and biochemical properties of strain ME27-1 [/fig_ref]. The morphological, physiological, and biochemical properties of the strain ME27-1 were found to be mostly similar to those of P. terrae [bib_ref] Paenibacillus kribbensis sp. nov. and Paenibacillus terrae sp. nov., bioflocculants for efficient..., Yoon [/bib_ref]. Thus, the strain ME27-1 was identified as P. terrae.
To our knowledge, till date, no study has reported about CMCase production by P. terrae, although other species of Paenibacillus have been found to produce cellulase. Some CMCase genes cloned from Paenibacillus polymyxa GS01, Paenibacillus barcinonensis, Paenibacillus xylanilyticus KJ-03, and Paenibacillus cookii SS-24 have been expressed in Escherichia coli and Saccharomyces cerevisiae [bib_ref] A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in..., Cho [/bib_ref] [bib_ref] Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae, Mormeneo [/bib_ref] [bib_ref] Gene cloning of endoglucanase Cel5A from cellulose-degrading Paenibacillus xylanilyticus KJ-03 and purification..., Park [/bib_ref] [bib_ref] Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the..., Shinoda [/bib_ref]. On the other hand, CMCases from Paenibacillus curdlanolyticus B-6, Paenibacillus campinasensis BL11, Paenibacillus sp. B39, and P. polymyxa have been purified [bib_ref] Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides, Pason [/bib_ref] [bib_ref] Paenibacillus campinasensis BL11: a wood materialutilizing bacterial strain isolated from black liquor, Ko [/bib_ref] [bib_ref] Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39, Wang [/bib_ref] [bib_ref] Production and characterization of carboxymethyl cellulase from Paenibacillus polymyxa using mango peel..., Kumar [/bib_ref].
## Effect of initial ph, temperature, carbon and nitrogen
Sources, Inoculum Size, and Incubation Time on CMCase Production by P. terrae ME27-1. The best incubation conditions were pH 8.0 and 28 ∘ C [fig_ref] Figure 3: Effect of initial pH and temperature on enzyme production by the strain... [/fig_ref]. The CMCase activity declined when the initial pH and incubation temperature were not optimal. There have been diverse reports on the optimal initial pH and temperature for cellulolytic enzyme production by Paenibacillus sp. In a previous study, P. curdlanolyticus B-6 was cultivated for enzyme production at pH 7.0 and 37 ∘ C [bib_ref] Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus..., Waeonukul [/bib_ref]. Furthermore, Kumar et al. reported that the optimal initial pH and temperature for CMCase production by P. polymyxa were 5.5 and 37 ∘ C, respectively [bib_ref] Production and characterization of carboxymethyl cellulase from Paenibacillus polymyxa using mango peel..., Kumar [/bib_ref]. Yoon et al. accounted that the optimal growth temperature for P. terrae was 30 ∘ C, which is similar to that observed for optimal CMCase production by the strain ME27-1 [bib_ref] Paenibacillus kribbensis sp. nov. and Paenibacillus terrae sp. nov., bioflocculants for efficient..., Yoon [/bib_ref].
Various cellulosic materials have been used to induce microorganisms to produce cellulase. When fructose and glucose were used as the sole carbon source, no CMCase activity was detected. Wheat bran induced the highest CMCase activity, which was about 2.5-fold higher than that observed in the basal medium containing CMC-Na [fig_ref] Figure 4: Effect of carbon and nitrogen sources on CMCase production by the strain... [/fig_ref]. The optimal concentration of wheat bran in the medium was found to be 50 g/L [fig_ref] Figure 4: Effect of carbon and nitrogen sources on CMCase production by the strain... [/fig_ref]. Da Vinha et al. used steampretreated sugarcane bagasse (or wheat bran) as the main carbon source and found that wheat bran was the best inducer for CMCase production by S. viridobrunneus SCPE-09 [bib_ref] Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate, Da [/bib_ref]. Gao et al. demonstrated that rice bran was the optimal carbon source for CMCase production by Cellulophaga lytica LBH-14, while Kumar et al. reported that high CMCase production by P. polymyxa was obtained when using mango peel as substrate [bib_ref] Production and characterization of carboxymethyl cellulase from Paenibacillus polymyxa using mango peel..., Kumar [/bib_ref] [bib_ref] Enhanced carboxymethylcellulase production by a newly isolated marine bacterium, Cellulophaga lytica LBH-14,..., Gao [/bib_ref]. In addition, wheat straw, rice straw, and xylan have been reported to be good carbon sources for CMCase production by Cellulomonas sp. and Cellulosimicrobium cellulans [bib_ref] Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic..., Lo [/bib_ref] [bib_ref] Optimization of liquid fermentation of microbial consortium WSD-5 followed by saccharification and..., Wen [/bib_ref].
Furthermore, maximum CMCase activity was noted when using NH 4 Cl as the sole nitrogen source [fig_ref] Figure 4: Effect of carbon and nitrogen sources on CMCase production by the strain... [/fig_ref] , and the best concentration of NH 4 Cl in the medium was observed to be 3 g/L [fig_ref] Figure 4: Effect of carbon and nitrogen sources on CMCase production by the strain... [/fig_ref]. Many reports have shown that organic nitrogen sources are better than inorganic nitrogen sources [bib_ref] Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate, Da [/bib_ref] [bib_ref] Isolation, screening, and optimization of the fermentation conditions of highly cellulolytic bacteria..., Sheng [/bib_ref] [bib_ref] Optimization of nutrient medium containing agricultural waste for xylanase production by Bacillus..., Geetha [/bib_ref] [bib_ref] Statistical optimization of fermentation conditions and comparison of their influences on production..., Kim [/bib_ref]. In the present study, the CMCase activity of the strain ME27-1 was higher when inorganic nitrogen sources were used as the sole nitrogen source. Likewise, Kumar et al. and Kalogeris et al. also observed a similar phenomenon in their studies [bib_ref] Production and characterization of carboxymethyl cellulase from Paenibacillus polymyxa using mango peel..., Kumar [/bib_ref] [bib_ref] Production and characterization of cellulolytic enzymes from the thermophilic fungus Thermoascus aurantiacus..., Kalogeris [/bib_ref]. In addition, use of an inoculum size of 2% resulted in maximum CMCase activity after incubation of the strain for 60 h [fig_ref] Figure 5: Effect of inoculum size and incubation period on CMCase production by the... [/fig_ref]. There has been increasing interest in cellulase-producing bacteria because of their ability to grow fast [bib_ref] Isolation and characterization of mesophilic cellulose-degrading bacteria from flower stalks-vegetable waste co-composting..., Lu [/bib_ref]. In the present study, the strain ME27-1 produced the highest CMCase activity after 60 h of incubation. On the other hand, in previous studies, maximum CMCase activity of Pseudomonas sp. HP207 and S. viridobrunneus SCPE-09 was observed after 24 and 48 h of incubation, respectively, which is much earlier than that noted for the strain ME27-1 [bib_ref] Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate, Da [/bib_ref] [bib_ref] Isolation, screening, and optimization of the fermentation conditions of highly cellulolytic bacteria..., Sheng [/bib_ref]. However, different results have been reported in various studies. Maximum CMCase activity of C. lytica LBH-14 was obtained after 72 h of incubation, whereas that of Brevibacillus sp. DUSELG12 and Geobacillus sp. DUSELR7 was noted after days 9 and 7, respectively [bib_ref] Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the..., Rastogi [/bib_ref] [bib_ref] Enhanced carboxymethylcellulase production by a newly isolated marine bacterium, Cellulophaga lytica LBH-14,..., Gao [/bib_ref].
## Properties of cmcase produced by p. terrae me27-1.
The optimum pH and temperature of CMCase produced by strain ME27-1 were found to be 5.5 and 50 ∘ C, respectively [fig_ref] Figure 6: Properties of CMCase produced by the strain ME27-1 [/fig_ref]. The CMCase produced by the strain ME27-1 was stable from pH 4.0 to 11.0, with more than 60% CMCase activity being retained [fig_ref] Figure 6: Properties of CMCase produced by the strain ME27-1 [/fig_ref]. Furthermore, the enzyme maintained 65% activity after incubation at 4 ∘ C and pH 11.0 for 24 h. The temperature profiles demonstrated that more than 95% CMCase activity was retained at 30-45 ∘ C for 1 h [fig_ref] Figure 6: Properties of CMCase produced by the strain ME27-1 [/fig_ref]. However, the enzyme activity was reduced at temperatures above 50 ∘ C. In fact, approximately 77% residual activity was maintained after preincubating the enzyme at 50 ∘ C for 1 h.
Similar results were observed for cellulases produced by S. viridobrunneus SCPE-09 and P. cookii SS-24, with an optimal pH of 5.0 and 5.1 and an optimal temperature of 50 ∘ and 55 ∘ C, respectively [bib_ref] Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate, Da [/bib_ref] [bib_ref] Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the..., Shinoda [/bib_ref]. However, maximum CMCase activity of bacteria at pH lower than 6.0 has been rarely observed, and the maximum CMCase activities of P. campinasensis BL11, P. polymyxa GS01, Paenibacillus sp. B39, and Bacillus mycoides S122C were observed at neutral or alkaline conditions [bib_ref] Paenibacillus campinasensis BL11: a wood materialutilizing bacterial strain isolated from black liquor, Ko [/bib_ref] [bib_ref] Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39, Wang [/bib_ref] [bib_ref] Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison..., Kye [/bib_ref] [bib_ref] Purification and biochemical characterization of a novel thermo-stable carboxymethyl cellulase from Azorean..., Balasubramanian [/bib_ref]. In the present study, the CMCase produced by the strain ME27-1 was stable at pH 5.0-9.5, and almost 85% residual activity was retained. Only a few studies have reported that CMCase was stable at such a wide pH range; for example, Da Vinha et al. reported the 60% CMCase activity was retained within the pH range of 3.0-8.0 [bib_ref] Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate, Da [/bib_ref].
## Comparison of cmcase production by p. terrae me27-1 and other microorganisms.
When measured at the optimal pH and temperature of CMCase, P. terrae ME27-1 produced CMCase activity of 2.08 U/mL under the optimized cultivation conditions, which was a 12-fold improvement in the CMCase production. This yield of CMCase production was higher than most of the aerobic bacterial strains but less than some of aerobic bacterial strains that have been exploited previously [fig_ref] Table 3: Comparison of CMCase production by Paenibacillus terrae ME27-1 with other bacterial and... [/fig_ref]. However, the CMCase production by P. terrae ME27-1 was lower than that by several anaerobic bacterial strains, for example, Clostridium papyrosolvens CFR-703, C. thermocellum YM4, C. thermocopriae JT3-3 [fig_ref] Table 3: Comparison of CMCase production by Paenibacillus terrae ME27-1 with other bacterial and... [/fig_ref]. Some anaerobic bacteria can degrade lignocellulosic substrates efficiently by producing multienzyme complex termed cellulosome [bib_ref] Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides, Pason [/bib_ref]. The carbohydrate binding modules and different proteins in the cellulosome allow the whole enzyme complex to bind to the substrates, which avoids the wasteful expenditure of energy of bacteria releasing large amounts of individual enzymes and makes lots of advantages over singleenzyme system [bib_ref] The prospects of cellulaseproducing bacteria for the bioconversion of lignocellulosic biomass, Maki [/bib_ref] [bib_ref] Bacterial genomes: what they teach us about cellulose degradation, Brumm [/bib_ref]. Furthermore, the CMCase produced by P. terrae ME27-1 was lower than that by most aerobic fungal strains while it was higher than that by anaerobic fungal strains [fig_ref] Table 3: Comparison of CMCase production by Paenibacillus terrae ME27-1 with other bacterial and... [/fig_ref].
The CMCase production by most bacteria was usually lower than that by aerobic fungal strains. Genomic analysis showed that less glycosyl hydrolases existed in aerobic bacterial strains than aerobic fungal strains, which may explain why aerobic bacteria usually produce lower CMCase activity [bib_ref] Bacterial genomes: what they teach us about cellulose degradation, Brumm [/bib_ref].
# Conclusion
Ten genera of bacteria hydrolyzing cellulose were isolated from different natural reserves in the subtropical region of China, and the genus Burkholderia was found to be the most prevalent and predominant. The strain ME27-1, identified to be P. terrae, showed the highest CMCase activity among the 22 strains isolated, and after optimization of the cultivation conditions, the enzyme activity was significantly improved to 2.08 U/mL. This bacterial species has been rarely found to produce cellulase. Thus, this study revealed the diversity of cellulose-degrading bacteria in the subtropical region of China and found that P. terrae ME27-1 was a good CMCase producer.
[fig] Figure 1: Hydrolyzing zones produced by bacterial strains on agar plates containing CMC after Congo-red staining. (a) Strain BS16-3, (b) strain FCD1-3, (c) strain FCD2-1, (d) strain FCD3-5, (e) strain FCD7-2, (f) strain SK3-4, and (CK) Escherichia coli DH5 . [/fig]
[fig] Figure 2: Phylogenetic tree for the 22 strains and related bacterial strains. The accession numbers of the strains are given in brackets. [/fig]
[fig] Figure 3: Effect of initial pH and temperature on enzyme production by the strain ME27-1. (a) Initial pH. (b) Temperature ( ). [/fig]
[fig] Figure 4: Effect of carbon and nitrogen sources on CMCase production by the strain ME27-1. (a) Different carbon sources: 1 ∼ 9 represented glycerol, lactose, sucrose, maltose, CMC-Na, filter paper, Avicel, soluble starch, and wheat bran, respectively. (b) The concentration of wheat bran. (c) Different nitrogen sources: a ∼ j represented (NH 4 ) 2 SO 4 , NH 4 NO 3 , NaNO 3 , KNO 3 , NH 4 Cl, urea, soybean, yeast extract, tryptone, and beef extract, respectively. (d) The concentration of NH 4 Cl. [/fig]
[fig] Figure 5: Effect of inoculum size and incubation period on CMCase production by the strain ME27-1. 2% (empty triangle); 4% (filled triangle); 6% (filled circle); 8% (filled square); and 10% (empty square). Error bars show the standard deviation of experimental point ( = 3). [/fig]
[fig] Figure 6: Properties of CMCase produced by the strain ME27-1. (a) Effect of pH on CMCase activity. (b) Effect of temperature on CMCase activity. (c) Effect of pH on the stability of CMCase. (d) Thermostability of CMCase. The different buffers used are as follows (100 mM):sodium citrate buffer (empty square; pH 3.0-6.5), Na 2 HPO 4 -NaH 2 PO 4 buffer (filled square; pH 6.5-7.5), Tris-HCl buffer (empty triangle; pH 7.5-8.5), and glycine-NaOH buffer (filled triangle; pH 8.5-11.0). Error bars show the standard deviation of experimental point ( = 2). [/fig]
[table] Table 1: Cellulose-degrading bacteria isolated from different natural reserves of subtropical region in China. [/table]
[table] Table 2: Physiological and biochemical properties of strain ME27-1. [/table]
[table] Table 3: Comparison of CMCase production by Paenibacillus terrae ME27-1 with other bacterial and fungal strains. CMC: carboxymethyl cellulose; OPEFB: oil palm empty-fruit-bunch fibres; CP-123: cellulose powder 123. [/table]
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Acute orexin antagonism selectively modulates anticipatory anxiety in humans: implications for addiction and anxiety
Research indicates that heightened anticipatory anxiety underlies several forms of psychopathology. Anticipatory anxiety can be reliably and objectively measured in the laboratory using the No-Predictable-Unpredictable (NPU) threat paradigm. The NPU paradigm is an ideal research tool for the NIH 'Fast-Fail' approach of screening promising compounds and testing human target engagement. Evidence from preclinical studies suggests that the hypocretin/orexin (ORX) hypothalamic neuropeptide system is a potential means for modulating anticipatory anxiety and disrupting stress-related alcohol use. The current study tested this question using a psychophysiological probe of the ORX system in humans. We examined whether a single dose of suvorexant (SUV; 10 mg; dual ORX receptor antagonist) can effectively and selectively target a well-validated human laboratory index of exaggerated anticipatory anxiety using a within-subjects placebo-controlled design. A total of twenty-one volunteers completed two laboratory sessions during acute administration of 10 mg SUV or placebo. Across sessions, we administered the NPU paradigm probing sustained anticipatory anxiety and fear while startle eyeblink was recorded as an index of aversive reactivity. Questionnaires assessing mood states and subjective drug effects were also collected. Results indicated SUV was well-tolerated. Compared with placebo, SUV was associated with decreased startle reactivity during anticipatory anxiety but not fear or no-threat conditions. Therefore, SUV selectively and effectively reduced objective indicators of anticipatory anxiety in humans and engaged our laboratory target of psychopathology. ORX antagonism may be a promising strategy for modulating human anxiety and potentially, stress-related alcohol use.Translational Psychiatry (2022) 12:308 ; https://doi.
# Introduction
Converging research indicates that heightened reactivity to uncertain threat (U-threat) underlies several forms of psychopathology. U-threat is typically defined as a threat that is unpredictable in its temporality, intensity, frequency, or duration. U-threat elicits a generalized feeling of apprehension and hypervigilance that is not associated with an identifiable source, referred to as anticipatory anxiety [bib_ref] Unraveling the mysteries of anxiety and its disorders from the perspective of..., Barlow [/bib_ref] [bib_ref] Phasic vs. sustained fear in rats and humans: Role of the extended..., Davis [/bib_ref]. U-threat is in contrast with predictable threat (P-threat), which is signaled by a discrete cue and elicits a phasic response to an identifiable stimulus that is time-locked to the threat [bib_ref] Unraveling the mysteries of anxiety and its disorders from the perspective of..., Barlow [/bib_ref] [bib_ref] Phasic vs. sustained fear in rats and humans: Role of the extended..., Davis [/bib_ref]. U-threat and P-threat produce distinguishable aversive states [bib_ref] The benzodiazepine alprazolam dissociates contextual fear from cued fear in humans as..., Grillon [/bib_ref] that are mediated by overlapping, but separable, neural circuits [bib_ref] Phasic and sustained fear in humans elicits distinct patterns of brain activity, Alvarez [/bib_ref] [bib_ref] Neural systems involved in fear and anxiety measured with fearpotentiated startle, Davis [/bib_ref]. Fear and anxiety, and U-threat and P-threat, are related though accumulating evidence points to the fact that individual differences in reactivity to U-threat (but not P-threat) play a role in the onset and maintenance of multiple forms of psychopathology, particularly fear-based anxiety disorders (e.g., panic disorder, social anxiety disorder, phobias) [bib_ref] Startle potentiation to uncertain threat as a psychophysiological indicator of fear-based psychopathology:..., Gorka [/bib_ref] and addiction [bib_ref] Behavioral and neural sensitivity to uncertain threat in individuals with alcohol use..., Gorka [/bib_ref].
In order to induce anticipatory anxiety and fear in the laboratory, Grillon and colleagues developed the No-Predictable-Unpredictable (NPU) threat task [bib_ref] Assessing fear and anxiety in humans using the threat of predictable and..., Schmitz [/bib_ref]. The NPU is a translational paradigm with three within-subjects conditions: no-shock (N), predictable electric shock (P), and unpredictable electric shock (U).
The task includes bursts of white noise used to elicit and record the startle eyeblink reflex, a highly reliable and easy-to-record index of aversive reactivity [bib_ref] A review of the modulation of the startle reflex by affective states..., Grillon [/bib_ref] [bib_ref] Emotion, attention, and the startle reflex, Lang [/bib_ref]. Using an adapted version of the NPU task, our lab has repeatedly demonstrated that fear-based anxiety disorders and alcohol use disorder (AUD) are characterized by exaggerated startle eyeblink potentiation to U-threat, but not P-threat, compared with matched and healthy controls [bib_ref] Startle potentiation to uncertain threat as a psychophysiological indicator of fear-based psychopathology:..., Gorka [/bib_ref] [bib_ref] Behavioral and neural sensitivity to uncertain threat in individuals with alcohol use..., Gorka [/bib_ref] [bib_ref] A psychophysiological investigation of threat and reward sensitivity in individuals with panic..., Shankman [/bib_ref]. Startle magnitude during U-threat correlates with severity of anxiety and AUD symptoms and stress-related coping motives for alcohol use [bib_ref] Startle potentiation to uncertain threat as a psychophysiological indicator of fear-based psychopathology:..., Gorka [/bib_ref] [bib_ref] Behavioral and neural sensitivity to uncertain threat in individuals with alcohol use..., Gorka [/bib_ref]. Meanwhile, alcohol intoxication selectively and effectively dampens reactivity to U-threat [bib_ref] Alcohol selectively reduces anxiety but not fear: startle response during unpredictable versus..., Moberg [/bib_ref] [bib_ref] Alcohol stress response dampening: selective reduction of anxiety in the face of..., Hefner [/bib_ref].
We have also shown the NPU paradigm is sensitive, reliable, and reflective of frontolimbic brain function [bib_ref] Psychometric properties of startle and corrugator response in NPU, affective picture viewing,..., Kaye [/bib_ref] [bib_ref] Association between neural reactivity and startle reactivity to uncertain threat in two..., Gorka [/bib_ref]. Consequently, the NPU paradigm has been identified as an ideal research tool for mechanistic medication development [bib_ref] Orexin modulation of stress reactivity as a novel targeted treatment for anxiety..., Gorka [/bib_ref] [bib_ref] A way forward for anxiolytic drug development: testing candidate anxiolytics with anxiety-potentiated..., Grillon [/bib_ref]. Recent recommendations urge researchers to use biologically-plausible, valid, and reliable human laboratory measures of pathophysiology to test promising compounds from preclinical studies to directly inform the go/no-go decision to move to a clinical trial. This strategy is intended to accelerate the pace of drug discovery and bring forth novel mechanistic pharmacotherapies. With regard to anxiety and AUD, though there are available medications to treat these disorders, they are modestly effective and do not work for everyone. The development of new and more efficacious pharmacotherapies for anxiety disorders and AUD are urgently needed and a top public health priority.
To this end, one neurochemical system that has emerged as a promising target for next-generation medication development is the orexin (ORX) system. Orexins (also known as hypocretins) are peptides that are produced in the hypothalamus and regulate a range of behavioral and physiological processes including wakefulness, arousal, energy metabolism, and stress reactivity [bib_ref] Orexins and orexin receptors: a family of hypothalamic neuropeptides and G proteincoupled..., Sakurai [/bib_ref] [bib_ref] The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity, De Lecea [/bib_ref] [bib_ref] Motivational activation: a unifying hypothesis of orexin/hypocretin function, Mahler [/bib_ref]. There are currently two known types of ORX receptors (OX1R and OX2R) that are distributed throughout the brain including networks involved in stress, fear, and anxiety [bib_ref] The role of orexin/hypocretin in the central nervous system and peripheral tissues, Tsunematsu [/bib_ref]. Preclinical studies show that ORX is involved in several key aspects of chronic anxiety including arousal and hypervigilance. For instance, ORX neurons are activated by acute threat, and infusion of orexin directly into the brain induces anxiety-like behaviors [bib_ref] The role of the orexin system in stress response, Sargin [/bib_ref] [bib_ref] Hypocretin/orexin in arousal and stress, Berridge [/bib_ref]. Meanwhile, decreases in ORX signaling blunts stress and anxiety responses, particularly during challenges that elicit arousal [bib_ref] Evaluation of JNJ-54717793 a novel brain penetrant selective orexin 1 receptor antagonist..., Bonaventure [/bib_ref] [bib_ref] Orexins (hypocretins) contribute to fear and avoidance in rats exposed to a..., Chen [/bib_ref] [bib_ref] A key role for orexin in panic anxiety, Johnson [/bib_ref]. In humans, individuals with anxiety disorders have higher serum and cerebrospinal fluid ORX levels compared with controls [bib_ref] A key role for orexin in panic anxiety, Johnson [/bib_ref] [bib_ref] Orexin A in adolescents with anxiety disorders, Akça [/bib_ref]. A recent study in those with panic disorder reported a gene variant linked to ORX system functioning (HCRTR1) is assocaited with increased avoidance and arousal-based anxiety symptoms [bib_ref] Orexin in the anxiety spectrum: association of a HCRTR1 polymorphism with panic..., Gottschalk [/bib_ref]. In sum, evidence clearly indicates ORX plays a critical role in arousal-based anxiety; though there are only a few studies in humans testing these theories.
Accumulating preclinical data also indicates the ORX system is involved in alcohol abuse. Antagonism of ORX receptors has been repeatedly shown to decrease alcohol self-administration and relapse-like behavior [bib_ref] The hypocretin/orexin system as a target for excessive motivation in alcohol use..., Moorman [/bib_ref] [bib_ref] Recent perspectives on orexin/hypocretin promotion of addictionrelated behaviors, Hopf [/bib_ref]. For example, administration of SB334867 (SB) (ORX1R antagonist) was found to decrease alcohol consumption, block alcohol condition place preference, and prevent cue-induced reinstatement of alcohol seeking [bib_ref] The orexin system regulates alcohol-seeking in rats, Lawrence [/bib_ref] [bib_ref] Involvement of the orexin/hypocretin system in ethanol conditioned place preference, Voorhees [/bib_ref]. In mouse models, ORX antagonism similarly decreases binge-like alcohol consumption and escalated alcohol intake due to cycles of chronic intermittent ethanol exposure [bib_ref] Orexin receptor 1 signaling contributes to ethanol binge-like drinking: pharmacological and molecular..., Carvajal [/bib_ref] [bib_ref] Binge-like consumption of ethanol and other salient reinforcers is blocked by orexin-1..., Olney [/bib_ref]. In many studies, the effect of OXR antagonism was most robust in animals exhibiting high motivation for alcohol, such as those genetically selected for high alcohol preference [bib_ref] Orexin-1 and orexin-2 receptor antagonists reduce ethanol self-administration in high-drinking rodent models, Anderson [/bib_ref] or rats with natural high propensity for alcohol [bib_ref] Orexin-1 receptor antagonism decreases ethanol consumption and preference selectively in high-ethanol-preferring Sprague-Dawley..., Moorman [/bib_ref]. Given that high motivation for alcohol is characteristic of human AUD, these data are promising for human translation. There are also studies showing that SB treatment is particularly effective at reducing stress-induced alcohol relapse-like behavior [bib_ref] Inhibition of orexin-1/hypocretin-1 receptors inhibits yohimbine-induced reinstatement of ethanol and sucrose seeking..., Richards [/bib_ref] , and that ORX is involved in the emotional dysregulation that occurs during alcohol withdrawal [bib_ref] Orexin A expression and promoter methylation in patients with alcohol dependence comparing..., Bayerlein [/bib_ref]. These data are consistent with the role of ORX in regulating both arousal and alcohol behavior and the interplay between anxiety and AUD symptoms.
ORX antagonism may be an effective strategy for reducing chronic hyper-arousal and disrupting the negative reinforcement cycle of AUD and anxiety; though no study has directly addressed this question. To date, the dual ORX receptor antagonist suvorexant (Belsomra™), used to treat insomnia, is an FDA-approved ORX receptor antagonist. Consistent with the NIH 'Fast-Fail' mission, the present study was a small, proof-ofconcept psychophysiological probe of the ORX system in humans. We examined whether a single dose of suvorexant (SUV; 10 mg) can effectively and selectively target a wellvalidated human laboratory index of exaggerated anticipatory anxiety using a within-subjects placebo-controlled design. A dose of 10 mg was selected because it is the lowest effective clinical dose, has negligible cognitive and emotional side effects, and has not been associated with adverse events [bib_ref] Safety, tolerability, and pharmacokinetics of suvorexant: a randomized rising-dose trial in healthy..., Yee [/bib_ref]. If ORX antagonism selectively engages our U-threat laboratory target, without modifying reactivity to P-threat, there would be compelling human evidence that ORX is a neurochemical modulator of anticipatory anxiety, and ORX antagonism would be a promising pharmacological target for anxiety disorder and/ or AUD medication development.
# Method participants
Volunteers were recruited from the community via flyers and online advertisements for an open-label pharmacological challenge laboratory study. To be included in the study, individuals were required to be between ages 18 and 30 years, generally medically healthy, and able to provide written informed consent. Exclusion criteria included: (a) clinically significant medical or neurologic condition, (b) active suicidal ideation, (c) current or recent (past 2 months) use of any psychoactive medications or antihistamines, (d) current use of strong CYP3A live enzymes or moderate CYP3A inhibitors or strong CYP3A inducers, (e) current use of digoxin, (f) currently pregnant, not using contraception, nursing, or trying to become pregnant, (g) obesity as defined by a body-mass index equal or greater than 30, (h) current substance use disorder, (i) current or past major depressive disorder, psychotic disorder, bipolar disorder, or obsessivecompulsive disorder, (j) deafness in either ear, (k) moderate to severe traumatic brain injury, (l) smoke 5 or more cigarettes (or electronic equivalent) per day and susceptible to acute nicotine withdrawal, (m) positive breath alcohol screen the lab of the lab visits, and (n) unwillingness to abstain from driving or engaging in any activities requiring full alertness within 24 hours of lab visits. All participants provided written informed consent and the protocol was approved by the university Institutional Review Board. Participants were monetarily compensated for their time.
A total of 21 participants (9 males, 12 females) enrolled in the study. Participants were 21.6 (±2.2) years old. Ethnic and racial composition of the sample included the following: 4.8% Hispanic, 71.4% Caucasian, 23.8% Asian, and 4.8% 'biracial'. No participant met diagnostic criteria for any current mood, anxiety, or substance use disorder. All participants completed both laboratory sessions and had good quality startle eyeblink data (i.e., at least 4/10 good blinks in each task condition).
## Drug and placebo
Capsules were obtained from a compound pharmacy and dispensed by the university Investigational Drug Service (IDS). All capsules were opaque and identical in appearance. Drug capsules included 10 mg of SUV (i.e., Belsomra®; Merck & Co., Inc.) with dextrose filler. Placebo capsules included only dextrose filler. Capsules were administered to subjects in double-blind conditions under medical supervision. Drug order was randomized by the university IDS prior to data collection. A total of 11 subjects received SUV at their first session and 10 received placebo. Randomization was therefore successful.
## Procedure
Each participant completed three study visits including a video-conference screen and two in-person lab visits. Study visits were 2-7 days apart. The screen included informed consent, a short battery of questionnaires, and the Mini International Neuropsychiatric Interview (MINI).
Each lab visit began at 8:30 am. Participants were instructed to abstain from food and beverage other than water 2-hours prior to the session. Upon arrival, participants provided a negative breath and urine screen for alcohol, pregnancy (women), and recent illicit drug use. The placebo or drug capsule was administered while nursing staff monitored vital signs every 30-mins. Peak plasma concentration for SUV is 2 hours postingestion. All psychophysiological laboratory assessments were administered during peak concentration. At 240-minutes post-ingestion, participants were discharged following medical clearance from nursing staff.
## Subjective mood and drug effects
Standardized questionnaires were used to assess mood states and subjective drug effects throughout the lab sessions. Specifically, participants completed a 21-item visual analog scale (VAS) [bib_ref] Reliability, validity, and clinical application of the Visual Analogue Mood Scale, Folstein [/bib_ref] , Drug Effects Questionnaire (DEQ) [bib_ref] Drug preference and mood in humans: d-amphetamine, Johanson [/bib_ref] , and the Profile of Mood States (POMS) [bib_ref] Manual Profile of Mood States, Mcnair [/bib_ref]. The VAS ratings were made using a 0 (not at all) to 100 (extremely) scale and included the following scales of interest: anxious, tired, drowsy, and nauseous. The DEQ required participants to rate the extent to which they: (1) feel any drug effect; (2) like the drug effect; (3) feel high; and (4) would like more drug. DEQ items were rated on a 5-point Likert scale except for like drug effect which was a 9-point scale. The POMS is a 72-item adjective checklist rated on a five-point Likert Scale. The POMS yields eight subscales and two composite scales of negative arousal and positive mood. The VAS and DEQ were collected immediately before capsule ingestion and every 30-minutes afterwards. The POMS was collected immediately before S.M. Gorka et al.
capsule ingestion and every 60-minutes afterwards. At the end of each session, participants were asked to indicate which capsule they believed they received. Participants' scores on VAS, DEQ, and POMS were averaged across each session, consistent with prior our studies [bib_ref] Striatal activation to monetary reward is associated with alcohol reward sensitivity, Radoman [/bib_ref].
## Self-reported symptoms
During the screen session participants completed the Depression, Anxiety, and Stress Scale (DASS-21) [bib_ref] The structure of negative emotional states: comparison of the Depression Anxiety Stress..., Lovibond [/bib_ref]. The DASS-21 is comprised of 21 items and three subscales measuring current symptoms of depression, anxiety, and stress. Respondents rate the severity of each symptom during the past week on a 0-3 scale. Participants also completed the Alcohol Use Disorders Identification Test (AUDIT), which was developed by the World Health Organization (WHO) to assess hazardous and harmful alcohol use. The AUDIT includes 10-items that are combined to create a total score.
## Npu threat test
The NPU startle task and laboratory procedures have been extensively described by our group [bib_ref] Startle response to unpredictable threat in comorbid panic disorder and alcohol dependence, Gorka [/bib_ref] [bib_ref] Association between problematic alcohol use and reactivity to uncertain threat in two..., Gorka [/bib_ref]. In brief, shock electrodes were placed on participants' left wrist and a shock work-up procedure was completed to identify the level of shock intensity each participant described as "highly annoying but not painful" (between 1-5 mA). Participants then completed a 2-min startle habituation task to reduce early, exaggerated startle potentiation. The task itself was modeled after Grillon and colleagues NPU threat task and thus included three within-subjects conditions: no shock (N), predictable shock (P), and unpredictable shock (U). Text at the bottom of the computer monitor informed participants of the current condition. Each condition lasted 145-s, during which a 4-s visual countdown (CD) was presented six times. The interstimulus intervals (ISIs; i.e., time between CDs) ranged from 15 to 21-s during which only the text describing the condition was on the screen. No shocks were delivered during the N condition. A shock was delivered every time the CD reached 1 during the P condition. Shocks were delivered at random during the U condition (both during the CD and ISI). Startle probes were administered during both the CD and ISI, and there was always at least 10-s between two probes or a shock and a probe. Each condition was presented two times in a randomized order (counterbalanced). Participants received 24 total electric shocks (12 in P; 12 in U) and 60 total startle probes (20 in N; 20 in P; 20 in U).
## Startle data collection and processing
Startle data were acquired using BioSemi Active Two system (BioSemi, Amsterdam, The Netherlands) and stimuli were administered using Presentation (Albany, CA). Electric shocks lasted 400-ms and acoustic startle probes were 40-ms duration, 103-dB bursts of white noise with near-instantaneous rise time presented binaurally through headphones.
Startle responses were recorded from two 4-mm Ag/AgCl electrodes placed over the orbicularis oculi muscle below the left eye. The ground electrode was located at the frontal pole (Fpz) of an electroencephalography cap that participants were wearing as part of the psychophysiological set-up. One startle electrode was placed 1-cm below the pupil and the other was placed 1-cm lateral of that electrode. Data were collected using a bandpass filter of DC-500-Hz at a sampling rate of 2000-Hz.
Blinks were processed and scored according to published guidelines: [bib_ref] Committee report: guidelines for human startle eyeblink electromyographic studies, Blumenthal [/bib_ref] applied a 28 Hz high-pass filer, rectified, and then smoothed using a 40 Hz low-pass filter. Peak amplitude was defined within 20-150-ms following the probe onset relative to baseline (i.e., average activity for the 50-ms preceding probe onset). Each peak was identified by software but examined by hand to ensure acceptability. Blinks were scored as nonresponses if activity during the post-stimulus time frame did not produce a peak that is visually differentiated from baseline. Blinks were scored as missing if the baseline period was contaminated with noise, movement artifact, or if a spontaneous or voluntary blink began before minimal onset latency. Blink magnitude values (i.e., condition averages include values of 0 for non-responses) were used in all analyses.
# Data analysis plan
We first examined session differences in subjective drug effects using a series of paired samples t-tests. We focused on the 10 specific scales hypothesized to be affected by SUV. Next, to test our specific hypotheses, we conducted a session (2: SUV, placebo) by task condition (3: no-threat, Pthreat, U-threat) repeated measures analysis of variance (ANOVA) with startle magnitude as the dependent variable. Biological sex was entered as a between-subjects covariate given that it is unknown whether SUV has differential sex effects on threat reactivity. Drug capsule order was also dummy coded and entered as a covariate to account for potential treatment order effects.
An a priori power analysis was conducted using G*Power version 3.1.9.7 to determine the minimum sample size required to test the study hypothesis. Results indicated the required sample size to achieve a large effect (f = 0.40) with 90% power, α = 0.05, and a conservative correlation of .40 between observations on the same subject was 20 subjects. A large effect was estimated based on prior studies demonstrating a robust modulation of startle magnitude by known anxiolytics (e.g., benzodiazepines [bib_ref] The benzodiazepine alprazolam dissociates contextual fear from cued fear in humans as..., Grillon [/bib_ref]. A large effect also serves as justification for sufficient target engagement and subsequent clinical trial testing. Overall, the obtained sample size of 21 individuals was adequate to test the study hypothesis.
Post-hoc we conducted two exploratory Pearson's correlations to examine whether SUV-related change in U-threat reactivity was associated with baseline anxiety symptoms and/or current problem alcohol use. Specifically, we correlated DASS-21 anxiety subscale scores and AUDIT total scores with percent change in startle magnitude (((placebo-SUV)/ placebo) × 100))).
# Results
## Subjective drug effects
Mean subjective ratings for each scale, averaged across each session, are presented in [fig_ref] Table 1: Subjective reports during each session [/fig_ref]. Administration of SUV was associated with increased reports of feeling drowsy and tired relative to placebo. Participants also reported they could 'feel' drug effects to a greater extent during administration of SUV compared with placebo. SUV was associated with reductions in negative arousal and positive mood relative to placebo. Overall, administration of SUV was well-tolerated with no adverse events
## Startle effects
Results of the omnibus repeated measures ANOVA are presented in [fig_ref] Table 2: Results of omnibus repeated measures ANOVA [/fig_ref]. There was a main effect of session such that startle was lower during SUV relative to placebo. There was also the expected main effect of task condition. Startle during U-threat (F[1,17] = 9.15, p = 0.008, η G 2 = 0.350) and P-threat (F[1,17] = 6.03, p = 0.025, η G 2 = 0.262) was greater than startle during No-threat. In addition, startle during U-threat was greater than startle during P-threat (F[1,17] = 5.17, p = 0.036, η G 2 = 0.233). These main effects were qualified by a significant session by task condition interaction [fig_ref] Figure 1: Bar graph displaying mean startle magnitude during each task condition, across both... [/fig_ref]. Startle was lower during SUV relative to placebo during U-threat (F[1,17] = 13.12, p = 0.002, η G 2 = 0.435). However, there was no effect of session on startle during P-threat [bib_ref] Unraveling the mysteries of anxiety and its disorders from the perspective of..., Barlow [/bib_ref] [bib_ref] A way forward for anxiolytic drug development: testing candidate anxiolytics with anxiety-potentiated..., Grillon [/bib_ref] [fig_ref] Figure 2: Scatter plot displaying the significant association between current anxiety symptoms and drug-related... [/fig_ref]. There was no association between baseline AUDIT scores and SUV-related changes in startle magnitude (r = 0.16, p = 0.494).
We also explored whether SUV-related decreases in startle magnitude during U-threat were associated with self-reported session changes in feeling drowsy and tired.
Correlational analyses indicated there were no significant associations between startle change and tiredness (r = 0.19, p = 0.398) or drowsiness (r = 0.24, p = 0.300), further highlighting the pattern of results was not entirely driven by general sedation.
# Discussion
The current study was designed to test whether a single dose of SUV effectively and selectively modulates an objective indicator of anticipatory anxiety in humans. There is a robust animal literature suggesting the ORX system is involved in arousal-based anxiety and the motivational drive for alcohol use [bib_ref] Activation of the orexin 1 receptor is a critical component of CO2-mediated..., Johnson [/bib_ref] [bib_ref] The role of orexins/hypocretins in alcohol use and abuse, Walker [/bib_ref]. ORX antagonism has been identified as a promising next-generation therapeutic for anxiety and AUD [bib_ref] Experimental drugs for panic disorder: an updated systematic review, Caldirola [/bib_ref] [bib_ref] Orexin receptor antagonists as emerging treatments for psychiatric disorders, Han [/bib_ref]. Surprisingly, very few studies on human volunteers have directly probed the ORX system. It is therefore unclear whether prior preclinical findings translate to clinical populations and whether ORX antagonism is S.M. likely to succeed in clinical trials. In hopes of ultimately bringing forth novel mechanistic pharmacotherapies for arousal-based disorders, we aimed to implement a 'Fast-Fail' design using an objective laboratory target and a small unselected sample. Our results supported our hypotheses and corroborate preclinical data. Compared with placebo, a single dose of SUV decreased startle reactivity to U-threat, while sparing changes in startle reactivity to P-threat and no-threat. The magnitude of the within-subjects effect was large and specific. The study is the first to show in humans that ORX is a neurochemical modulator of objective anticipatory anxiety.
The effect of SUV administration on startle reactivity was specific to the U-threat condition. This is important given that excessive reactivity to U-threat is theorized to underlie the onset and maintenance of multiple forms of psychopathology. Pharmacotherapies that specifically dampen U-threat reactivity, without impacting other forms of threat sensitivity, are of extreme interest. Benzodiazepines dampen startle eyeblink potentiation to U-threat, but not P-threat, during the NPU paradigm and are known as effective anxiolytics [bib_ref] The benzodiazepine alprazolam dissociates contextual fear from cued fear in humans as..., Grillon [/bib_ref]. Although SUV targets a different neurotransmitter system, present results suggest it has a similar selective impact on human anticipatory anxiety. ORX antagonists have limited abuse potential and a relatively safe medication profile making them attractive therapeutics [bib_ref] Retrospective assessment of toxicity following exposure to Orexin pathway modulators modafinil and..., Russell [/bib_ref] [bib_ref] Suvorexant poisoning in a patient with cirrhosis and renal failure, Ito [/bib_ref].
The current findings also revealed a significant correlation between current DASS-21 anxiety scores and SUV-related decreases in startle during U-threat. This indicates that individuals with higher levels of anxiety may gain increased benefit from SUV, or greater acute decreases in anticipatory anxiety. This relationship further highlights the potential utility of targeting the ORX system in the context of clinical anxiety. However, by design, the current sample was unselected and reported generally low levels of anxiety. The correlational finding is therefore noteworthy but must be interpreted with utmost caution.
This study represents an initial, systematic step in bringing forth ORX antagonism to the clinic. The ultimate goal was to demonstrate target engagement and inform decisions to advance this line of work. The findings are a clear 'Go' signal to push forward in several important directions. First, additional laboratory studies are needed to determine whether SUV (or other ORX antagonists) selectively decreases reactivity to U-threat within clinical populations. Our data indicate SUV modulates anticipatory anxiety in relatively healthy adults. Given the multifaceted differences between healthy adults and patients, it remains to be tested whether the present findings will generalize, as well as the magnitude of SUV effects in high anxiety patients with or without a history of heavy alcohol use. Second, it is presently unclear whether SUV, and other dual orexin receptor antagonists, will be the ideal orexin-based therapies for psychiatric disorders. SUV is a readily available probe of the ORX system that was leveraged in the present study for systematic human investigation. It is important to note that the literature on functional differences between ORX1R and ORX2R is somewhat mixed. There is evidence that ORX1R plays a more prominent role in arousal, anxiety-like behavior, and alcohol use compared with ORX2R [bib_ref] The highly selective orexin/ hypocretin 1 receptor antagonist GSK1059865 potently reduces ethanol..., Lopez [/bib_ref] [bib_ref] Orexin, stress, and anxiety/ panic states, Johnson [/bib_ref]. A few studies have specifically shown that ORX1R inhibition blocks the anxiolytic-like effects of acute alcohol exposure [bib_ref] Intra-accumbal orexin-1 receptor inhibition prevents the anxiolytic-like effect of ethanol and leads..., Morales-Mulia [/bib_ref]. Accordingly, there is widespread enthusiasm for the development of selective ORX1R receptors for human translation. Some evidence has also pointed to the role of ORX2R in alcohol-seeking behavior [bib_ref] Nucleus incertus Orexin2 receptors mediate alcohol seeking in rats, Kastman [/bib_ref] and alcohol reinforcement [bib_ref] Selective blockade of the orexin-2 receptor attenuates ethanol self-administration, place preference, and..., Shoblock [/bib_ref]. Thus, additional human research is needed to better understand whether ORX1R, ORX2R, or dual receptor antagonism is most effective, and for whom.
The current study had numerous strengths including the withinsubjects placebo-controlled design and the use of a well-validated laboratory paradigm. There were also several important limitations. We administered a single dose of SUV (10 mg) and examined startle reactivity during peak drug concentration. The dose-dependent effects and time course of SUV on startle reactivity to U-threat are therefore unclear. Future studies are needed to identify optimal dosing and to what extent SUV's impact on startle is maintained over time.
The study was openlabel and there was no active control. Related, capsule blinding was only partially successful based on participant self-report. The study also lacked additional anxiety-related biomarkers and a finegrained assessment of drug mechanisms. Lastly, the sample size was small, by intention, given the Fast-Fail, proof-of-concept design. The effect of SUV on startle reactivity to U-threat was robust; though it is possible the study was underpowered to detect drug-related differences in the other task conditions.
The current study is the first to demonstrate that a single dose of SUV dampens objective measures of anticipatory anxiety in humans. SUV had no impact on baseline (No-threat) startle nor startle reactivity to threats that are predictable (P-threat). ORX antagonism, therefore, targets and modifies our laboratory measure and may be a promising strategy for alleviating chronic arousal and disrupting stress-related alcohol use. The current findings serve as a preliminary 'Go' signal to pursue ORX antagonism as a potential novel pharmacological intervention for anxiety and addiction.
[fig] Figure 1: Bar graph displaying mean startle magnitude during each task condition, across both lab sessions. PBO placebo, SUV 10 mg suvorexant. Bars reflect two standard errors. [/fig]
[fig] Figure 2: Scatter plot displaying the significant association between current anxiety symptoms and drug-related change in startle magnitude during unpredictable threat. DASS Depression, Anxiety, and Stress Scale (21 items). [/fig]
[table] Table 1: Subjective reports during each session. DEQ Drug Effects Questionnaire, VAS Visual Analog Scale, POMS Profile of Mood States. [/table]
[table] Table 2: Results of omnibus repeated measures ANOVA. [/table]
|
Genetic and environmental contributions to population group differences on the Raven's Progressive Matrices estimated from twins reared together and apart
We carried out two studies to test the hypothesis that genetic and environmental influences explain population group differences in general mental ability just as they do individual differences within a group. We estimated the heritability and environmentality of scores on the diagrammatic puzzles of the Raven's Coloured and/or Standard Progressive Matrices (CPM/SPM) from two independent twin samples and correlated these estimates with group differences on the same items. In Study 1, 199 pairs of 5-to 7-year-old monozygotic (MZ) and dizygotic (DZ) twins reared together provided estimates of heritability and environmentality for 36 puzzles from the CPM. These estimates correlated with the differences between the twins and 94 Serbian Roma (both rsZ0.32; NsZ36; ps!0.05). In Study 2, 152 pairs of adult MZ and DZ twins reared apart provided estimates of heritability and environmentality for 58 puzzles from the SPM. These estimates correlated with the differences among 11 diverse samples including (i) the reared-apart twins, (ii) another sample of Serbian Roma, and (iii) East Asian, White, South Asian, Coloured and Black high school and university students in South Africa. In 55 comparisons, group differences were more pronounced on the more heritable and on the more environmental items (mean rsZ0.40 and 0.47, respectively; NsZ58; ps!0.05). After controlling for measurement reliability and variance in item pass rates, the heritabilities still correlated with the group differences, although the environmentalities did not. Puzzles found relatively difficult (or easy) by the twins were those found relatively difficult (or easy) by the others (mean rZ0.87). These results suggest that population group differences are part of the normal variation expected within a universal human cognition.
# Introduction
Ever sinceand his cousin, there has been debate over whether general mental ability (GMA) is an innate, cultural universal or is specific to population, time and place. With the growth of evolutionary psychology, innate universalism has regained ground after six decades of being out of fashion. One question this paper addresses is 'How universal are psychological theories?' Specifically, 'Are group differences influenced by the same transaction of genetic and ecological factors as individual differences within a group?' It is well established that individual differences in GMA, at least within the White populations of the First World, are 50-80% heritable [bib_ref] Genetic and environmental influences on human psychological differences, Bouchard [/bib_ref]. The smaller amount of data available for East Asian populations, and for the Black population of the US, yield similar values [bib_ref] Thirty years of research on race differences in cognitive ability, Rushton [/bib_ref]. However, the mean differences between groups are often postulated to be due to specific ecological factors and specialized cognitive styles.
As the trend towards a more global economy continues, mean group differences in GMA are likely to become more salient, both within and across countries.
In this paper, two studies based on independent twin samples are used to calculate estimates of heritability (an indicator of genetic influence) and environmentality (an indicator of non-genetic influence) for scores on the diagrammatic puzzles that make up the Raven's Progressive Matrices. These estimates are then correlated with differences calculated between diverse groups on the same test items. Strong inference is possible [bib_ref] Strong inference, Platt [/bib_ref]. (i) Genetic theory predicts a positive association between heritabilities and group differences, (ii) ecological theory predicts a positive association between environmentality and group differences, and (iii) many models predict that both genetic and environmental factors contribute independently. However, extreme culture-only theories, which emphasize non-universality, predict a zero relationship between heritability and group differences.
To simplify, it is assumed that monozygotic (MZ) twins share 100% of their genes, while dizygotic (DZ) twins share only 50%. When the twins are reared together, they are assumed to share environmental influences, but when reared apart, they are not. Heritabilities and environmentalities are then estimated from these twin similarities and differences [bib_ref] Genetic and environmental influences on human psychological differences, Bouchard [/bib_ref]. In Study 1, the twins were reared together (MZT/DZT). Heritability was estimated by 2!(MZTrKDZTr), i.e. doubling the difference between the MZT and DZT similarities, and two environmentalities were estimated: shared family effects by MZTr-heritability and non-shared family effects by SjMZT1-MZT2j, i.e. the sum of all the MZT pair differences, with the differences between the twins assumed to be due to the environment. In Study 2, the twins were reared apart (MZA/DZA). Four heritability estimates were calculated: (i) 2!(MZArKDZAr), i.e. doubling the difference between the MZA and DZA similarities, (ii) the MZAr itself, (iii) 2!DZAr, and (iv) the average of the three. Environmentality was estimated by SjMZA1KMZA2j, the sum of all the MZA pair differences.proposed the method of correlated vectors for determining whether there is an association between a column of quantified elements (such as heritabilities and environmentalities) and any parallel column of independently derived scores (such as differences between groups). Previous studies have taken vectors of heritabilities and environmentalities from twins and other family members beyond the immediate data and found, in studies of mate choice, liking and friendship, that similarity between partners was more pronounced on the more heritable items within the sets of homogeneous traits [bib_ref] The importance of heritability in psychological research: the case of attitudes, Tesser [/bib_ref] [bib_ref] Mate choice and friendship in twins: evidence for genetic similarity, Rushton [/bib_ref]. Among anthropometric measures, for example, wrist size is more heritable than biceps size because osseous parts of the body are less susceptible to environmental modification than muscular parts. Other studies have investigated US group differences in GMA. For example, P. L. unpublished data found a correlation of rZ0.67 ( p!0.05) between heritabilities for 13 tests estimated from twins and the magnitude of mean White-Black differences.found an inverse relation of rZK0.70 ( p!0.01) between environmentality for 16 tests estimated from siblings and the mean White-Black differences. [bib_ref] Japanese inbreeding depression scores: predictors of cognitive differences between Blacks and Whites, Rushton [/bib_ref] found a correlation of rZ0.48 ( p!0.05) between genetic influence on 11 tests estimated from inbreeding depression in cousin marriages in Japan and White-Black differences in the US.
# Material and methods
The Raven's Progressive Matrices are the most well-known and best researched of all culture-reduced tests of GMA. Two versions of the test are used. Both consist of diagrammatic puzzles, each with a missing part, which the test taker attempts to identify from several options. The Coloured Progressive Matrices (CPM) consist of 36 puzzles presented in colour. Since this test spreads out the scores of the bottom 20% of the general population, it is typically given to young children. The Standard Progressive Matrices (SPM) consists of 60 non-coloured puzzles suitable for a middle range of ability. The first 24 puzzles are the same in both the CPM and the SPM (although the CPM presents them in colour). Reliability and validity remain high across a wide variety of cultural groups, regardless of whether a timed or untimed assessment is administered. Both the CPM and the SPM are good measures of g, the general factor of GMA. These tests have been described as measuring 'analogical thinking', 'the ability to identify relationships' and to 'think clearly'.
(a) Study 1: 199 pairs of 5-to 7-year-old Canadian twins reared together Two samples were compared on the CPM. The first sample consisted of 199 pairs of 5-to 7-year-old Canadian twins reared together (MZT/DZT) from the Western Ontario Twin Project, an ongoing longitudinal study initiated in 1987 [bib_ref] The Western Ontario Twin project: an overview, Vernon [/bib_ref]. The sample was selected from a larger pool of 3-to 7-year-olds on the assumption that scores below 5 years would be less reliable. Only twins with complete information (e.g. zygosity) were included. One year after the initial testing, 108 participants were tested a second time.
(The most recent score was used.) There were 58 MZT pairs (29 female pairs and 29 male pairs) and 141 DZT pairs (31 female pairs, 42 male pairs and 68 opposite-sex pairs), with 148 5-year-olds, 208 6-year-olds and 42 7-year-olds. The second sample consisted of 92 16-to 66-year-old Roma (Gypsies) in Serbia, previously studied by [bib_ref] General mental ability in South Asians: data from three Roma (Gypsy) communities..., Rushton [/bib_ref]. They were a subset of 323 who had been allotted the CPM after it was found that the SPM produced very low scores for this population.
(b) Study 2: 152 pairs of twins from the University of Minnesota Study of Twins Reared Apart Eleven samples were compared on the SPM. The first sample consisted of 152 pairs of adult twins reared apart (MZA/DZA) from the University of Minnesota Study of Twins Reared Apart (MISTRA). This research project was initiated in 1979 and many results reported [bib_ref] Sources of human psychological differences: the Minnesota study of twins reared apart, Bouchard [/bib_ref] [bib_ref] Genetic and environmental influences on human psychological differences, Bouchard [/bib_ref]. Most of the twins were separated early in life, reared in adoptive families and then reunited only in adulthood. They were assessed with a week-long battery of tests evaluating medical and physical traits as well as psychological characteristics that included GMA, personality, interests and attitudes. The SPM were presented through slides and individually administered on an untimed basis [bib_ref] Research with twins: the concept of emergenesis, Lykken [/bib_ref]. Eight to twelve years after the initial testing, 87 participants returned for a second assessment. The full sample consisted of 385 people (142 males and 243 females), 16-to 77-year-olds (meanZ44 years). There were 92 MZ pairs (57 female pairs and 35 male pairs) and 60 DZ pairs (33 female pairs, 12 male pairs and 15 opposite-sex pairs), as well as 33 spouses of twins and 48 other adopted and biological family members. (This was a subset of the fuller MISTRA sample because not all twins completed the SPM.) There were 10 other samples. One of these comprised the 231 16-to 66-year-old Roma in Serbia remaining from study 1; four were from [bib_ref] The suitability of Raven's standard progressive matrices for various groups in South..., Owen [/bib_ref] who administered the SPM to 1093 White, 778 South Asian, 1063 Coloured and 1056 Black 14-to 16-year-old high school students in South Africa; and five were from [bib_ref] Construct validity of Raven's Advanced Progressive Matrices for African and non-African engineering..., Rushton [/bib_ref] [bib_ref] General mental ability in South Asians: data from three Roma (Gypsy) communities..., Rushton [/bib_ref]
# Results
(a) Study 1: 199 pairs of 5-to 7-year-old Canadian twins reared together In the electronic supplementary material, sheet 1 summarizes the results for the twins and the Roma on the 36 puzzles of the CPM. Column A lists the item numbers. Columns B and C give the proportion of the twins and Roma who answered each item correctly. Column D gives the twin-Roma differences in item pass rates (kept positive by subtracting the lower scoring group from the higher). Column E gives the tetrachoric item test-retest correlation, with a minimum score of zero, calculated from the 108 twins tested twice (mean item reliabilityZ0.16). Items relatively difficult or easy for the twins were those found relatively difficult or easy for the Roma (rZ0.90; NZ36; p!0.001), indicating construct similarity across the two groups. Columns F and G give the item-total correlations, which are the biserial correlations of each item's pass or fail status (0 or 1) with the total score on the test. They indicate the extent to which a particular item measures the same construct measured by the test as a whole, as well as how well the item discriminates between testees within each group. Those with high values among the twins had high values among the Roma (rZ0.64; NZ36; p!0.001). Columns H and I show the intraclass correlations for the MZT and DZT pairs with a minimum set at zero and the inclusion of opposite-sex pairs in the DZT column. Column J shows the heritabilities calculated by 2!(MZTrKDZTr), with a minimum score of zero. Column K shows the shared family environmentality measured by MZTr-heritability and Column L gives the non-shared environmentalities measured by the differences within twin pairs, i.e.
## Sjmzt1kmzt2j.
The vectors of both heritability and non-shared environmentality (columns J and L) significantly correlated with the vector of standardized twin-Roma differences (rsZ0.32; NsZ36; ps!0.05), but the vectors of shared environmentalities did not (rZK0.10). Two possible confounding effects were considered: item reliability and the degree of variance in the twins' item pass rates. Given that items with more reliability and more variance enable higher heritabilities and larger group differences to be calculated, a spurious relation could be found between vectors of heritability and environmentality on the one hand and of group differences on the other, owing to the relation between both these sets and variance and reliability. To examine this possibility, we used partial correlations to statistically control for item reliability (using the test-retest correlation in column E) and item variance (measured by each item's deviation from the maximally variant pass rate of 50% in column B, i.e. jitem pass rate-50j). Partialling out the reliability did not alter the results, whereas partialling out the variation in item pass rate caused the correlation between heritability and group differences to increase (rZ0.40; p!0.01), the correlation with non-shared environmentality to decrease (rZ0.20; ns) and the correlation with shared environmentality to remain null (rZK0.16).
We also examined whether the twin-Roma differences were on g, the general factor of mental ability. Since the total score on the Raven's is a good measure of g, the item-total correlations (columns F and G) provide an estimate of each item's g loading. These item-totals were correlated with the standardized twin-Roma differences, first using the itemtotals for the twin group and then those for the Roma. The results were rZ0.47 ( p!0.01) and 0.31 ( p!0.05), respectively, indicating that the twin-Roma differences were on the more g-loaded items. (Note: it would have been incorrect to use the item-total correlations from the combined samples because these would reflect the betweengroups variance in addition to the within-groups variance and thus inflate the effect.)
## (b) study 2: 152 pairs of twins from the university of minnesota study of twins reared apart
In the electronic supplementary material, sheet 2 summarizes the results for the Minnesota twin sample on the 60 puzzles of the SPM. Column A lists the item numbers. Columns B and C give the proportion of the twins who passed each item and the sample size on which it was based. (The first two items were given as practice and not scored.) Column D gives the tetrachoric test-retest correlation for each item, with a minimum score of zero, calculated from data on 87 twins tested twice (mean item reliabilityZ0.40). Column E gives the item-total correlations, which indicate each item's g loading, as described in Study 1. Columns F and G give the intraclass correlations for the MZA and DZA twin pairs with a minimum of zero and the inclusion of opposite-sex pairs in the DZ column. Columns H-L provide the four heritabilities and the measure of environmentality (mean item heritabilityZ0.20 and mean item environmentalityZ0.21). Sheet 3 gives the proportion of each non-twin sample that selected the correct answer on the items. Column A repeats the listing of SPM item numbers. Column B gives the item pass rates for the Roma. Columns C-F show the item pass rates for the South African high school students (White, South Asian, Coloured and Black). Columns G-K show the item pass rates for the South African undergraduates (East Asian, White, South Asian, Coloured and Black). The average item pass rates ranged widely: 93% for East Asian undergraduates, 69% for the twins and 49% for the Roma. Sheet 4 gives the 55 combinations of group comparisons with each group's mean pass rate (column A, with column B giving the mean difference). Columns C-L provide the results of correlating the four heritability vectors and the one of environmentality with those of the group differences in standardized pass rates, along with the levels of significance. (The correlations were kept positive by subtracting the lower scoring group from the higher.)
The vectors of both heritability and environmentality were found to be associated with the magnitude of the group differences (mean rsZ0.40 and 0.47, respectively; NsZ58; ps!0.05). As in study 1, two possible confounding effects were considered: the item reliabilities (sheet 2, column E) and the degree of variance in the twins' item pass rates (the deviation from the maximally variant pass rate of 50% in column B). The results did not change when item reliability was statistically controlled. However, when controlling for item pass rate variance, the average heritability correlation with the average group difference was reduced to rZ0.21 ( p!0.05) and the environmentality correlation was no longer significant (rZ0.08).
The item pass rates were very similar for all 11 samples (mean rZ0.87). Those items found relatively difficult (or easy) by one group were found relatively difficult (or easy) by the others, indicating construct validity across the groups. These high correlations occurred despite marked differences in mean levels of passing the items. Moreover, as in study 1, the item-total scores for the twin sample (sheet 2, column E) correlated with the standardized differences in pass rates for all the twin/non-twin comparisons (mean rZ0.38; NZ58; p!0.05), indicating Twins together and apart J. P. that the twin/non-twin differences were on g, as in study 1. When correlating the 55 group comparisons with the relevant item-total correlations (not shown), the mean rZ0.36 (total N of correlationsZ110).
# Discussion
We found that vectors of heritability and environmentality calculated from two independent twin samples on tests of GMA were associated with vectors of population group differences on the same tests and, prior to correction, at about the same level of magnitude. The results were robust despite marked heterogeneity in age range across samples, a lack of power due to small Ns in some groups, and many non-optimal item pass rates. Heritabilities and environmentalities estimated from 5-to 7-year-old twins reared together in Canada generalized to a sample of 16-to 66-year-old Roma in Serbia, while those estimated from 17-to 77-year-old twins reared apart in the Minnesota Study of Twins Reared Apart generalized to another sample of Serbian Roma as well as to high school and university students from South Africa. Thus, these results join other data to suggest that genetic as well as environmental influences contribute to group differences in GMA [bib_ref] Thirty years of research on race differences in cognitive ability, Rushton [/bib_ref]. They appear to confirm what has long been referred to as the 'default hypothesis' by those psychometricians who have studied the issue most intensely, i.e. that, by adulthood, genetic and environmental factors carry the same weight in causing population group differences in GMA as they do in causing individual differences (say 50% each).
Item reliabilities and item variance in the twins' pass rates were considered as potential sources of contamination because each could affect item heritability as well as the magnitude of the group differences, thereby producing a spurious relation between them. When item reliability and item variance were statistically controlled, the correlation between heritability and group differences remained intact, although the correlation between environmentality and group differences went to zero. This led one reviewer to suggest that the results could be interpreted in terms of a 100% genetic-0% environmental model. However, in the case of the item reliabilities, there may have been an under-correction as the reliabilities themselves were based on 1-year retests in 5-to 7-yearolds and 10-year retests in adults. In the case of the item variances, there may have been an over-correction and it is always possible that an (unmeasured) methodological factor that affected heritability might also affect the group differences and thus reduce that correlation to zero too.
A range of interpretations concerning the strength of the effects in these data, ranging from 'weak' to 'strong', is possible. The more stringent conclusion would emphasize that the findings are based only on correlational analyses, which do not prove causality. There may be (unmeasured) gene-environment interactions that can make heritabilities and environmentalities more dependent on each other than is typically assumed ( Johnson in press). For example, identical twins reared apart may experience similar environments owing to the similar way they select from the array of possible alternatives, thereby making the phenotypic variance apportioned to heritability partly environmental in origin. Conversely, identical twin differences, apportioned to environmentality, may occur because each twin inherits an equally vulnerable (or resilient) personality and thus suffer a similar level of setback to separate events. However, it is difficult to see how these (unmeasured) potential interactions could explain away our finding that the test items measured the same construct across twins reared together and apart and across very diverse groups, as indicated by their similar levels of item pass rate (rZ0.87), item-total correlation (rZ0.39) and item-total association with the magnitude of the group differences (rZ0.38).
Rough-hewn though our heritability and environmentality estimates may have been, as well as our corrections for item reliability and item variance, the results call into question three widely held assertions that, in various circles, have become dogma: (i) test takers must be similar in cultural, educational and social background to those on whom the test was standardized, (ii) heritability estimates are only specific to a population, and (iii) the differences between population groups in GMA are only due to ecological factors and only trivially, if at all, due to genetic influence.
The results found here are consistent with the preponderance of evidence from other studies on the crosscultural validity of GMA. Apart from the obvious example of language bias, there is little or no evidence of populationspecific cultural effects. For example, [bib_ref] The relationship between academic and practical intelligence: a case study in Kenya, Sternberg [/bib_ref] found that GMA in 12-to 15-year-old Kenyans predicted school grades at about the same level as they do in the West with a mean rZ0.40 ( p!0.001). [bib_ref] Construct validity of Raven's Advanced Progressive Matrices for African and non-African engineering..., Rushton [/bib_ref] found that GMA predicted university performance equally well in African and non-African engineering students in South Africa (rw0.30; p!0.05). [bib_ref] International validity generalization of GMA and cognitive abilities: a European community meta-analysis, Salgado [/bib_ref] demonstrated the international generalizability of GMA across 10 member countries of the European Community (EC), thus contradicting the view that criterion-related validity is moderated by differences in a nation's culture, religion, language, socioeconomic level or employment legislation. He found that scores predicted job performance ratings at rZ0.62 and training success at rZ0.54.
Twin designs are an underused resource in the human sciences [bib_ref] Genetic and environmental influences on human psychological differences, Bouchard [/bib_ref]. The present study demonstrates their usefulness in showing that a similar transaction of genetic and non-genetic influence applies across a wide range of population groups growing up in diverse cultures. There appears to be a set of human psychological adaptations underlying the cognitive problem solving required for the type of GMA test used here, with individual and group differences comprising normal variants. |
Influence of shift work on cardiovascular disease risk in Southern African long-distance truck drivers: a cross-sectional study
## Linear regression
## Log (frs)
## Univariable
Unstandardized β
[formula] coefficient (95% CI) P Multivariable Unstandardized β coefficient (95% CI) P Day/night shift (2-3 nights) -0.03 (-0.10-0.04) 0.34 -0.03 (-0.06-0.01) 0.15 [/formula]
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Fiberoptic bronchoscopy for the rapid diagnosis of smear-negative pulmonary tuberculosis
Background: This study was aimed to investigate the diagnostic value of fiberoptic bronchoscopy (FOB) with chest high-resolution computed tomography (HRCT) for the rapid diagnosis of active pulmonary tuberculosis (PTB) in patients suspected of PTB but found to have a negative sputum acid-fast bacilli (AFB) smear.Methods:We evaluated the diagnostic accuracy of results from FOB and HRCT in 126 patients at Gangnam Severance Hospital (Seoul, Korea) who were suspected of having PTB.Results: Of 126 patients who had negative sputum AFB smears but were suspected of having PTB, 54 patients were confirmed as having active PTB. Hemoptysis was negatively correlated with active PTB. Tree-in-bud appearance on HRCT was significantly associated with active PTB. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FOB alone was 75.9%, 97.2%, 95.3%, and 84.3%, respectively, for the rapid diagnosis of active PTB. The combination of FOB and HRCT improved the sensitivity to 96.3% and the NPV to 96.2%.Conclusions: FOB is a useful tool in the rapid diagnosis of active PTB with a high sensitivity, specificity, PPV and NPV in sputum smear-negative PTB-suspected patients. HRCT improves the sensitivity of FOB when used in combination with FOB in sputum smear-negative patients suspected of having PTB.
# Background
Tuberculosis (TB) is a major health problem around the world, with an approximate incidence of 9.4 million (8.9-9.9 million) and with about 1.7 million TB-related deaths in 2009. The global strategy to control TB is prompt diagnosis, notification, and successful treatment of patients with active, transmissible disease. Early diagnosis of active pulmonary tuberculosis (PTB) is critical for TB control.
Unfortunately, diagnosis of active PTB is often delayed because fewer than half of these patients have a positive sputum smear (29-39% in 2009 in Korea), and isolation of Mycobacterium tuberculosis (MTB) takes a long time. Acid-fast bacilli (AFB) smears of respiratory specimens (at least two or more specimens) are important for the prompt diagnosis of PTB, but AFB smears have poor sensitivity (30-70%) despite high specificity (98-99%).
Mycobacterial cultures are more sensitive than AFB smears (80-85%), but culture results usually require 3-8 weeks [bib_ref] New tools for the diagnosis of tuberculosis: the perspective of developing countries, Foulds [/bib_ref]. The diagnosis of TB and the decision to start treatment against sputum smear-negative TB is usually dependent on clinical features, but 20% of PTB patients are completely asymptomatic whereas 42-86% of PTB patients may be symptomatic. Sputum smear-negative PTB patients are especially likely to show no or mild respiratory symptoms and systemic manifestations [bib_ref] The roles of HRCT and clinical parameters in assessing activity of suspected..., Tozkoparan [/bib_ref].
Chest x-ray is another method often used in PTB screening, but in sputum smear-negative TB, many cases showed atypical x-ray patterns or normal findings [bib_ref] Identifying pulmonary tuberculosis in patients with negative sputum smear results, Kanaya [/bib_ref]. In addition, this method has the limitation of a high false positive rate due to previous PTB infection in an intermediate or high TB burden country. To assist in the diagnosis of sputum smear-negative PTB and latent TB infection (LTBI), clinicians use the tuberculin skin test (TST). However, the TST is limited in application due to cross-reactivity with non-tuberculous mycobacteria (NTM) species and Bacillus Calmette-Geurin (BCG) vaccine strains, and its sensitivity is affected by both malnutrition and immunosuppression [bib_ref] An Update on the Diagnosis of Tuberculosis Infection, Richeldi [/bib_ref].
Chest high-resolution computed tomography (HRCT) provides information about the extent and distribution of PTB and can be a great help in identifying its activity. In sputum smear-negative cases, HRCT is superior to chest x-ray in the diagnosis of PTB and in the determination of its extent and distribution. Further, it confirms the presumptive diagnosis of active PTB and allows antituberculosis treatment to commence more quickly. Despite some disadvantages such as high cost and radiation exposure, HRCT is widely used when traditional methods have failed to diagnose PTB in sputum smearnegative patients. Typical radiological patterns of PTB reactivation such as upper lobe involvement or cavity formation are rarely observed in sputum smear-negative cases due to the smaller burden of mycobacterium [bib_ref] The roles of HRCT and clinical parameters in assessing activity of suspected..., Tozkoparan [/bib_ref] ; therefore, HRCT alone is limited in diagnosing PTB in sputum smear-negative patients.
Fiberoptic bronchoscopy (FOB) can provide alternative respiratory specimens for diagnosis [bib_ref] Diagnostic role of fiberoptic bronchoscopy in suspected smear negative pulmonary tuberculosis, Charoenratanakul [/bib_ref] [bib_ref] Use of the flexible fibreoptic bronchoscope in diagnosis of sputum-negative pulmonary tuberculosis, Willcox [/bib_ref] , especially from specific sites that are suspected by radiological testing for involvement of PTB when sputum expectoration has repeatedly failed because sputum is absent [bib_ref] Diagnosing sputum/smearnegative pulmonary tuberculosis: Does fibre-optic bronchoscopy play a significant role, Bachh [/bib_ref]. FOB is also more useful in the diagnosis of endobronchial TB, which can be seen as normal in HRCT, and FOB may be superior in the differential diagnosis of tuberculosis with other commonly encountered diseases such as pneumonia or lung cancer [bib_ref] The diagnostic value of bronchoscopy in smear negative cases with pulmonary tuberculosis, Araz [/bib_ref]. Despite the fact that it is more aggressive and relatively expensive, FOB is considered useful for the diagnosis of sputum smear-negative PTB because of these advantages. Results of bronchial washing specimens with AFB smears or MTB polymerase chain reaction (PCR) and tissue specimens from bronchoscopic or transbronchial biopsies are usually received within one week, thus enabling rapid diagnosis before the availability of confirmation from sputum cultures of sputum smear-negative PTB patients.
The aim of this study was to evaluate the diagnostic value of FOB with HRCT in the diagnosis of active PTB in patients suspected of PTB but presenting with a negative sputum smear in an intermediate TB burden country.
# Methods
## Study setting and subjects
We retrospectively reviewed the clinical records and FOB and HRCT results of all patients with suspected PTB who visited the pulmonary clinic of Gangnam Severance Hospital, Seoul, Korea from January 2009 to December 2010.
Inclusion criteria were (1) age of at least 18 years with clinical or radiographic suspicion of active PTB,negative AFB smear results of 2 or more sputum pairs or failure to expectorate sputum (in other words, could not expectorate sputum voluntarily, did not submit any sputum specimen) and (3) having received both FOB and HRCT within 1 month due to suspected PTB before initiation of anti-TB treatment. When FOB was performed, a bronchial washing from the affected lung was acquired for AFB smear, mycobacterial culture and MTB-PCR for all patients. A bronchoscopic biopsy was performed on patients with an endobronchial lesion. Exclusion criteria were (1) AFB smear-positive patients, (2) patients with clinically diagnosed PTB according to clinical and radiological tuberculosis findings who showed no clinical improvement with empirical antibiotics but did show clinical and radiological improvement with anti-tuberculosis medication, and (3) patients with inconclusive diagnoses due to loss of follow-up.
From January 2009 to December 2010, 182 patients who were suspected of having PTB visited our clinic and underwent FOB and chest CT. HRCT was performed with contrast CT, simultaneously. After excluding smear-positive PTB patients and patients with other exclusion criteria, we evaluated the diagnostic accuracy of FOB and HRCT results in 126 patients. The recorded clinical information on these patients included age, gender, cough, sputum, fever, hemoptysis, chest pain, and weight loss.
Bronchoscopic procedure is performed according to our institute's infection regulation and instruction guideline. Inspectors wear N95 masks, goggles and gown during procedure, bronchosopy room is equipped with negative pressure isolation and air disinfection system, Bronchoscopy is performed according to manufacturer's guidelines plus scope surveillance.
## Diagnostic definition
Active PTB was confirmed when (1) MTB was cultured or (2) a caseating granuloma was found in the lung tissue by bronchoscopic biopsy or transthoracic needle biopsy, and when the PTB showed appropriate response to treatment. In cases diagnosed by pathology, active PTB was confirmed only if MTB tissue culture or tissue MTB-PCR was positive, for excluding NTM or other granulomatous diseases. A final diagnosis of 'non-TB' was accepted when an alternative diagnosis was reached.
A rapid diagnosis of sputum smear-negative PTB by FOB was defined as a diagnosis of PTB through methods that yielded results within 1 week: (1) a positive AFB smear, (2) positive MTB-PCR, or (3) a caseating granuloma upon biopsy.
CT findings were reviewed by two experienced thoracic radiologists, without clinical information or final diagnosis in each case. CT findings were described as having more than one of the following characteristics: (1) consolidation, (2) cavities, (3) centrilobular air space nodules, (4) tree-in-bud appearance and (5) hilar and/or mediastinal lymph node enlargement. After reviewing the CT findings independently, the two radiologists met, discussed and reached a final diagnosis by consensus.
The definition of an immunocompromised condition included the following: (1) having a diagnosis of diabetes mellitus, (2) undergoing chemotherapy for an underlying malignancy at the time of TST and QuantiFERON W -TB Gold In-Tube (QFT-IT, Cellestis Ltd; Carnegie, Australia) testing, (3) having had received either a solid organ transplant or bone marrow transplant, (4) having a diagnosis of end-stage renal disease and on renal replacement therapy, (5) having a diagnosis of advanced liver cirrhosis with Child-Pugh class C, [bib_ref] Identifying pulmonary tuberculosis in patients with negative sputum smear results, Kanaya [/bib_ref] being seropositive for human immunodeficiency virus, or (7) undergoing daily administration of systemic corticosteroids (at least 15 mg prednisone per day for more than 1 month) or combination therapy with low dose corticosteroids and other immunosuppressants including azathioprine, mycophenolate, methotrexate, cyclosporine, or cyclophosphamide.
For the combination of FOB and HRCT, the results of the combination were considered positive when at least one test was positive.
# Analysis
Data were analyzed with SPSS statistical software (version 18.0; SPSS; Chicago, IL, USA). Univariate comparisons between active PTB and non-TB patients were performed using Fisher's exact test for categorical variables and the Mann-Whitney test for continuous variables, where appropriate. Associations of clinical and radiological parameters with active PTB diagnosis were analyzed using univariate or multivariate logistic regression modeling. For multivariate analysis, variables were incorporated into the model in a stepwise manner. All tests of significance were two-sided and a P value < 0.05 was considered statistically significant. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of active PTB disease were calculated for each diagnostic test.
# Ethical issues
# Results
## Demographic characteristics
Of the 145 patients with suspected sputum smearnegative TB, five patients were excluded due to clinically diagnosed PTB, and 14 patients were excluded due to inconclusive diagnoses [fig_ref] Figure 1: Study flowchart [/fig_ref].
Baseline characteristics of the non-excluded 126 patients are summarized in [fig_ref] Table 1: Demographic and clinical characteristics of all sputum smear-negative TB suspects [/fig_ref]. Active PTB was diagnosed in 54 patients (42.9%), of which 48 (88.9%) had positive TB cultures and 6 (11.1%) cases were confirmed by pathology and MTB-PCR. Of 48 cultureconfirmed cases, 28 were culture positive by both sputum and bronchial washing specimens, 6 by sputum only, and 14 by bronchial washing specimens only.
Alternative (non-TB) diagnoses were made for 72 patients (34 pneumonia, 5 NTM colonization, 15 sequelae of a previous TB infection, 12 lung cancers, and 6 interstitial lung disease).
The mean patient age was 51.0 ± 18.2 years, and the mean age of the non-TB group was higher than that of the TB group (55.4 ± 16.6 years vs. 45.3 ± 18.8 years, respectively, P = 0.002). Immunocompromised conditions were found in 11 patients (6 diabetes, 2 chemotherapy, 2 hematologic malignancies, and 1 systemic corticosteroid use). There was no difference between the TB and non-TB groups in terms of immunocompromised status, but previous TB history was significantly different between the TB and non-TB groups (P = 0.014, [fig_ref] Table 1: Demographic and clinical characteristics of all sputum smear-negative TB suspects [/fig_ref].
## Clinical characteristics and hrct findings in sputum smear-negative tb suspects
Chest x-rays showed a higher positive rate in the TB group than in the non-TB group (53.7% vs. 20.8%, respectively, P < 0.001), and QFT-IT also showed significantly higher positive rate in the TB group than in the non-TB group (87.0% vs. 46.4%, respectively, P = 0.009) [fig_ref] Table 1: Demographic and clinical characteristics of all sputum smear-negative TB suspects [/fig_ref]. Only hemoptysis (P = 0.009, OR = 0.30 [0.12-0.76, 95% CI]) was negatively correlated with active PTB in sputum smear-negative suspects [fig_ref] Table 2: First presenting symptoms of patients with sputum smear-negative TB Definition of abbreviations [/fig_ref].
Fifty-three patients were suspected of having active PTB by HRCT, and 38 (71.7%) were correctly diagnosed with active PTB. Of the 73 patients suspected to be non- TB, 58 (79.5%) were correctly excluded. HRCT findings in the diagnosis of PTB showed a significant difference between TB and non-TB patients (P < 0.001) (data not shown). The most frequently observed CT findings (in order of decreasing frequency) were consolidation, mediastinal lymphadenopathy, tree-in-bud appearance, and nodules. Tree-in-bud nappearance was the only significant finding associated with active PTB status (P < 0.001, OR = 5.10 [2.37-10.99, 95% CI]) [fig_ref] Table 3: Multivariate regression analysis of CT findings in sputum smear-negative TB suspects [/fig_ref].
## Bronchoscopic characteristics in ptb patients
Of the 54 patients confirmed with active PTB, 41 patients had TB and 13 patients did not have TB according to rapid diagnosis via bronchoscopic methods. As a result, two non-TB patients were misdiagnosed as having TB by bronchoscopic rapid diagnosis (Additional file 1: [fig_ref] Table 1: Demographic and clinical characteristics of all sputum smear-negative TB suspects [/fig_ref]. Baseline characteristics were not different between TB and non-TB patients divided by bronchoscopic diagnosis (Additional file 1: [fig_ref] Table 2: First presenting symptoms of patients with sputum smear-negative TB Definition of abbreviations [/fig_ref].
The bronchoscopic findings are summarized in [fig_ref] Table 4: Bronchoscopic findings in 54 patients confirmed to have active PTB [/fig_ref]. Of the 54 PTB-confirmed patients, 42 patients had positive bronchial washing cultures [fig_ref] Table 4: Bronchoscopic findings in 54 patients confirmed to have active PTB [/fig_ref] , 34 patients had positive sputum cultures, and 28 patients showed a positive result on both the sputum and bronchial washing cultures, but 14 patients had positive only in bronchial washing cultures (Additional file 1: [fig_ref] Table 3: Multivariate regression analysis of CT findings in sputum smear-negative TB suspects [/fig_ref].
Of the 54 PTB-confirmed patients, 41 (75.9%) patients were rapidly diagnosed in less than 1 week by bronchial washing AFB smear, MTB-PCR and/or biopsy, and 13 patients were confirmed several weeks later by bronchial washing or sputum culture results. AFB smears produced by bronchial washing showed positive results for 25.9% (14/54) of the PTB-confirmed patients, and bronchoscopic biopsy results were positive in 22 of 23 patients who underwent a biopsy [fig_ref] Table 4: Bronchoscopic findings in 54 patients confirmed to have active PTB [/fig_ref]. Of 23 patients who underwent a biopsy, a transbronchial biopsy was performed in 5 patients and a bronchial biopsy was performed in 18 patients with an endobronchial lesion (data not shown). MTB-PCR from a bronchial washing showed positive results in 61.1% (33/54) of patients [fig_ref] Table 4: Bronchoscopic findings in 54 patients confirmed to have active PTB [/fig_ref] , and 2 of the finally diagnosed 72 non-TB patients showed falsepositive results (Additional file 1: [fig_ref] Table 1: Demographic and clinical characteristics of all sputum smear-negative TB suspects [/fig_ref].
## Diagnostic accuracy of fob and hrct in sputum smearnegative tb suspects
The diagnostic accuracy of FOB, HRCT, and the combination of FOB and HRCT in sputum smear-negative TB suspects is summarized in
# Discussion
In this retrospective study, we found that FOB showed high sensitivity, specificity, PPV, and NPV in the rapid diagnosis of sputum smear-negative PTB, and the combination of FOB and HRCT increased the sensitivity and NPV. Several previous studies evaluated clinical characteristics and scoring systems for the diagnosis of sputum smear-negative PTB [bib_ref] Methods for diagnosing tuberculosis among in-patients in Eastern Africa whose sputum smears..., Samb [/bib_ref] [bib_ref] An evaluation of the diagnostic value of clinical and radiological manifestations in..., Tessema [/bib_ref] [bib_ref] HRCT and whole-blood interferon-gamma assay for the rapid diagnosis of smear-negative pulmonary..., Lee [/bib_ref]. Samb et al. [bib_ref] Methods for diagnosing tuberculosis among in-patients in Eastern Africa whose sputum smears..., Samb [/bib_ref] reported independent predictors of active PTB including a chronic cough lasting longer than 3 weeks, chest pains longer than 15 days, absence of sputum, and absence of dyspnea, and Lee et al. [bib_ref] HRCT and whole-blood interferon-gamma assay for the rapid diagnosis of smear-negative pulmonary..., Lee [/bib_ref] reported that the lack of sputum was a positive predictor of active PTB. But even in these studies, the specificity of the clinical predictors or scoring system was low, and the PPV was reported at a mere 50%. In our study, hemoptysis was the only negative predictor in sputum smear-negative TB suspects, and other clinical characteristics did not help in the prediction of active PTB.
We discovered that tree-in-bud appearance was a meaningful HRCT finding in the diagnosis of sputum smear-negative PTB. Recently, several studies have reported that tree-in-bud appearance, lobular consolidation and large nodules were positively associated with active PTB in sputum smear-negative TB suspects, but the diagnostic accuracy was not satisfactory [bib_ref] HRCT and whole-blood interferon-gamma assay for the rapid diagnosis of smear-negative pulmonary..., Lee [/bib_ref] [bib_ref] Utility of high-resolution computed tomography for predicting risk of sputum smear-negative pulmonary..., Nakanishi [/bib_ref]. Matsuoka et al. [bib_ref] Relationship between CT findings of pulmonary tuberculosis and the number of acid-fast..., Matsuoka [/bib_ref] stated that CT findings in sputum smear-negative patients differed from those in smearpositive patients and suggested that CT findings are not helpful in judging sputum smear-negative TB suspects.
Despite being less infectious than sputum smear-positive PTB, smear-negative PTB serves as an important cause of transmission in communities by delaying diagnosis and precluding initiation of treatment and often leads to complications of irreversible lung damage in infected individuals [bib_ref] New tools for the diagnosis of tuberculosis: the perspective of developing countries, Foulds [/bib_ref]. Therefore, sputum smear-negative PTB often requires more invasive diagnostic tools to be distinguished from other diseases such as lung cancer. In addition, in intermediate or high TB burden countries such as South Korea, beginning unnecessary anti-TB treatment prior to Definition of abbreviations: CT = computed tomography, TB = tuberculosis, n = number, OR = odds ratio, 95% CI = 95% confidence interval, LAP = lymphadenopathy. receiving results of mycobacterial culture may cause unnecessary economic burden to the community and drug side effects to the patient. Lee et al. [bib_ref] HRCT and whole-blood interferon-gamma assay for the rapid diagnosis of smear-negative pulmonary..., Lee [/bib_ref] reported that the sensitivity and the specificity of sputum TB-PCR was 43.2% (95% CI, 27-60%) and 97.7% (95% CI, 86-99%), respectively. Because of its low sensitivity and NPV (66.7% [95% CI, 54-78%]), sputum TB-PCR alone was limited in ruling out TB. In a recent meta-analysis study [bib_ref] Interferon-gamma release assays for the diagnosis of active tuberculosis: a systematic review..., Sester [/bib_ref] , the sensitivity and the specificity of QFT-IT was 80% (95% CI, 75-84%) and 79% (95% CI, 78-84%), respectively, and the sensitivity and the specificity of T-SPOT TB was 81% (95% CI, 78-84%) and 59% (95% CI, 56-62%), respectively. The diagnostic sensitivity of both interferon-gamma release assays (IGRAs) was higher than that of the TST, but the sensitivity of IGRAs was still not high enough for IGRAs to be used as a rule out test for TB. In addition, the specificity of the IGRAs was not sufficient for the diagnosis of active TB disease with differentiation from latent TB infection.
In previous studies, FOB had a sensitivity of 80-93% and a specificity of 70-95% [bib_ref] Diagnostic role of fiberoptic bronchoscopy in suspected smear negative pulmonary tuberculosis, Charoenratanakul [/bib_ref] [bib_ref] Use of the flexible fibreoptic bronchoscope in diagnosis of sputum-negative pulmonary tuberculosis, Willcox [/bib_ref] [bib_ref] Detection of Mycobacterium tuberculosis in bronchoalveolar lavage from patients with sputum smear-negative..., Liam [/bib_ref] [bib_ref] Rapid diagnosis of smear-negative pulmonary tuberculosis via fibreoptic bronchoscopy: utility of polymerase..., Wong [/bib_ref] for rapid diagnosis of sputum smear-negative PTB, and HRCT had a sensitivity of 60-80% and a specificity of 50-70% [bib_ref] HRCT and whole-blood interferon-gamma assay for the rapid diagnosis of smear-negative pulmonary..., Lee [/bib_ref] [bib_ref] The role of 67gallium scintigraphy and high resolution computed tomography as predictors..., Lai [/bib_ref] [bib_ref] The rapid diagnosis of smearnegative pulmonary tuberculosis: a cost-effectiveness analysis, Lim [/bib_ref]. Our results indicate that HRCT has a similar sensitivity of 85.2% and specificity of 72.2%, but HRCT alone is limited as usual in the diagnosis of PTB in sputum smear-negative TB suspects due to its low PPV of 69.7% (positive likelihood ratio (LR+), 3.067 [95% CI, 2.131-4.117]). We found that FOB alone is useful for the rapid diagnosis of sputum smearnegative PTB with a high PPV of 95.3% (LR + = 27.333 [95% CI, 8.674-99.257]). FOB is also useful for the exclusion of non-TB cases from patients suspected of having active PTB with a high specificity and NPV in our setting. The combination of FOB with HRCT increased the sensitivity to 96.3% and NPV to 96.2%.
The high cost and concerns about the invasiveness of FOB and radiation exposure of HRCT limit the usefulness of these tests, but FOB is a safe and widely performed procedure. Previous studies [bib_ref] Complications of fiberoptic bronchoscopy, Credle [/bib_ref] [bib_ref] Bronchoscopy in the intensive care unit, Jolliet [/bib_ref] have reported that with the help of oxygenation, adequate premedication, and performance by experienced physicians, FOB shows very low complication rates and few life-threatening side effects. One previous study investigating the cost-effectiveness of FOB and HRCT in the diagnosis of PTB [bib_ref] The rapid diagnosis of smearnegative pulmonary tuberculosis: a cost-effectiveness analysis, Lim [/bib_ref] reported that FOB and HRCT play significant roles in the moderate or high PTB probability setting compared with the low PTB probability setting. Considering the high specificity of our study, FOB and/ or HRCT would be useful in a high TB burden country as well as in an intermediate TB burden country such as South Korea.
There are several limitations in this study. First, it was a retrospective study and the study population and clinical setting were selective and limited, so it is difficult to generalize this result to other settings. Second, the commonly used TST was not evaluated in our study. This was based on previous studies showing the limitation of TST for evaluating TB in South Korea due to BCG vaccination [bib_ref] The effect of previous tuberculin skin test on the follow-up examination of..., Choi [/bib_ref] [bib_ref] Discrepancy between the tuberculin skin test and the whole-blood interferon gamma assay..., Kang [/bib_ref]. Third, the use of FOB in the diagnosis of PTB is not clinically available worldwide. To improve diagnostic accuracy and ensure safety, a well-trained pulmonologist is essential. High cost and the complication risk limit the use of FOB in other situations. However, our study demonstrates that FOB and FOB with HRCT can play an important role in the rapid diagnosis of active PTB in sputum smear-negative patients suspected of TB.
# Conclusions
In conclusion, our results suggest that FOB has good sensitivity, specificity, PPV, and NPV and was useful in the rapid diagnosis of sputum smear-negative PTB and the exclusion of non-TB in sputum smear-negative TB-suspected patients. HRCT alone was limited for the diagnosis of active PTB, but the combination of FOB and HRCT may improve the sensitivity of FOB in the rapid diagnosis of sputum smear-negative PTB. Based on our results, we suggest that physicians actively consider performing FOB and HRCT in sputum smear-negative patients suspected of TB.
## Additional file
Additional file 1: Tables S1-S3. Bronchoscopic findings of all subjects. Tables S2. Diagnostic and clinical characteristics of active PTB confirmed 54 patients according to bronchoscopic diagnosis. Tables S3. Bronchoscopic findings in 54 active PTB patients according to sputum mycobacteria culture results. Definition of abbreviations: TB = tuberculosis, PPV = positive predictive value, NPV = negative predictive value, +LR = positive likelihood ratio, -LR = negative likelihood ratio, 95% CI = 95% confidence interval, FOB = fiberoptic bronchoscopy, CT = computed tomography.
[fig] Figure 1: Study flowchart. Definition of abbreviations: FOB = fiberoptic bronchoscopy, CT = computed tomography, N = number, TB = tuberculosis. [/fig]
[table] Table 1: Demographic and clinical characteristics of all sputum smear-negative TB suspects [/table]
[table] Table 2: First presenting symptoms of patients with sputum smear-negative TB Definition of abbreviations: TB = tuberculosis, n = number, OR = odds ratio, 95% CI = 95% confidence interval. [/table]
[table] Table 3: Multivariate regression analysis of CT findings in sputum smear-negative TB suspects [/table]
[table] Table 4: Bronchoscopic findings in 54 patients confirmed to have active PTB [/table]
[table] Table 5: Diagnostic accuracy in all sputum smear-negative TB suspects [/table]
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What predicts regression from pre-diabetes to normal glucose regulation following a primary care nurse-delivered dietary intervention? A study protocol for a prospective cohort study
# Introduction
The prevalence of diabetes and concomitant costs continue to increase. [bib_ref] National, regional, and global trends in fasting plasma glucose and diabetes prevalence..., Danaei [/bib_ref] [bib_ref] Global, regional, and national incidence, prevalence, and years lived with disability for..., Vos [/bib_ref] Diabetes now affects an estimated 8.8% of adults globally. [bib_ref] IDF diabetes atlas: global estimates for the prevalence of diabetes for 2015..., Ogurtsova [/bib_ref] Pre-diabetes, a condition in which blood glucose levels are higher than normal, but not high enough to be classified as diabetes, is more common, affecting as many as 30%-50% of some adult populations. [bib_ref] Cardiovascular and renal burdens of prediabetes in the USA: analysis of data..., Ali [/bib_ref] [bib_ref] Prevalence of diagnosed and undiagnosed diabetes and prediabetes in New Zealand: findings..., Coppell [/bib_ref] [bib_ref] Number and characteristics of US adults meeting prediabetes criteria for diabetes prevention..., Lee [/bib_ref] [bib_ref] Prevalence of diabetes and prediabetes in adults from a third-tier city in..., Song [/bib_ref] Without treatment, about 5%-10% of those with pre-diabetes will progress to type 2 diabetes mellitus (T2DM) annually, and most will eventually develop T2DM. Lifestyle modification and weight loss can prevent or delay progression, [bib_ref] Reduction in the incidence of type 2 diabetes with lifestyle intervention or..., Knowler [/bib_ref] [bib_ref] Prevention of type 2 diabetes by lifestyle intervention: a Japanese trial in..., Kosaka [/bib_ref] [bib_ref] A randomized lifestyle intervention with 5-year follow-up in subjects with impaired glucose..., Lindahl [/bib_ref] [bib_ref] Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects..., Tuomilehto [/bib_ref] [bib_ref] Effects of diet and exercise in preventing NIDDM in people with impaired..., Pan [/bib_ref] [bib_ref] The Indian diabetes prevention programme shows that lifestyle modification and metformin prevent..., Ramachandran [/bib_ref] [bib_ref] Factors affecting the decline in incidence of diabetes in the diabetes prevention..., Hamman [/bib_ref] with the overall risk being approximately halved. [bib_ref] Pharmacological and lifestyle interventions to prevent or delay type 2 diabetes in..., Gillies [/bib_ref] Medication strengths and limitations of this study ► This mixed-methods study follows a pragmatic nonrandomised pilot study which showed that a primary care nurse-delivered 6-month dietary intervention for people with pre-diabetes and body mass index >25 kg/m 2 was feasible in a primary care setting, and weight loss was achieved in the intervention group. ► The study is set in a geographical region where approximately 21% of the population is Māori, the indigenous peoples of New Zealand, who have high rates of pre-diabetes and type 2 diabetes mellitus. ► Following the 6-month dietary intervention, participants will be reviewed every 3 months by primary care nurses for 18 months. ► A possible limitation is that the study is taking place in a real-world setting, and health sector changes influencing the primary care environment may influence the study outcomes. ► This study is limited as it will not include a control group.
## Open access
has also been shown to prevent T2DM in people with prediabetes enrolled in clinical trials. Implementing diabetes prevention lifestyle interventions in primary care: the challenges Implementing diabetes prevention lifestyle clinical trial evidence into real-world settings is challenging, particularly in primary care settings, [bib_ref] Obesity in the US: what is the best role for primary care?, Ard [/bib_ref] [bib_ref] Steps toward the meaningful translation of prevention strategies for type 2 diabetes, Cefalu [/bib_ref] [bib_ref] Nutrition counseling in clinical practice: how clinicians can do better, Kahan [/bib_ref] where those with pre-diabetes are typically a more heterogeneous group compared with those participating in clinical trials. [bib_ref] Considerations for diabetes translational research in real-world settings, Garfield [/bib_ref] Further, the cost of delivering lifestyle interventions in primary care can be a barrier. [bib_ref] Clinical outcomes and costeffectiveness of strategies for managing people at high risk..., Eddy [/bib_ref] The Diabetes Prevention Program (DPP) lifestyle intervention costs US$1399 per participant in the first year. 27 A significant proportion of this cost was specialist lifestyle intervention staff, who are often employed in diabetes prevention lifestyle programmes, irrespective of the setting. Few studies have specifically employed primary care nurses to deliver lifestyle advice, [bib_ref] A systematic review of the effectiveness of promoting lifestyle change in general..., Ashenden [/bib_ref] [bib_ref] The impact of a team-based intervention on the lifestyle risk factor management..., Chan [/bib_ref] [bib_ref] Randomised trial of lipid lowering dietary advice in general practice: the effects..., Neil [/bib_ref] [bib_ref] A two-year randomized trial of obesity treatment in primary care practice, Wadden [/bib_ref] although dietary advice given by an appropriately trained nurse can be as effective as that given by a dietitian in the primary care setting, [bib_ref] Randomised trial of lipid lowering dietary advice in general practice: the effects..., Neil [/bib_ref] and is potentially more sustainable and cost-effective. [bib_ref] Barriers to providing nutrition counseling by physicians: a survey of primary care..., Kushner [/bib_ref] Furthermore, as pre-diabetes increases the risk of not only T2DM, but also cardiovascular disease, and the microvascular complications associated with diabetes, the goal of pre-diabetes screening and treatment should arguably be regression to normoglycaemia. [bib_ref] Prediabetes: a high-risk state for diabetes development, Tabák [/bib_ref] Even if transient, regression to normoglycaemia is associated with a significantly reduced risk of developing T2DM, independent of whether this occurs spontaneously or in response to lifestyle advice or metformin therapy. [bib_ref] Effect of regression from prediabetes to normal glucose regulation on long-term reduction..., Perreault [/bib_ref] Greater insulin secretion, lower baseline glucose concentrations, younger age, weight loss and intensive lifestyle modification predict restoration of normoglycaemia, [bib_ref] Diabetes prevention program Research Group. regression from pre-diabetes to normal glucose regulation..., Perreault [/bib_ref] [bib_ref] Diabetes prevention program Research Group. early response to preventive strategies in the..., Maruthur [/bib_ref] [bib_ref] Early improvement predicts reduced risk of incident diabetes and improved cardiovascular risk..., Nanditha [/bib_ref] but other factors such as sociocultural, economic and health service delivery, which have not been previously examined, may also be important.
## Pre-diabetes lifestyle interventions have been implemented in primary care
The primary care-based Pre-diabetes Intervention Package (PIP) is a feasible primary care nurse-delivered 6-month pre-diabetes lifestyle intervention with a focus on diet, sociocultural context and goal setting, for overweight and obese patients with pre-diabetes. [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] In this pragmatic mixed-methods non-randomised pilot study conducted in the Hawke's Bay region, New Zealand (NZ), intervention implementation fidelity was high and after adjustment, the intervention group lost a mean 1.3 kg more than the control group (p<0.0.001). The mean glycated haemoglobin (HbA1c) decreased in the intervention group and increased in the control group, but the difference was not significantly different. Extending the study and offering the intervention to all study practices, alongside exploring clinical, as well as sociocultural, economic and genetic variables, provides an opportunity to determine if there are clinically relevant differences among those with pre-diabetes who regress to normoglycaemia, who have persistent pre-diabetes and who progress to T2DM. It is anticipated that these prospectively collected data will contribute to limited international data on sociocultural factors that may be associated with regression from prediabetes to normoglycaemia, [bib_ref] Diabetes prevention program Research Group. regression from pre-diabetes to normal glucose regulation..., Perreault [/bib_ref] and to clinical decisionmaking risk assessments in the primary care setting. The primary aim of this study is [bib_ref] National, regional, and global trends in fasting plasma glucose and diabetes prevalence..., Danaei [/bib_ref] to determine if there are clinically relevant differences among those with pre-diabetes who regress to normoglycaemia, those who have persistent pre-diabetes and those who progress to T2DM at 6 months following participation in a structured primary care nurse-delivered pre-diabetes dietary intervention in the primary care setting. The secondary aims are (2) to quantify the reduction in incident T2DM at 2 years in participants who regressed to normoglycaemia at 6 months compared with the other participants; (3) to qualitatively explore and examine barriers, challenges and facilitators of clinically meaningful lifestyle changes between those who regress to normoglycaemia and those who do not; (4) to explore whether a T2DM genetic risk score of common genetic variants might influence regression to normoglycaemia at 2 years in a sub-sample, (5) to describe changes in HR-QoL at 2 years and (6) to estimate the potential cost savings to the health sector associated with a reduction in T2DM.
# Methods and analysis
This extension of the PIP pilot study is a mixed-method prospective cohort study with pre-intervention and postintervention measures and a qualitative study. nZ context In NZ, among adults aged 15 years and over, the prevalence of diabetes is 7.0% and the prevalence of pre-diabetes is 25.5%. [bib_ref] Prevalence of diagnosed and undiagnosed diabetes and prediabetes in New Zealand: findings..., Coppell [/bib_ref] Māori, the indigenous people of NZ, and Pacific people have higher rates than the NZ European and Other ethnic group, with diabetes rates of 9.8%, 15.4% and 6.1%, respectively, and pre-diabetes rates of 30.4%, 29.8% and 24.6%, respectively. 7 Screening for diabetes and pre-diabetes is recommended as part of cardiovascular risk assessment.For those with no symptoms and no known risk factors, screening is recommended from 45 years for men and 55 years for women, with Māori, Pacific, Indo-Asian peoples, and those with known cardiovascular risk factors or at high risk of developing diabetes to be screened 10 years earlier, or even younger if there is particular clinical concern regarding unfavourable risk factors.
## Study setting
The study will be conducted in general practices in the urban zones of two neighbouring provincial cities (Napier and Hastings) in the Hawke's Bay region, NZ. Napier had a population of 57 240 (18.2% Māori) and Hastings District had a population of 73 245 (23.0% Māori) in 2013.Māori comprise 15% of the NZ population.The general practices will be members of the Health
## Open access
Hawke's Bay Primary Health Organisation (PHO). PHOs are not-for-profit organisations that provide primary health services either directly or through their provider members. Health Hawke's Bay is the only PHO in the study region.
## Patient and public involvement
The PIP was designed in collaboration with the Health Hawke's Bay PHO. [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] The intervention was first pre-tested with members of the public, then primary care nurses reviewed and contributed to the implementation and research protocols at a meeting. The process evaluation by an independent researcher 48 enabled views about the intervention and its implementation from patient participants, participating nurses and Health Hawke's Bay PHO staff involved in the design and implementation to be incorporated into the planning of this study. Results of the pilot study and process evaluation were presented and discussed at a forum to which all participants (patients and professionals) were invited.
## Participant eligibility
General practices who use the Medtech patient management system and employ primary care nurses will be invited to participate in the study. The reason for including general practices using Medtech is that this is the dominant patient management system in Hawke's Bay, and study data collection systems had been modified for Medtech only in the pilot study. The demographic description of practice populations will be reviewed to ensure that patients with different sociocultural backgrounds are offered the opportunity to participate.
Adults aged 18-69 years with pre-diabetes (defined as HbA1c 41-49 mmol/mol according to the NZ diagnostic criteria)and a body mass index (BMI) ≥25 kg/m 2 will be included. Pre-diabetes will have been diagnosed following screening (at the time of a recommended cardiovascular risk assessment or because they are deemed to be at risk).We set a lower age limit of 18 years to reflect the high prevalence of pre-diabetes in young adults in NZ, particularly in high-risk ethnic groups including indigenous Māori and Pacific. [bib_ref] Prevalence of diagnosed and undiagnosed diabetes and prediabetes in New Zealand: findings..., Coppell [/bib_ref] Those with a history of diabetes, prescribed metformin, unable to communicate in English, with a terminal illness or planning to move from the area during the first 6 months of the study and women pregnant at the time of study enrolment will be excluded.
## Identification of potential participants and recruitment
General practice patient management systems will be used to identify potential eligible participants. Queries will be conducted using the HbA1c, BMI, age and metformin status criteria to generate lists of potential participants. The queries will not be time-constricted. Primary care nurses will review these lists to check eligibility status including co-morbidities, current medications, confirmation of English-speaking ability and pregnancy status. The lists will be generated in no particular order. Potential participants will be recruited sequentially according to the random lists generated, in order to minimise selection bias. The research nurse will check these lists regularly to assess who has been invited, who has declined and who is yet to be invited. A letter inviting participation and information about the study will be sent to potential participants in blocks to manage nurse's workload. Potential participants will be contacted by their primary care nurse 5-7 days later to determine participation status and to make a baseline study appointment for those choosing to participate. For those who decline to participate, the nurse will record the reason(s). Those diagnosed with pre-diabetes after recruitment begins and those who meet the eligibility criteria will be invited to participate at the time of their diagnosis.
## Intervention
The intervention will be delivered by trained primary care nurses in the general practice setting. The key underpinning principle of the intervention is to provide participants (and their family) with an understanding of the principles of healthy eating and to deliver consistent evidence-based dietary messages, so they are empowered to make good dietary choices; that is, the dietary advice will not be prescriptive. This pragmatic intervention package has six components 48 :
Health Professional Training and Support Primary care nurses will undertake an intensive 3-4 hour theoretical and practical training course. The course content will include the rationale for the study, key nutritional concepts, dietary assessment in the primary care setting (using Starting the Conversation (STC) [bib_ref] Starting the conversation performance of a brief dietary assessment and intervention tool..., Paxton [/bib_ref] modified for the NZ context), [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] healthy conversations and goal setting. The nurses will be instructed how to use the PIP study-structured dietary tool, [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] and about the necessary research processes (informed consent and data collection). They will also be instructed on standard practices for measuring anthropometry and blood pressure. The course will be delivered by study investigators and a local dietitian. A training manual will provide reference material for both primary care nurses and participants, as well as research protocols. A half-day update course will be run at 6 months.
The local dietitian will be available by email and phone to answer any queries or discuss specific clinical cases. The dietitian will also arrange monthly clinical case review meetings for 6 months at each practice. Both the primary care nurses and dietitian found that dietitian's support and guidance was only required for up to 6 months in the pilot study. [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] A research nurse will also visit participating practices to provide support and advice, as needed, to maximise intervention fidelity.
## Individualised dietary assessment, goal setting and dietary advice sessions
The intervention goal is a 10% wt loss over 6 months. After providing informed written consent, participants Open access will be offered an initial 30 min individualised dietary session with their primary care nurse. They will be encouraged to bring family to the session. A brief structured dietary assessment will be undertaken. STC:Diet, a validated eight-item simplified food frequency instrument designed for use in primary care and health-promotion settings, will be used. 55 STC:Diet was minimally modified, with permission, for the NZ context. Specifically, the word 'sodas' was changed to 'soft drinks', and a traffic light system was added to indicate healthy, not-so healthy and unhealthy dietary habits. The nurse will review the STC:Diet responses; ask additional dietary prompt questions; seek additional contextual information, such as household membership and budget, who purchases household foods and specific dietary requirements/ choices such as vegetarianism and take anthropometric measures (height, weight and waist circumference) using calibrated equipment. The additional questions are called the Detailed Dietary Assessment (DDA). A 10% wt loss goal over 6 months will be calculated. Responses to the STC:Diet and DDA will inform three dietary goals negotiated with the participant, and individualised dietary advice. Participants will be given the standard 'Be Active Every Day' pamphlet, which advises 30 min of physical activity of moderate intensity on most, if not all, days of the week.Follow-up intervention appointments will be arranged at 2-3 weeks, 6 weeks, 3 months, 4 months and 6 months, then 3-monthly appointments for a 'weigh-in' and provision of ongoing brief targeted dietary advice and support for 18 months. Intervention appointments at 6 weeks and 4 months were added, following feedback from both participants and nurses in the feasibility study. [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] All study appointments will be at no cost to the participant.
## Key messages and consistent opportunistic reminders
Each participant's three dietary goals will be recorded in the practice patient management system. These goals and accompanying clinical notes will facilitate opportunistic targeted advice and guidance by GPs, thus reinforcing dietary advice and support provided by the nurses. The goals will be reviewed and updated accordingly at follow-up nurse appointments.
Nutritionally supportive primary care environment Prior to the beginning of this study, each intervention practice will be visited to discuss ways to enhance dietary messages provided by nurses. Specifically, the dietary information provided in pamphlets, magazines and posters in the waiting rooms will be reviewed and updated, if necessary, so dietary messages are appropriate and consistent. Provision of magazines that support reputable dietary messages and active living, hobbies and sports and posters promoting fruit and vegetables, such as those offered by Vegetables. co. nz ( www. vegetables. co. nz), will be encouraged.
Community-based group education for participants and their family At the 2-3 week intervention appointment, nurses will refer participants to the Kia Ora programme, a community group nutrition education course consisting of six weekly sessions of 2.5 hours. The Kia Ora programme is an approved Stanford chronic disease self-management programme. [bib_ref] Chronic disease selfmanagement program: 2-year health status and health care utilization outcomes, Lorig [/bib_ref] [bib_ref] Evidence suggesting that a chronic disease self-management program can improve health status..., Lorig [/bib_ref] The education sessions will be delivered by lay trainers, and topics will include healthy eating, label reading, problem solving and making action plans.
## Written patient resources
Readily available patient resources will be utilised. The key resource will be the Diabetes New Zealand booklet, Diabetes and healthy food choices, 59 a clearly presented and easily understood source of dietary information. The booklet was used successfully in the LOADD study, 60 and the PIP pilot study. [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] data collection Data will be collected as part of the intervention appointments with primary care nurses, as was done in the pilot study. [bib_ref] The effectiveness of a primary care nursing-led dietary intervention for prediabetes: a..., Coppell [/bib_ref] Study data collection forms have been incorporated into the Medtech patient management system. Additional data will be collected by a research nurse at separate study-specific visits. Recruitment began August 2017, and it is expected that data collection will be completed December 2021. The type and frequency of measurements are shown in table 1. These include:
Routine clinical data Clinical data will be collected by primary care nurses. These data will include demographics, medical history, family history, social history, brief dietary assessment, height, weight, waist circumference, blood pressure and bloods (HbA1c, total cholesterol, high-density lipoprotein cholesterol, triglycerides, liver enzymes (alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transferase) and urate. Known data such as date of birth and ethnicity will pre-populate the study data collection sheets and additional pre-diabetes clinical management data (eg, brief dietary assessment details) will be added by the primary care nurses during the intervention consultations.
Additional study-specific quantitative data A research nurse will administer a questionnaire and arrange for a fasting blood sample to be taken. The questions will explore areas known to influence pre-diabetes lifestyle management. Data will be collected on selfmonitoring activities (self-weighing at home, food diaries and self-initiated attendance at support groups), social support systems, sleep patterns, 63 stages and processes in weight management, physical activity (the International Physical Activity Questionnaire (IPAQ) long) [bib_ref] International physical activity questionnaire: 12-country reliability and validity, Craig [/bib_ref] and HR-QoL using the EQ-5D. [bib_ref] EuroQol-a new facility for the measurement of health-related quality of life, Group [/bib_ref] [bib_ref] Which health-related quality of life score? A comparison of alternative utility measures..., Glasziou [/bib_ref] The additional non-routine blood tests will include fasting insulin and fasting glucose (to enable calculation of homeostatic model assessment (HOMA) [bib_ref] Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose..., Matthews [/bib_ref] Overview of the quantitative data to be collected, and timing and frequency of data collection Includes Social Support Systems, Sleep Quality, [bib_ref] The Pittsburgh sleep quality index: a new instrument for psychiatric practice and..., Buysse [/bib_ref] Stages and Processes in Weight Management, IPAQ (long), [bib_ref] International physical activity questionnaire: 12-country reliability and validity, Craig [/bib_ref] EQ-5D-5L. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; HbA1c, glycated haemoglobin; IPAQ, International Physical Activity Questionnaire.
of insulin resistance), and whole genome sequencing (WGS) in a sub-sample.
## Genetic testing
The genomes from participants who, in addition to the main study, provide consent to genetic testing, and are from the opposite ends of the response spectrum will be sequenced, with the intention of sequencing genomes from all consenting participants as the cost of sequencing reduces. Using genome-wide single nucleotide polymorphism (SNP) genotyping platforms would not be adequate as these platforms use common genetic variants identified in other populations. WGS is the only practical approach as it will allow identification of uncommon (including Māori-specific) variants predicted to have a strong functional effect in the suite of genes to be analysed for a burden of functional variation. WGS also allows evaluation of regulatory variants which would not be possible using an exome sequencing approach. Sequencing will be done at 30-fold coverage.
## Outcome measures
The primary outcome measures will be HbA1c (mmol/ mol) and weight (kg). Other outcome measures will include waist circumference, BMI, social support score, behavioural change score and EQ-5D scores.
## Sample size calculations
The sample size calculation used information from the Voglibose study. It was assumed that 25% of those with pre-diabetes at baseline will regress to normoglycaemia at 6 months. Assuming 5% loss to follow-up at 6 months, a sample of 400 people with pre-diabetes will provide 80% power to detect factors associated with regression at 6 months with relative risks for regression of 2.2 or higher, for any two groups each comprising between 20% and 80% of the sample using a two-sided test at p<0.05 level of significance.
Planned statistical analysis STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) [bib_ref] Strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for..., Evon [/bib_ref] and TRIPOD (Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis) [bib_ref] Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis..., Collins [/bib_ref] reporting standards will be used. Descriptive statistics will be provided for all measures of interest, as appropriate. For inferential analyses, all results will be reported showing effect sizes, 95% CIs and p values. Two-sided p<0.05 will be considered statistically significant. Analyses will be conducted using R V.3.5.1 and Stata V.15.1 (or later versions). Poisson regression with robust standard errors and clustered by practice (using Froot's extension to Huber-White estimators 74 ) will be used to identify predictors of regression to normoglycaemia and progression to T2DM at 6 months. Similar models will be used to examine risk factors for developing T2DM at 2 years. Exploratory modelling will use Markov models to identify variables associated with transitions between the three glycaemic states of interest, defined according to the NZ definitions:
Open access normoglycaemia (HbA1c<41 mmol/mol), pre-diabetes (41 mmol/mol≥HbA1 c ≤ 49 mmol/mol) and diabetes (HbA1c>49 mmol/mol).Providing the number of transitions between each pair of states is sufficient, multistate models will initially be used to describe transition probabilities between these states over all measurement times (all three transitions are possible) with an absorbing state of death also reachable from any of these three states. Factors associated with transitions between these three glycaemic states (but not between these states and death) are the focus of this study and these prognostic variables will be used to construct models of the relative risks for transitions between each pair of stages. Initially, these factors will be evaluated independently and Hosmer-Lemeshow's criteria for logistic regression of unadjusted p<0.25 will be used to identify candidate predictors for further (adjusted) modelling.The number of potential predictors included in adjusted models will be limited by the number of transitions providing data for parameter estimates. Peduzzi et al's heuristic for logistic regression of 10 events (and 10 non-events) per parameter estimated will be used to determine the maximum model complexity when identifying predictors for further examination. [bib_ref] A simulation study of the number of events per variable in logistic..., Peduzzi [/bib_ref] If more factors are identified than can be modelled using the Hosmer-Lemeshow screening heuristic, selection will be based on favouring modifiable factors which could be used to inform interventions over non-modifiable factors. Similarly, where multiple predictors are identified that are highly correlated, only one will be retained for further modelling. If data allow estimation of the necessary number of parameters, time-dependent transition probabilities will be estimated in similar ways.
A genetic risk score will be compiled from common variants identified as T2DM-associated from a NZ genomewide association study of T2DM currently in progress. The genetic risk score will be tested for association with the outcomes of the intervention in the sub-sample of participants with genetic information.
## Qualitative component
Inclusion of qualitative approaches in the design and reporting of evaluations of complex health interventions is relatively uncommon and rarely reported, although qualitative components offer opportunities to enhance the interpretation of the quantitative outcome data. [bib_ref] Use of qualitative methods alongside randomised controlled trials of complex healthcare interventions:..., Lewin [/bib_ref] [bib_ref] Process evaluation in randomised controlled trials of complex interventions, Oakley [/bib_ref] [bib_ref] What can qualitative research do for randomised controlled trials? A systematic mapping..., O'cathain [/bib_ref] The qualitative component of this study will provide important insight into the psychosociocultural factors that influence lifestyle change in relation to regression to normoglycaemia. It will specifically explore the experience of being diagnosed with pre-diabetes and how both those who regress to normoglycaemia at 6 months and those who do not describe the process in relation to barriers to and facilitators of lifestyle changes.
A purposive sample of 60 participants who have completed the 6-month intervention will be sought (30 people who regressed to normoglycaemia and 30 people who did not). The sample will be stratified to include equal numbers of participants by gender and half of the sample will comprise indigenous Māori to ensure that a meaningful level of analysis can be undertaken. Participants will be selected from those who agreed to participate in further research when first recruited into the main study. Those purposively selected will be contacted shortly after completion of the 6-month measures to arrange a face-toface interview if willing to participate in this part of the study. Written consent will be obtained immediately prior to this interview, which will last approximately 60 min. The interviewer (SA), who has, with co-author (DT-L), worked and published extensively in projects where the cross-cultural context is central, will ensure cultural safety throughout the interview process with Māori and Pacific participants. With permission, all interviews will be audio recorded and transcribed.
Analysis will be completed by a sub-team (SA, LW, DT-L and KC) using thematic analysis. [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref] This will involve multiple reading of transcriptions followed by the development and description of a priori and emergent themes and concepts. Further analysis will explore interconnections between themes. The thematic findings will also be triangulated with the quantitative data to assist interpretation of results and provide descriptive context.Economic analysis Using the EQ-5D scores, the number and percentage of participants reporting each level of problem on each dimension of the EQ-5D will be described by age, gender and ethnicity, and compared across time and groups (ie, those who regress to normoglycaemia, those who have persistent pre-diabetes and those who progress to T2DM). Participants' self-reported health status as measured on the EQ Visual Analogue Scale will also be reported. An estimate of potential cost savings (acknowledging that any change in glycaemic status cannot be attributed to the programme with certainty) will be calculated from a funder's perspective using average cost estimates and the expected rate of progression to T2DM.
## Ethics and dissemination
Local consultation with Māori was facilitated via Health Hawke's Bay's Operational Māori Advisor and was undertaken at the outset of study planning, particularly in relation to the genetic studies. The study was endorsed by the Priority Population Committee of the regional primary health organisation, Health Hawke's Bay. This committee is tasked with ensuring programmes contribute to equitable access and health gains for Māori, Pacific and other populations living in high deprivation areas (NZ deprivation score 9-10).Study author, DT-L (Māori public health physician), performed an advisory role across all appropriate planning components of the study and will be in-field cultural advisor across the project, in particular for the research nurse and the qualitative researcher. The study was also endorsed by the University of Otago Ngāi Tahu Research Consultation Committee.
## Open access
Further consultation was undertaken specifically with respect to taking blood for genetic analysis. Families will be interested in learning if there is a genetic link with a higher risk of T2DM. The most obvious outcome would include a more responsive health system with early detection, destigmatisation, early 'no blame' intervention and prevention programmes for family who are at risk of developing T2DM. As part of the study, participants will be given a choice on consenting as to the disposal of body fluids/parts as per the University of Otago's and Hawke's Bay District Health Board's Policies and Procedures.
The main study results will be first presented to participating GPs and participating patients. The results of the study will be written and published in peer-reviewed journals and presented at relevant conferences. A written summary of the results will also be distributed to participants, participating GPs, and published on Health Hawke's Bay website for their members.
Coppell K, et al. BMJ Open 2019;9:e033358. doi:10.1136/bmjopen-2019-033358 |
Body Mass Index, Waist-circumference and Cardiovascular Disease Risk Factors in Iranian Adults: Isfahan Healthy Heart Program
# Introduction
Chronic non-communicable diseases (CNCDs), such as cardiovascular diseases (CVDs), cancers, (WC) which is a better predictor than BMI for obesity outcomes [bib_ref] Are waist circumference and body mass index independently associated with cardiovascular disease..., Wildman [/bib_ref] [bib_ref] Obesity, regional body fat distribution, and the metabolic syndrome in older men..., Goodpaster [/bib_ref]. According to the National Institute of Health guidelines, it is supposed that BMI and WC have independent effects on obesityrelated diseases. It is clear that combination of WC and BMI predicts health risks better than does BMI alone [bib_ref] AusDiab Steering Committee. Waist circumference, waist-hip ratio and body mass index and..., Dalton [/bib_ref] ; however, the reverse is uncertain.
According to Adult Treatment Panel III (ATP III) criteria, about 33.8% of Iranian adults (more than 10.6 million) were centrally obese. It was about 4 times in females compared to males [bib_ref] Third national surveillance of risk factors of non-communicable diseases (SuRFNCD-2007) in Iran:..., Esteghamati [/bib_ref].
Considering the importance of CNCDs and the main effect of obesity on these diseases, this study was performed to assess the association between BMI, WC, and major cardiometabolic risk factors and to corroborate whether either or both BMI and WC are independently associated with these risk factors in a sample of Iranian adults who participated in the baseline survey of Isfahan Healthy Heart Program (IHHP). IHHP was a long-term community-based interventional programme for health promotion through reduction of CVD risk factors, and hence, reduction of morbidity and mortalitydue to CVDs. The study was conducted in 3 central counties of Iran [bib_ref] Isfahan Healthy Heart Program: a comprehensive integrated community-based programme for cardiovascular disease..., Sarraf-Zadegan [/bib_ref].
# Materials and methods
## Sampling
This is an analytical study done with data of the baseline survey of IHHP [bib_ref] Isfahan Healthy Heart Program: a comprehensive integrated community-based programme for cardiovascular disease..., Sarraf-Zadegan [/bib_ref] [bib_ref] Isfahan Healthy Heart Program: evaluation of comprehensive, community-based interventions for non-communicable disease..., Sarrafzadegan [/bib_ref]. IHHP was undertaken in 3 counties of Isfahan, Najafabad, and Arak in the central part of Iran. According to the 2000 National Census, the population was 1,895,856, 275,084, and 668,531 in Isfahan, Najafabad, and Arak respectively [bib_ref] Isfahan Healthy Heart Program: a comprehensive integrated community-based programme for cardiovascular disease..., Sarraf-Zadegan [/bib_ref] [bib_ref] Isfahan Healthy Heart Program: evaluation of comprehensive, community-based interventions for non-communicable disease..., Sarrafzadegan [/bib_ref]. Multistage random-sampling technique was employed based on sex, age, and settlement distributions in each community to select 12,600 adults aged ≥19 years. Approximately 5-10% of households within these clusters were randomly selected. One individual aged ≥19 years per household was randomly selected. The selection criteria were: Iranian and mentally-competent individuals and also non-pregnant women [bib_ref] Isfahan Healthy Heart Program: a comprehensive integrated community-based programme for cardiovascular disease..., Sarraf-Zadegan [/bib_ref] [bib_ref] Isfahan Healthy Heart Program: evaluation of comprehensive, community-based interventions for non-communicable disease..., Sarrafzadegan [/bib_ref].
Single eligible subject within the household was selected randomly from one of the six age-groups: 19-<25, 25-<35, 35-<45, 45-<55, 55-64, or 65 years and more [bib_ref] Isfahan Healthy Heart Program: a comprehensive integrated community-based programme for cardiovascular disease..., Sarraf-Zadegan [/bib_ref].
## Data collection
Eligible individuals had a 30-minute home interview by trained health professionals. The questionnaire included questions on socioeconomic and demographic characteristics, health knowledge, cardiovascular risk-related attitudes and behaviours regarding dietary practice, smoking, and physical activity [bib_ref] Isfahan Healthy Heart Program: a comprehensive integrated community-based programme for cardiovascular disease..., Sarraf-Zadegan [/bib_ref]. Medical and drug history of participants were obtained for DLP, DM, and HTN by well-trained physicians.
Trained nurses obtained blood samples from the participants by venipuncture from the left antecubital vein after 12-14 hours of fasting. They kept all blood samples frozen at −20 °C to be assayed within 72 hours at the central laboratory of Isfahan Cardiovascular Research Center (ICRC) which meets the criteria of the National Reference Laboratory (a WHO-collaborating centre). Serum total cholesterol (TC) and triglyceride (TG) were determined by enzymatic method, using special kits (Immunodiagnostic, Germany) in an Elan 2000 auto-analyzer (Eppendorf, Germany). Also, HDL-C was measured by enzymatic method after precipitating the other lipoproteins with dextran sulphate magnesium chloride [bib_ref] Dextran sulfate-Mg 2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol, Warnick [/bib_ref]. LDL-C was calculated by using the Friedewald formula [bib_ref] Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use..., Friedewald [/bib_ref]. Direct measurement of LDL-C was performed with a turbidimetric method for those with TG ≥400 mg/dL. Blood sugar, including fasting blood glucose (FBG) and 2-hour postload plasma glucose (2hpp), were determined by glucose oxidase enzymatic method. To measure 2hpp, the blood sugar of non-diabetic participants was measured 2 hours after giving a syrup containing 75 g of glucose powder.
Physical measurements were done by trained medical staff with standardized methods. The weight was measured by a digital scale, with minimum necessary clothing, and recorded to the nearest 0.5 kg. Height was measured in a standing position, without shoes, to the nearest 0.5 cm, using a non-elastic stadiometer while the shoulders were in a normal state. BMI was calculated and recorded as weight in kg divided by height in metre squared. While the subjects were standing, WC and hip-circumference were measured by a tape at a level midway between the lower rib margin and iliac crest and at the point yielding the maximum circumference over the buttocks respectively. The blood pressure (BP) was measured by trained physicians based on standard criteria [bib_ref] National High Blood Pressure Education Program Coordinating Committee. Seventh report of the..., Chobanian [/bib_ref]. These physicians were trained for a week to teach how to use sphygmomanometer and how to measure BP in a seated position. BP was measured two times in a seated position with a random-zero sphygmomanometer and an appropriate cutoff after a 5-minute rest. Average of the two measures was recorded for both systolic and diastolic BP (SBP and DBP).
## Ethics
Written informed consents were obtained from all participants, and the study was approved by the Ethical Committee of the ICRC (15).
## Definitions of risk factors and diseases
DLP was defined as having at least one of the following situations: TC ≥240 mg/dL and/or TG ≥200 mg/dL and/or LDL-C ≥160 mg/dL and/or HDL-C <40 mg/dL in men or HDL-C <50 mg/dL in womenand/or medications for hypolipidaemia. DM was defined as FBG ≥126 mg/dL and/or a 2hpp level of ≥200 mg/dL and/or medications for hypoglycaemia, using the WHO criteria. Individuals having SBP ≥140 mmHg and/or DBP ≥90 mmHg and/or using medications for BP were considered hypertensive patients [bib_ref] National High Blood Pressure Education Program Coordinating Committee. Seventh report of the..., Chobanian [/bib_ref].
# Statistical analysis
Statistical analyses were performed using SPSS for Windows software (version 15; SPSS, Chicago, IL, USA) and STATA software (version 10). The significance level was set at p value of <0.05. Comparison of the mean of variables with different quartiles of BMI and WC was done by analysis of covariance (ANCOVA), adjusting for the effect of age, smoking status (ever-smoker/non-smoker), total daily physical activity (METS per minute), and socioeconomic status (SES), including education and monthly income in each sex. The comparison of the mean value of BMI and WC in DM, HTN, and DLP patients who had either no other risk factors, one risk factor, or two risk factors were done by the analysis of variance (ANOVA). The chi-square test was used for comparing the prevalence of the risk factors in different quartiles of BMI and WC. The quantitative and qualitative basic characteristics were analyzed by t-test and chi-square test respectively.
Logistic regression analysis was also used for examining the independent and combined effects of BMI and WC on CVD risk factors. The odds ratios (ORs) in the 2 nd , 3 rd , and 4 th quartile of BMI and WC were compared with the 1 st quartile as reference. ORs were adjusted for the above potential confounding variables. BMI and WC were entered into the regression model as continuous variables.
Preliminarily, the models with and without interaction effect were compared by likelihood ratio test (LR), Akaike Information Criterion (AIC), and Bayesian Information Criterion (BIC) tests. The results of these analyses confirmed that the combined model with BMI and WC should be reported without their interaction effect.
To test for linear trend of ORs and determine p for trend across quartile of BMI or WC, we assigned the median BMI or WC to varying individuals as continuous variables in logistic regression for hypertensive vs non-hypertensive, diabetic vs non-diabetic hyperlipidaemic vs normolipidaemic subjects.
# Results
The study participants comprised 12,514 adults. We limited the current analysis to 12,416 individuals, including 6,081 men and 6,335 women because we did not have enough data for 98 individuals to be included in this analysis. The basic characteristics of the participants are presented in .
The adjusted mean values of BMI, WHR, FBG, 2hpp, TC, serum lipids, and BPs in different quartiles of WC based on sex are shown in . Increase in WC had significant positive relationship with increase in the mean of BMI, WHR, FBG, 2hpp, TC, TG, LDL-C, SBP, and DBP but with decrease in HDL-C (p<0.001 for all). Comparison of DM, HTN, and HLP prevalence in WC quartiles according to sex is presented in . The DM, HTN, and DLP prevalence had a significant relationship with increase in WC (p<0.001 for all). indicates that rise in BMI had a significant direct association with increase in the mean adjusted values of WC, WHR, FBG, 2hpp, TC, TG, LDL-C, SBP, and DBP and decrease in HDL-C. DM, HTN and DLP prevalence had a significant relationship with increase in BMI (p<0.001 for all) . Adjusted ORs for HTN, DLP, and DM according to WC and BMI quartiles are presented in [fig_ref] Table 6: Adjusted* odds ratio [/fig_ref].
When both BMI and WC were entered in the model, the ORs for all risk factors, in men, according to BMI were comparable to WC. Moreover, ORs for DLP based on BMI were more than on WC in both the sexes. Conversely to men, ORs for DM and HTN in WC quartiles were more compared to BMI quartiles in women. The trends in ORs for all risk factors were gradually increased significantly by enhancing both BMI and WC quartiles (p for trend <0.05 for all). The use of BMI or WC alone in the models caused to enhance all ORs (data not shown).
In total population, the mean values of WC significantly increased [90.6±14.4, 98±12.2, and 103.5±11.6 (p for trend <0.001)] in those with DM and no other risk factor, with one risk factor, and with two risk factors respectively. These were 93.2±13.7, 98±13.3, and 103.6±11.6 respectively in hypertensive subjects (p for trend <0.001). In dyslipidaemic subjects, the mean level of WC were 90.5±12.7, 98.1±13.2, and 103.6±11.6 respectively (p for trend <0.001). BMI (mean±SD) in diabetes and no other risk factor, with one risk factor, and with two risk factors were 24.5±4.2, 27.6±4.7, and 29.4±4.5 respectively (p for trend <0.001). These were 27.8±4.5, 24.9±5.1, and 26.4±4.5 in hypertension respectively (p for trend <0.001). In dyslipidaemic subjects, BMI (mean±SD) was 25.7±4.6, 27.9±5, and 29.4±4.5 respectively (p for trend <0.001). Morevover, the mean value of WC and BMI in diabetic, hypertensive and dyslipidaemic subjects with different numbers of CVD risk factors based on sex are illustrated in and 2. All values were significant (p<0.001).
# Discussion
The results indicated that higher BMI and WC were significantly associated with HTN, DLP, and DM. Adjusted odds ratio of CVD risk factors by age, smoking, SES, and total daily physical activity showed that the occurrence of DM, HTN, and DLP is significantly related with increase in obesity indicators. As the odds ratio in the combined models with BMI and WC were less than that in the models with BMI or WC alone, we concluded that assessing both BMI and WC may be a better predictor of CVD risk factors compared to BMI or WC alone. Ying et al. reported similar results in young and middle-aged Chinese women [bib_ref] Body mass index, waist circumference, and cardiometabolic risk factors in young and..., Ying [/bib_ref].
Our results are in conformity to similar studies in Korea, India, Australia, and Singapore. In these studies, the incidence of DM and HTN was found to increase with higher levels of BMI even in those whose BMI was within normal range [bib_ref] AusDiab Steering Committee. Waist circumference, waist-hip ratio and body mass index and..., Dalton [/bib_ref] [bib_ref] Elevated body fat percentage and cardiovascular risks at low body mass index..., Deurenberg-Yap [/bib_ref] [bib_ref] Cutoff values for normal anthropometric variables in Asian Indian adults, Snehalatha [/bib_ref] [bib_ref] Non-obese (body mass index <25 kg/m 2 ) Asian Indians with normal..., Vikram [/bib_ref] [bib_ref] Obesity, abdominal obesity, and clustering of cardiovascular risk factors in South Korea, Park [/bib_ref].
A study in Japan performed by Ito and his colleagues showed that the risk of DLP, including high levels of LDL-C, and TG was significantly more in those placed in the highest one-third of WC compared to those placed in the lowest onethird of this index [bib_ref] Excess accumulation of body fat is related to dyslipidaemia in normal-weight subjects, Ito [/bib_ref]. Our study illustrated that the risk of DLP is significantly higher in the highest quartile of obesity indicators, especially BMI in both the sexes. In general, there was a positive significant relationship between the quartiles and the mean of serum lipids and the prevalence of DLP. In another study done by Tanaka and his colleagues in Japan, the same finding was reported in such a way that individuals with WC in the upper quartile had significantly higher prevalence of CVD risk factors. This study noted that at least one of the CVD risk factors increased with higher levels of WC [bib_ref] Is adiposity at normal body weight relevant for cardiovascular disease risk?, Tanaka [/bib_ref].
Obesity may cause insulin resistance, leading to DM, HTN, and DLP [bib_ref] Non-obese (body mass index <25 kg/m 2 ) Asian Indians with normal..., Vikram [/bib_ref] [bib_ref] Obesity, abdominal obesity, and clustering of cardiovascular risk factors in South Korea, Park [/bib_ref] [bib_ref] Excess accumulation of body fat is related to dyslipidaemia in normal-weight subjects, Ito [/bib_ref]. These results can be confirmed by significant relationship between BMI and WC, and these CVD risk factors are presented in our study. [bib_ref] The Canadian Heart Health Surveys Research Group. A comparative evaluation of waist..., Dobbelsteyn [/bib_ref] [bib_ref] Waist circumference and abdominal sagittal diameter: best simple anthropometric indexes of abdominal..., Pouliot [/bib_ref] [bib_ref] Overweight and obesity in relation to cardiovascular disease risk factors among medical..., Bertsias [/bib_ref]. Furthermore, according to the multiple logistic regression analysis reported herein, WC in women and BMI in men were the better predictor of DM and HTN. It may occur since the cut-points of central obesity in men based on both ATP III and International Diabetes Federation (IDF) criteriawere higher than the 1 st quartile of WC, which was considered the reference value to estimate the ORs in this study. However, various studies had different results. In some of these, WC was a better and accurate measure of CVD risk [bib_ref] Isfahan Healthy Heart Program: evaluation of comprehensive, community-based interventions for non-communicable disease..., Sarrafzadegan [/bib_ref] [bib_ref] Dextran sulfate-Mg 2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol, Warnick [/bib_ref] [bib_ref] Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use..., Friedewald [/bib_ref]. For instance, Vazquez et al. reported that abdominal obesity was a stronger predictor of DM incidence than overall obesity [bib_ref] Comparison of body mass index, waist circumference, and waist/hip ratio in predicting..., Vazquez [/bib_ref]. However, in the US and European Caucasians, overall obesity was a better predictor of DM [bib_ref] Obesity, fat distribution, and weight gain as risk factors for clinical diabetes..., Chan [/bib_ref]. Also, Knowles et al. noted that WC was the best predictor of HTN in men and DM in women [bib_ref] Waist circumference, body mass index, and other measures of adiposity in predicting..., Knowles [/bib_ref]. In several studies, WC had a much stronger relationship with DM, HTN, and DLP [bib_ref] Body mass index (BMI) and waist circumference (WC) as screening tools for..., Foucan [/bib_ref] [bib_ref] Body fat distribution and risk of coronary heart disease in men and..., Canoy [/bib_ref] [bib_ref] Anthropometric measures and absolute cardiovascular risk estimates in the Australian Diabetes, Obesity..., Chen [/bib_ref] while in some others, both WC and BMI were worthy predictors of the CVD risk factors [bib_ref] Are waist circumference and body mass index independently associated with cardiovascular disease..., Wildman [/bib_ref] [bib_ref] Body mass index and waist circumference: association with cardiovascular risk factors, Rezende [/bib_ref] [bib_ref] Comparison of waist circumference, body mass index, percent body fat and other..., Paniagua [/bib_ref]. Furthermore, a meta-analysis illustrated that BMI and WC were equally good in forecasting DM [bib_ref] Comparison of body mass index, waist circumference, and waist/hip ratio in predicting..., Vazquez [/bib_ref]. Thus, measurement of both BMI and WC can improve CVD risk stratification [bib_ref] Are waist circumference and body mass index independently associated with cardiovascular disease..., Wildman [/bib_ref].
In this study, along with the other study performed by
# Limitations
The use of cross-sectional data to determine the relationship between anthropometric indices and CVD risk factors is the limitation of this research work. Therefore, we were not exactly able to analyze the causal relationship between obesity and the risk factors in this study.
# Conclusions
We conclude that BMI is the better predictor of DM, HTN, and DLP in men compared to WC. Conversely in women, WC is a superior predictor than BMI, particularly for DM and HTN subjects. So, BMI alone might be a useful indicator in Iranian men if measuring WC is difficult. Furthermore, the measurement of both WC and BMI in Iranian adults may be a better predictor of
## Acknowledgements
This study was conducted by the ICRC (a WHOcollaborating centre) in collaboration with Isfahan Provincial Health Office, both of which are affiliat-
[fig] Figure 2, Figure 1: The mean of body mass index in diabetic, hypertensive and dyslipidaemic subjects with different numbers of risk factors based on [/fig]
[table] Table 1 Table 4: General characteristics of study participants based on gender Comparing the adjusted* mean values of cardiovascular risk factors in body mass index quartiles based on gender Variable [/table]
[table] Table 6: Adjusted* odds ratio (95% CI) of cardiovascular risk factors in waist-circumference and body mass index quartiles in comparison with 1 st *Adjusted for age, smoking status, daily physical activity, and socioeconomic status and combined model with waist-circumference and body mass index; Figures in parentheses are (95% confidence intervals traditional risk factors of CVDs than BMI or WC alone. Therefore, it is suggested that both BMI and WC be regular measures for identification of the high-risk obese population in epidemiological studies. [/table]
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Structural and Functional Relationships in Glaucoma Using Standard Automated Perimetry and the Humphrey Matrix
Purpose: To evaluate and compare correlations between structural and functional loss in glaucoma as assessed by optical coherence tomography (OCT), scanning laser polarimetry (GDx VCC, as this was the model used in this study), standard automated perimetry (SAP), and the Humphrey Matrix (Matrix). Methods: Ninety glaucomatous eyes identified with SAP and 112 eyes diagnosed using Matrix were independently classified into six subgroups, either S1/M1 (MD>-6dB), S2/M2 (-12<MD<-6dB) or S3/M3 (MD<-12dB), according to the mean deviation (MD) of each test. Average and sectoral retinal nerve fiber layer (RNFL) thickness and percentage of abnormal classifications using the internal normative databases of OCT and GDx VCC were compared among the six subgroups. Results: In the SAP subgroups, RNFL thickness values obtained by OCT in the nasal and temporal quadrants and the inferior averages of GDx VCC did not differ between the S1 and S2 subgroups (p=0.137, 0.738 and 0.149, respectively). In the Matrix subgroups, no measurement parameters differed between the M1 and M2 groups except for the overall mean and average inferior RNFL thickness given by OCT and the NFI values of GDx VCC (p=0.013, 0.016 and 0.029, respectively). When abnormal classifications were compared, all measurement parameters, without exception, were significantly different in both the SAP and the Matrix subgroups. Conclusions: SAP subgroups showed a good correlation of structural and functional defects when assessed using OCT and GDx VCC. These correlations were weaker in the Matrix subgroups, especially in the early stages of glaucoma.
Glaucoma is a progressive optic neuropathy characterized by the loss of retinal ganglion cells (RGCs) and axons. These structural changes are known to presage functional deficits, causing gradual visual field (VF) defects. [bib_ref] Number of ganglion cells in glaucoma eyes compared with threshold visual field..., Kerrgan-Baumrind [/bib_ref] [bib_ref] Optic nerve damage in human glaucoma. III. Quantitative correlation of nerve fiber..., Quigley [/bib_ref] Traditionally, glaucomatous structural changes have been assessed by detailed ophthalmoscopic examination. Recently, objective and analytic approaches to structural assessment have become available with the development of imaging devices, most notably optical coherence tomography (OCT) and scanning laser polarimetry (SLP).
Standard automated perimetry (SAP) (Carl Zeiss Meditec Inc., Dublin, CA, USA) is the gold standard for testing functional changes in glaucoma. This technique measures light sensitivity thresholds at each retinal location. However, the method is not selective for the detection of particular glaucoma-related RGC damage. Therefore, VF defects may be detected by SAP only after the death of some RGCs. [bib_ref] Retinal ganglion cell atrophy correlated with automated perimetry in human eyes with..., Quigley [/bib_ref] [bib_ref] Visual field defects and retinal ganglion cell losses in patients with glaucoma, Harwerth [/bib_ref] Humphrey Matrix Frequency doubling technology perimetry (Matrix) (Carl Zeiss Meditec Inc.) was developed to detect damage to magnocellular ganglion cells, which are preferentially affected in glaucoma patients. [bib_ref] Testing for glaucoma with the spatial frequency doubling illusion, Maddess [/bib_ref] In this context, the Matrix has been found to be more sensitive than SAP because it can detect VF loss earlier. [bib_ref] Standard achromatic perimetry, short wavelength automated perimetry, and frequency doubling technology for..., Soliman [/bib_ref] [bib_ref] Prediction of future scotoma on conventional automated static perimetry using frequency doubling..., Kogure [/bib_ref] [bib_ref] Frequency doubling technology perimetry abnormalities as predictors of glaucomatous visual field loss, Medeiros [/bib_ref] [bib_ref] Prediction of visual field defects on standard automated perimetry by screening C-20-1..., Kamantigue [/bib_ref] Using a variable corneal compensator (VCC) (GDx VCC Carl Zeiss Meditec Inc.), a test of structural change, the GDx VCC can estimate the thickness of the retinal nerve fiber layer (RNFL) by measuring the summed retardation of a polarized scanning laser beam induced by form-birefringent microtubules supporting RGC axons. [bib_ref] Linear birefringence of the retinal nerve fiber layer measured in vitro with..., Huang [/bib_ref] [bib_ref] Assessment of the retinal nerve fiber layer by scanning-laser polarimetry, Dreher [/bib_ref] [bib_ref] Optic nerve imaging: recent advances, Zangwill [/bib_ref] [bib_ref] Visualization of localized retinal nerve fiber layer defects with the GDx with..., Reus [/bib_ref] Stratus OCT (Carl Zeiss Meditec Inc.) is used to obtain data on a cross-section of the retina based on the reflectivity of different retinal layers; the technique can define the thickness of the circumpapillary RNFL. [bib_ref] Optical coherence tomography, Huang [/bib_ref] [bib_ref] Micrometer-scale resolution imaging of the anterior eye in vivo with optical coherence..., Izatt [/bib_ref] [bib_ref] Quantification of nerve fiber layer thickness in normal and glaucomatous eyes using..., Schuman [/bib_ref] Because the two imaging modalities use different technologies to measure distinct aspects of retinal biology, it is possible that measurements derived from these approaches might show different associations with functional change.
In general, damage to the optic nerve and RNFL may precede VF loss in early glaucoma. [bib_ref] The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular..., Kass [/bib_ref] However, recent clinical trials, including the Ocular Hypertension Treatment Study and the European Glaucoma Prevention Study, reported that the earliest damage in glaucoma patients can be either structural or functional in nature. [bib_ref] The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular..., Kass [/bib_ref] Therefore, the relationship between functional and structural damage in glaucoma is a topic of debate.
The purpose of this study was to compare the structural changes in glaucoma assessed by OCT and GDx VCC and also the functional relationships detected by SAP and Matrix. We categorized patients into three groups according to the severity of their VF field injury, and evaluated the structural and functional relationships using two different VF tests.
# Materials and methods
## Participants
The medical records of all patients examined at the glaucoma clinic from January 2007 to March 2008 by one glaucoma specialist were reviewed, and if patients met the inclusion criteria of this study they were consecutively enrolled. All participants underwent a comprehensive ophthalmic examination, including visual acuity measurement, slit-lamp biomicroscopy, intraocular pressure measurement by Goldmann applanation tonometry, central corneal thickness ultrasound pachymetry measurement, SAP, Matrix, GDx VCC, and OCT. Patients were eligible if they had a best-corrected visual acuity greater than 20/40 and a normal anterior chamber and open-angle on slit-lamp and gonioscopic examinations, respectively. Participants with any other ophthalmic disease that could result in VF defects or who had histories of diabetes mellitus were excluded. A glaucomatous optic disc was defined as a disc showing increased cupping (vertical cup-disc ratio >0.6), asymmetrical cupping (a betweeneye difference in the vertical cup-disc ratio of >0.2), diffuse or focal neural rim thinning, hemorrhage, or nerve fiber layer defects. Glaucomatous eyes defined by SAP (SAP group) were those with a glaucomatous optic disc appearance and glaucomatous VF defects defined by the glaucoma hemifield test (GHT) as outside 99% of age-specific normal limits and also with a pattern standard deviation (PSD) outside 95% of the normal limit with the 24-2 full threshold program or the 24-2 Swedish Interactive Threshold Algorithm (SITA) standard programs of the HFA.
The same VF criteria were used to define glaucomatous eyes identified by Matrix. Eyes with a glaucomatous optic disc appearance and a GHT result outside 99% of the age-specific normal limits with a PSD outside 95% of the normal limit with Matrix were categorized by Matrix as suffering from glaucoma (Matrix group). Since there were no generalized criteria in Matrix for diagnosing glaucoma, we arbitrarily applied the same criteria as in SAP to the Matrix results.
We included only those patients who, within one month of the initial evaluation, yielded a reliable VF measurement, defined as a false-positive error <15%, a false-negative error < 15% and a fixation loss <20% in both SAP and Matrix. One eye was randomly selected if both eyes were found to be eligible. Subsequently, 90 eyes were classified as glaucomatous in the SAP group and 112 eyes were identified in the Matrix group. All procedures conformed to the Declaration of Helsinki, and the study was approved by the Ethics Committee of the Asan medical center. Written informed consent was obtained from all patients.
## Optical coherence tomography (stratus oct)
OCT measures RNFL thickness with a low-coherence light source projected onto the retina. The time delay of the light backscatter from the RNFL compared with light reflected by a reference mirror is then calculated. Circumpapillary RNFL was measured in the fast RNFL mode using three 360-degree circular, high-resolution scans with a diameter of 3.4 mm that were centered on the optic disc. RNFL thicknesses of the collective quadrants (360° measure), as well as the individual temporal (316°-45°), superior (46°-135°), nasal (136°-225°), and inferior (226°-315°) quadrants, were obtained for analysis. Diagnostic categorization by Stratus OCT involves a softwarebased comparison of thickness parameters with an internal normative database of 328 eyes; we also evaluated these parameters in our study. Parameters on the Stratus OCT printout are annotated to indicate whether they fall outside of the 99% confidence interval (CI), between the 95% and 99% CIs (borderline) or within the 95% CI (normal). In this study, only wellfocused and centered scans with signal strengths ≥ seven were included, and a parameter was considered outside the normal limits if CI < 95% and within the normal limits if CI > 95%.
## Scanning laser polarimetry (slp)
SLP imaging was performed in a standardized fashion (GDx VCC software version 5.5.0) with a circular scan (3.2 mm in diameter) centered on the optic disc. The general principles of SLP with variable corneal polarization compensation (VCC) have been described in detail elsewhere. [bib_ref] Individualized compensation of anterior segment birefringence during scanning laser polarimetry, Zhou [/bib_ref] [bib_ref] Glaucoma detection using scanning laser polarimetry with variable corneal polarization compensation, Weinreb [/bib_ref] We only analyzed eyes with a scan quality score of eight or better. Images with atypical retardation patterns (ARPs) were excluded from the study. The SLP parameters examined were TSNIT (temporal, superior, nasal, inferior, and temporal) imaging, superior imaging, inferior average imaging, and the nerve fiber indicator (NFI). The GDx VCC printout provides probabilities of abnormality based on a comparison with an internal database containing information on 540 normal eyes. In this study, a parameter was considered outside the normal limits if p<0.05 and within the normal limits if p>0.05. For the NFI parameter, the manufacturer-suggested cutoff of < 30 was considered as within the normal limits, whereas values of 31-100 were considered abnormal.
## Study group
Participants were classified based on the severity of their functional damage as assessed by SAP and Matrix. Since no generalized criteria for categorizing glaucoma severity using Matrix exists, we used the same mean deviation (MD) values for both SAP and Matrix. With reference to the mean deviations of SAP and Matrix, each VF defect was classified as early (no worse than -6dB): groups S1 and M1; moderate (worse than -6 dB but not worse than -12dB): groups S2 and M2; and advanced (worse than -12dB): groups S3 and M3. [bib_ref] A clinical comparison of visual field testing with a new automated perimeter,..., Beck [/bib_ref] The S1, S2 and S3 subgroups were classified according to SAP severity, whereas subgroups M1, M2 and M3 were determined by Matrix severity scores.
# Data analysis
All statistical tests were performed using a statistical soft-ware package (SPSS 15.0 for Windows; SPSS Inc., Chicago, IL). RNFL thicknesses measured by OCT and GDx VCC were compared among the three groups categorized by MD scores from both SAP and Matrix using a one-way analysis of variance (ANOVA) with pairwise, post-hoc Tukey comparisons. The normative classifications determined by the internal databases of the OCT and GDx VCC instruments were compared among the three subgroups for each of SAP and Matrix using the Chisquare test. Differences with a value of p<0.05 were considered statistically significant [fig_ref] Figure 1: Overview of the division of the SAP and Matrix groups and Analyses... [/fig_ref].
# Results
The mean age of the 90 participants in the SAP group was 56.9±13.4 years, and the mean age of the 112 participants in [bib_ref] Visual functionspecificperimetry for indirect comparison of different ganglioncell populations in glaucoma, Sample [/bib_ref] (88.5%) SAP group=glaucoma group diagnosed using standard automated perimetry; S1, S2, S3=subgroups divided by mean deviation of standard automated perimetry; Matrix group=glaucoma group diagnosed using Humphrey Matrix perimetry; M1, M2, M3=subgroups divided by mean deviation of Humphrey Matrix perimetry; NFI=nerve fiber indicator; TSNIT=temporal-superior-nasal-inferior-temporal; SD=standard deviation; All data were analyzed using the Chi-square test. * statistically significant (p<0.05). 0.67). Overall, no RNFL thickness parameter measured using either OCT or GDx VCC differed, with statistical significance, between the SAP and Matrix groups.
In the SAP group, average, superior and inferior RNFL thicknesses measured by OCT were significantly different between the S1 and S2 groups (p=0.001, 0.011 and 0.000, respectively), whereas the average and inferior RNFL thicknesses were significantly different in the M1 and M2 groups (p=0.016 and 0.013, respectively). Nasal and temporal RNFL thicknesses did not differ when the S1 and S2 or the M1 and M2 subgroups were compared. TSNIT values, superior averages, TSNIT SDs, and NFIs assessed by GDx VCC were significantly different between the S1 and S2 subgroups, but only NFIs were significantly different between the M1 and M2 subgroups. All parameters of OCT and GDx VCC were significantly different between the S2 and S3 subgroups and also between the S1 and S3 subgroups. Matrix subgroup analysis also revealed significant differences between groups M2 and M3, and M1 and M3 [fig_ref] Table 4: Analysis 1 [/fig_ref].
## Analysis 2: sap group vs. matrix group with reference to normative classifications assessed by oct and gdx vcc
We used the Chi-square test to compare structural parameters interpreted with the internal normative databases. Compared to abnormal classifications, percentages of such classifications among the subgroups were significantly different in all parameters in both the SAP and Matrix subgroups [fig_ref] Table 5: Comparison of observed parameters with data in internal normative databases between each... [/fig_ref].
## D iscussion
SAP is considered to be a standard approach for detecting VF loss in glaucoma. [bib_ref] Number of ganglion cells in glaucoma eyes compared with threshold visual field..., Kerrgan-Baumrind [/bib_ref] However, SAP may not be able to detect early functional changes because of redundancies in RGCs and optic nerve fibers, which can mask damage. [bib_ref] Visual functionspecificperimetry for indirect comparison of different ganglioncell populations in glaucoma, Sample [/bib_ref] [bib_ref] Ganglion cell losses underlying visual field defects from experimental glaucoma, Harwerth [/bib_ref] As a screening device, Humphrey Matrix Frequency doubling technology perimetry (Matrix) is known to be similar to or better than the Humphrey field analyzer. [bib_ref] An examination of physiological mechanisms underlying the frequency-doubling illusion, White [/bib_ref] [bib_ref] Frequency doubling technology perimetry for detection of glaucomatous visual field loss, Cello [/bib_ref] [bib_ref] Screening for glaucomatous visual field loss with frequency-doubling perimetry, Johnson [/bib_ref] [bib_ref] Identification of glaucoma-related visual field abnormality with the screening protocol of frequency..., Quigley [/bib_ref] Frequency doubling technology (FDT) perimetry was developed as a method to detect early glaucoma by attempting to functionally isolate "My" cells, which may be selectively damaged in the early stages of glaucoma. [bib_ref] Number of ganglion cells in glaucoma eyes compared with threshold visual field..., Kerrgan-Baumrind [/bib_ref] [bib_ref] An examination of physiological mechanisms underlying the frequency-doubling illusion, White [/bib_ref] [bib_ref] Performance of nonlinear visual units in ocular hypertension and glaucoma, Maddess [/bib_ref] My cells are not functionally redundant (making up 3-5% of all ganglion cells); the FDT technique thus has the theoretical potential to identify early VF loss. [bib_ref] Frequency doubling perimetry in glaucoma early diagnosis, Brusini [/bib_ref] [bib_ref] Comparison of frequency doubling perimetry with Humphrey visual field analysis in a..., Burnstein [/bib_ref] Matrix perimetry is an updated version of FDT perimetry, employing a larger number of stimulus locations and smaller targets than those used in FDT perimetry. Matrix also provides the global indices of SAP, such as MD, PSD and GHT classification, in a standard printout. [bib_ref] Screening for glaucomatous visual field loss with frequency-doubling perimetry, Johnson [/bib_ref] [bib_ref] Algorithm for interpreting the results of frequency doubling perimetry, Patel [/bib_ref] [bib_ref] Identifying glaucomatous vision loss with visual-function-specific perimetry in the dia gnostic innovations..., Sample [/bib_ref] [bib_ref] Diagnostic capabilities of frequency doubling technology, scanning laser polarimetry and nerve fiber..., Paczka [/bib_ref] Comparison of Matrix and SAP threshold sensitivities is not appropriate because the two technologies measure different aspects of retinal sensitivity SAP assesses differential light sensitivity, and Matrix obtains data on contrast sensitivity. Although global indices were reported to be significantly correlated between SAP and Matrix, [bib_ref] Comparison of frequency doubling perimetry and standard achromatic computerized perimetry in patients..., Serguhn [/bib_ref] [bib_ref] Relationship between indices of Humphrey perimetry and frequency doubling technology perimetry in..., Fukushima [/bib_ref] there is no consensus for the definition of glaucomatous VF defects as determined by Matrix. Therefore, we used the same criteria (PSD < 5% and abnormal GHT) to define glaucoma identified by both SAP and Matrix, and we divided patients into three groups according to the severity of the MD.
Stratus OCT and GDx VCC are the two most recent commercial instruments available to analyze peripapillary RNFL thickness. In many studies, both GDx VCC and OCT showed relatively high diagnostic accuracies in glaucoma detection. [bib_ref] Clinical classification of glaucomatous visual field loss by frequency doubling perimetry, Sponsel [/bib_ref] [bib_ref] Fourier analysis of scanning laser polarimetry measurements with variable corneal compensation in..., Medeiros [/bib_ref] [bib_ref] Evaluation of retinal nerve fiber layer, optic nerve head, and macular thickness..., Medeiros [/bib_ref] [bib_ref] Sensitivity and specificity of the Stratus OCT for perimetric glaucoma, Budenz [/bib_ref] Average RNFL thickness by OCT demonstrated a strong correlation with VF defects. [bib_ref] Scanning Laser Polarimetry With Variable Corneal Compensation and Optical Coherence Tomography in..., Bagga [/bib_ref] The NFI data from GDx VCC also revealed a strong correlation with VF defects. However, RNFL thicknesses determined by OCT in the nasal and temporal quadrants have been reported to show high measurement varia-bilities. [bib_ref] Evaluation of the Glaucomatous Damage on Retinal nerve fiber layer thickness measured..., Kanamori [/bib_ref] [bib_ref] Reproducibility of nerve fiber layer thickness measurements by use of optical coherence..., Blumenthal [/bib_ref] [bib_ref] Reliability of nerve fiber layer thickness measurements using optical coherence tomography in..., Carpineto [/bib_ref] [bib_ref] Reproducibility of scanning laser polarimetry (GDx) of peripapillary retinal nerve fiber layer..., Kook [/bib_ref] Reus and Lemij suggested that glaucoma patients with mildto-moderate VF loss might be better monitored with the GDx VCC. [bib_ref] The relationship between standard automated perimetry and GDx VCC measurements, Reus [/bib_ref] Kim and Kook reported that abnormal scores obtained on FDT perimetry screening programs correlated negatively with RNFL retardation values, as measured in glaucomatous changes seen by SLP. [bib_ref] Correlation Between Frequency Doubling Technology Perimetry and Scanning Laser Polarimetry in Glaucoma..., Kim [/bib_ref] However, to our knowledge, no data confirming a relationship between VF loss and GDx and OCT Matrix data has been published.
In this study, we divided patients into subgroups using MD values from both SAP and Matrix. Our results showed that all structural parameters except nasal and temporal RNFL thicknesses obtained by OCT were significantly different among cases in the three different stages of glaucoma. These findings suggest good correlations between RNFL thickness parameters assessed by OCT and GDx VCC on the one hand, and SAP defects on the other, in patients in different stages of glaucoma. Only nasal and temporal quadrant data of OCT, and the inferior average of GDx VCC, showed no meaningful correlations between early (S1) and moderate (S2) glaucoma stages as assessed by SAP. This might be explained by high measurement variability. [bib_ref] Evaluation of the Glaucomatous Damage on Retinal nerve fiber layer thickness measured..., Kanamori [/bib_ref] [bib_ref] Reproducibility of nerve fiber layer thickness measurements by use of optical coherence..., Blumenthal [/bib_ref] [bib_ref] Reliability of nerve fiber layer thickness measurements using optical coherence tomography in..., Carpineto [/bib_ref] The fact that nasal and temporal quadrants are relatively more resistant to glaucomatous damage may also partly explain these findings.
However, not all structural parameters showed significant differences between early (M1) and moderate (M2) stages of glaucoma as assessed by Matrix. Average and inferior RNFL thicknesses by OCT and NFI by GDx VCC were significantly different between the M1 and M2 subgroups, but there were no differences in any other parameters. Although some parameters did not differ between the M1 and M2 subgroups, all parameters assessed by GDx VCC and OCT showed significant differences between the moderate (subgroup M2) and advanced (subgroup M3) patients.
Matrix is known to be superior to SAP for detecting the early stages of glaucoma. In this study, we observed that although the Matrix subgroups showed definite structural differences between cases of moderate and severe glaucoma as assessed by GDx and OCT parameters, not all parameters differed between patients with early and moderate glaucoma. This could indicate that Matrix can be an excellent screening device to detect early glaucoma, especially in the preperimetric stage, but it has difficulty in discriminating between the early and moderate stages of glaucoma. The other possible explanation for the poor correlation of structural damage with functional deficits as determined by Matrix in early-to-moderate glaucoma stages is related to the reliability of Matrix itself. Most enrolled participants were tested only once for each Matrix and SAP. The learning effect of Matrix may improve VF outcome in repeat tests. We acknowledge that this is a limitation of our study. Because the study design was retrospective, all participants underwent a comprehensive ophthalmic examination at the initial visit, including SAP, FDT, OCT, and GDx VCC. If reliability indices were poor during testing, the operator stopped the test, explained the process thoroughly, and then restarted the test. To maximize reliability and to minimize learning effect-related issues, we adopted strict VF inclusion criteria.
To our knowledge, this is the first study to examine the relationship between functional and structural damage in glaucoma subgroups of different severities using both SAP and Matrix global indices. We concluded that glaucoma status as defined by both SAP and Matrix correlates with structural loss as assessed by OCT and GDx VCC, but that such correlations are slightly weaker in the early-to-moderate glaucoma stages as defined by Matrix.
[fig] Figure 1: Overview of the division of the SAP and Matrix groups and Analyses 1 and 2. [/fig]
[table] Table 1: Patient Characteristics SAP=standard automated perimetry; Matrix=frequency doubling technology perimetry 24-2 performed with the Humphrey Matrix; SD=standard deviation; MD=mean deviation;PSD=pattern standard deviation; SAP group=laucoma group diagnosed by standard automated perimetry; Matrix group=laucoma group diagnosed by Humphrey Matrix perimetry; POAG=rimary open angle glaucoma; NTG= normal tension glaucoma; SOAG=econdary open angle glaucoma; PACG=rimary angle closure glaucoma. * statistically significant (p<0.05). [/table]
[table] Table 2: Subgroups divided by mean deviations of SAP and Matrix PSD=pattern standard deviation; No.=number of patients; SAP group: glaucoma group diagnosed using standard automated perimetry; S1, S2, S3=subgroups divided by mean deviation of standard automated perimetry; Matrix group=glaucoma group diagnosed using Humphrey Matrix perimetry; M1, M2, M3=subgroups divided by mean deviation of Humphrey Matrix perimetry. [/table]
[table] Table 3: Comparison of OCT and GDx VCC parameters between the SAP group and the Matrix group NFI=nerve fiber indicator; SAP group=glaucoma group diagnosed using standard automated perimetry; Matrix group=glaucoma group diagnosed using Humphrey Matrix perimetry; TSNIT=temporal-superior-nasal-inferior-temporal; SD=standard deviation. [/table]
[table] Table 4: Analysis 1: Comparison of RNFL thickness between each subgroup classified by SAP (S1, S2 and S3) and Matrix (M1, M2 and M3) Structural parameters assessed by OCT and GDx VCC were compared in the SAP subgroups and the Matrix subgroups (Table 3). Average RNFL thickness measured by OCT was 79.7±19.5 μm in the SAP group and 86.8±18.6 μm in the Matrix group (p= 0.41). The average NFI determined by GDx VCC was 54.9± 23.3 in the SAP group and 46.0±22.4 in the Matrix group (p= [/table]
[table] Table 5: Comparison of observed parameters with data in internal normative databases between each subgroup classified by SAP (S1, S2 and S3) and Matrix (M1, M2 and M3) [/table]
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Early Rehabilitation and Periprosthetic Bone Environment after Primary Total Hip Arthroplasty: A Randomized Controlled Trial
Objective: To investigate whether the periprosthetic bone environment could be affected by activity during the early rehabilitation period after primary total hip arthroplasty (THA) and to evaluate the safety and efficacy of activity during the early rehabilitation period. A total of 22 selected patients with advanced osteonecrosis of the femoral head (ONFH) who underwent primary unilateral THA were randomized (1:1) to a high activity level group (HA group) or a low activity level group (LA group). The HA group included nine men and two women, aged 53.18 AE 13.29 years. The LA group included five men and six women, aged 55.73 AE 11.73 years. The intervention was different postoperative daily walking distances guided by researchers: 1727.27 AE 564.08 m 0-2 months and 4272.73 AE 904.53 m 3-6 months postoperation for the HA group and 909.09 AE 583.87 m 0-2 months and 2409.09 AE 1068.13 m 3-6 months postoperation for LA group. The primary outcomes were radiographic evaluation (prosthetic stability and stress shielding based on the Engh scale) and bone mineral density (BMD) with a femoral prosthesis (individual and intergroup comparison using seven Gruen zones) at 6 months postoperatively. Secondary outcomes were set to confirm the safety and efficacy of activity during early rehabilitation, including day 1 erythrocyte sedimentation rate (ESR), day 1 hypersensitive C-reactive protein (CRP), length of hospital stay (LOS), and the Harris hip score (HHS) at discharge, 2 months postoperatively, and 6 months postoperatively.Results: Patients were followed up for 6 months after surgery. Regarding primary outcomes, all prostheses were assessed as stable, with bone in-growth. There were no adverse events in any cases. The HA group had a higher incidence of stress shielding than the LA group, but there was no statistical significance (63.64% vs 18.18%; P > 0.05). The degree of stress shielding had a different distribution for the two groups (P < 0.05). In the HA group and the LA group, the median percentage difference of the BMD on the operated side was −25% and was −13% in Zone 1, −8% and − 1% in Zone 2, +1% and 3% in Zone 3, +6% and + 6% in Zone 4, −2% and +2% in Zone 5, −3% and −1% in Zone 6, and −24% and −12% in Zone 7 compared with the unoperated side. The BMD was significantly reduced in the medial proximal femur (Zone 1) and the lateral proximal femur (Zone 7) in both groups (P < 0.05). Furthermore, it was increased in the distal femur (Zone 4) in the HA group (P < 0.05). No difference was found in the BMD when comparing between groups. Regarding secondary outcomes, there was no statistical difference in day 1 ESR and day 1 CPR. The average LOS was similar in the HA and LA groups (7.00 days vs 7.18 days, P > 0.05). The HHS on day of discharge was higher in the HA group than in the LA group (60.73 AE 5.37 points vs 51.18 AE 8.05 points, P < 0.05); however, no statistically significant difference was found in postoperative the HHS at 2 months (81.73 AE 6.92 points vs 78.36 AE 9.18 points, P > 0.05) and 6 months (90.45 AE 5.24 points vs 91.55 AE 4.03 points, P > 0.05).Conclusion: High activity levels during early rehabilitation after primary THA accelerate the process of bone remodeling and aggravate stress shielding, with no significant benefits for functional recovery.
# Introduction
O steonecrosis of the femoral head (ONFH) is defined as death of bone cells around the femoral head due to decreased blood flow, especially in lateral epiphyseal arteries branching from medial circumflex arteries [bib_ref] Nontraumatic necrosis of bone (osteonecrosis), Mankin [/bib_ref]. It can be caused by traumatic or non-traumatic events. Direct and indirect risk factors include femoral head fractures, hip dislocation, alcoholism, use of glucocorticoids, and radiation. However, the etiology and pathogenesis of ONFH remain unknown because this local bone ischemia derives from a complex combination of genetic factors, metabolic changes, and vascular impairment 2 . In China, at least 8.12 million people over the age of 15 years suffer from ONFH 3 . Individuals between 40 and 50 years of age are typically affected by ONFH [bib_ref] Multicentric epidemiologic study on six thousand three hundred and ninety five cases..., Cui [/bib_ref]. Incorporating X-rays, CT, MRI, and histological examinations, the Association Research Circulation Osseous (ARCO) staging system for ONFH is widely used to guide therapy. The system comprises five stages (stages 0 to 4), with subclassification based on the location and size of the lesion [bib_ref] Guidelines for clinical diagnosis and treatment of osteonecrosis of the femoral head..., Zhao [/bib_ref]. Conservative treatment and hip-preserving surgery can be used in the early stages (ARCO stage 0 to 1) and the middle stages (ARCO stage 2 to 3b); however, the progressive course of ONFH is rarely reversed. Persistent groin pain and decreased range of motion in the hip joint ultimately arise in the late stages (ARCO stage 3c to 4), which severely impacts quality of life. When the femoral head has collapsed, total hip arthroplasty (THA) is the only treatment that can relieve pain and restore function.
In the 1990s, application of THA for ONFH was not considered the optimum choice because of the short longevity of prostheses and the high postoperative complications compared to its use in other diseases [bib_ref] Total hip arthroplasty with cement. A long-term radiographic analysis in patients who..., Sarmiento [/bib_ref] [bib_ref] Total hip arthroplasty for osteonecrosis: matched-pair analysis of 188 hips with long-term..., Ortiguera [/bib_ref]. However, thanks to innovations in prosthetic materials, improvements in surgical technology, and developments in research over the past decade, the clinical benefits for ONFH patients have now been established as substantial [bib_ref] Total hip arthroplasty with dual mobility cup in osteonecrosis of the femoral..., Martz [/bib_ref] [bib_ref] Short stem total hip arthroplasty for osteonecrosis of the femoral head in..., Capone [/bib_ref]. Today, there are more than 1 million THA are performed worldwide each year [bib_ref] International variation in hip replacement rates, Merx [/bib_ref] , and the estimated proportion of THA based on ONFH is nearly 10% [bib_ref] Osteonecrosis of the femoral head: evaluation and treatment, Zalavras [/bib_ref].
Fast track surgery (FTS), namely enhanced recovery after surgery (ERAS), initiated with abdominal surgery by Dr Henrik Kehlet, is an evidence-based multidisciplinary perioperative protocol followed for surgical patients to restore postoperative function rapidly without additional' morbidity and mortality [bib_ref] Multimodal approach to control postoperative pathophysiology and rehabilitation, Kehlet [/bib_ref]. Orthopaedic surgeons pioneered this concept in joint replacement surgery in Denmark in 2003 [bib_ref] Fast track in total hip and knee arthroplasty-experiences from Hvidovre University Hospital,..., Husted [/bib_ref]. Since then, THA has continued to demonstrate positive results in terms of shortened length of hospital stay (LOS), reduced medical expenses, and high patient satisfaction with no increased complications [bib_ref] Enhanced recovery after surgery for hip and knee arthroplasty: a systematic review..., Zhu [/bib_ref] [bib_ref] Fasttrack recovery technique applied to primary total hip and knee replacement surgery...., Wilches [/bib_ref]. Early rehabilitation, as an important component of FTS, benefits patients, especially in terms of restoration of function [bib_ref] Intermediate and long-term quality of life after total knee replacement: a systematic..., Shan [/bib_ref]. However, there is little data available on how much activity is appropriate postoperatively taking into consideration the periprosthetic bone environment. One reason for this lack of information is that rather than specific patient factors, the process of bone remodeling has been supposed to be affected more by surgical factors, such as preoperative osteoporosis, the surgeon's technique, and the characteristic of prostheses [bib_ref] Radiological changes around the stem after cementless hip implantation in case of..., Roth [/bib_ref] [bib_ref] Clinical consequences of stress shielding after porous-coated total hip arthroplasty, Engh [/bib_ref].
To evaluate the variation of the periprosthetic bone environment, bone resorption and bone dissolution (osteolysis) are measured, generally using X-rays. They are two independent processes that persisting after arthroplasty. Bone resorption is an adapted change to stress variation and osteolysis is a result of complicated immunologic responses initiated by prosthesis wear particles. In THA, the dynamic balance between osteoblasts and osteoclasts is broken unavoidably because of the load transfer from the prosthesis and the bone resorption (i.e. stress shielding) commonly at the proximal femur [bib_ref] Quantifying bone loss from the proximal femur after total hip arthroplasty, Mccarthy [/bib_ref]. The reported incidence of stress shielding varies considerably, from 13.5% to 84% [bib_ref] Radiological changes around the stem after cementless hip implantation in case of..., Roth [/bib_ref] [bib_ref] The middle-aged patient with hip arthritis: the case for extensively coated stems, Valle [/bib_ref]. This variation is mainly due to subjectivity and judgments based on X-rays. To ensure accuracy, dual-energy X-ray absorptiometry (DEXA) has been introduced as a standard procedure for evaluating postoperative bone conditions [bib_ref] Fundamentals and pitfalls of bone densitometry using dual-energy X-ray absorptiometry (DXA), Watts [/bib_ref]. The earliest evidence of stress shielding could be found on X-ray in some patients at 3 weeks postoperatively [bib_ref] Radiological changes around the stem after cementless hip implantation in case of..., Roth [/bib_ref] , and reduction of bone mineral density (BMD) was seen in patients at 3 months through DEXA [bib_ref] Monitoring of periprosthetic BMD after uncemented total hip arthroplasty with dual-energy X-ray..., Venesmaa [/bib_ref].
In summary, ONFH is a public health concern worldwide, and the sole effective end-stage therapy is THA. Nevertheless, in ONFH patients, patients that undergo THA have relatively poor clinical outcomes compared to patients who undergo THA for other diseases. ONFH patients have high activity levels due to the younger age of onset of the disease. So far, no research has focused on the potential relationship between activity and periprosthetic bone pathophysiological changes. In this context, we designed this primary clinical trial using Xrays combined with DEXA to investigate whether early rehabilitation was related to the periprosthetic bone environment in ONFH patients undergoing THA. The hypothesis was that patients with high activity levels would have an enhanced periprosthetic bone remodeling process compared with those with low activity levels because, theoretically, their total load transfer over the same period should be larger. Our research had three main goals: (i) to test the hypothesis that high activity levels after THA lead to an accelerated bone remodeling process; (ii) to determine whether high activity levels are beneficial for postoperative functional recovery; and (iii) to confirm the safety of early rehabilitation in a fasttrack THA.
# Materials and methods
O ur randomized controlled trial complies with the Declaration of Helsinki and was registered in the Chinese Clinical Trail Registry (Reg. no. ChiCTR-INR-17010451). The trial was approved by the ethics committee at Jilin University, First Clinical Hospital (no. 2017-250) and carried out at the same institute. Written informed consent was obtained from all patients.
## Inclusion and exclusion criteria
Inclusion criteria were based on the PICOS principle: (i) patients diagnosed with unilateral advanced ONFH (ARCO stage IIIC-IV); (ii) patients treated with unilateral THA with a posterolateral approach and prescribed different postoperative daily walking distances (2000 m at 0-2 months and 4000 m at 3-6 months vs 1000 m at 0-2 months and 2000 m at 3-6 months); (iii) stress shielding based on X-rays and BMD based on dual-energy X-ray absorptiometer (DEXA) at 6 months after surgery were used to evaluate the periprosthetic bone environment and the postoperative erythrocyte sedimentation rate (ESR), hypersensitive C-reactive protein (CRP), LOS, and Harris hip score (HHS) to assess clinical outcomes; (iv) patients with longer postoperative daily walking distances were more likely to show stress shielding at 6 months postoperatively and had better HHS on day of discharge, and there were no significant differences between the two groups in terms of postoperative BMD, ESR, CRP, and LOS; and (v) this study was a randomized controlled trial.
The exclusion criteria were: (i) patients aged under 18 years or above 75 years; (ii) patients with extreme body mass index (BMI) (<18 or >30); (iii) preoperative complications with uncontrollable comorbidity; (iv) previous surgery on the affected hip; (v) history of smoking and alcoholism; (vi) patients lacking essential economic and social support; (vii) American Society of Anesthesiologists (ASA) physical status >III; and (viii) undesirable bone condition as determined by preoperative X-ray. The full experimental approach can be found at www.chictr.org.cn.
## General information
This trial was a single-center, prospective, randomized, double-blind, controlled clinical study that complied with the Consolidated Standards of Reporting Trials (CONSORT) guidelines [bib_ref] Extending the CONSORT statement to randomized trials of nonpharmacologic treatment: explanation and..., Boutron [/bib_ref]. From January to July 2017, 22 patients with ONFH underwent a single total hip arthroplasty participated in the experiment.
All surgeries were performed by the same experienced chief physician using a posterolateral approach and cementless prostheses (Exceed ABT Biomet Delta Ceramic Acetabular Liner; Exceed ABT Ceramic Acetabular Cup; Biomet Delta Ceramic Femoral Head; Echo Bi-Metric Titanium Alloy Femoral Stem), the stem was Echo Bi-Metric RPP stem (Reduced Proximal Profile; [fig_ref] Figure 1: The stem used in this study [/fig_ref]. The stem was a collarless 3 bi-planer tapered stem, which was made of titanium alloy. The neck angle of the stem was 135 . The proximal part was porous-coated with a plasma-sprayed HA layer, and the distal part was polished and bullet-shaped. Patients were monitored in a post-anesthesia care unit after surgery for 2-4 h until their vital signs were stable.
## Randomization
An assigned person not involved in the study used a random number table to determine whether patients should be allocated to the HA group or the LA group and then saved the randomization sequence into numbered, opaque sealed envelopes.
## Blinding
Only the person who set the sequence (not involved in the study) knew the allocation. They would notify the chief researcher after surgery immediately so that a different rehabilitation schedule could be provided to patients in each group. All subjects and personnel in this study were blinded to the treatment group until the trial had been completed. Once the patients left hospital, the staff who collected followup data were blinded regarding the treatment group, as were the evaluators who were responsible for checking and grading the outcomes.
## Surgical procedure
Anesthesia and Position All patients received general anesthesia. The patient was placed in the lateral decubitus position with supports. The involved leg was disinfected preoperatively and kept free to move during surgery.
## Approach and exposure
A posterolateral approach to the hip was used to expose the posterior hip capsule. The 12-cm curved incision rounded the greater trochanter from the posterior to the lateral side with its proximal 6 cm pointed to the posterior superior iliac spine and distal 6 cm along the femoral axis. In line with the skin incision, a sharp dissection of the fascia lata and gluteal muscle was made across the greater trochanter. The tendinous insertions of the short external rotators were bluntly dissected, and nonabsorbable sutures were placed in the piriformis. Obturator and gemellus tendons were prepared for final repair. After reflection of the short rotator muscles, a full thick, broad-based flap of the posterior hip capsule was necessary to expose the hip joint. Surgical dislocation was performed by flexion, adduction, and slight internal rotation of the hip joint for the subsequent THA.
## Pathological changes and resection
To confirm ARCO stages 3a and 4 in ONFH patients, femoral head collapse and acetabular arthritis were verified in each operation after surgical dislocation. Because preoperative measurement for the prosthesis had been done, the femoral head was directly cut using a swing saw accordingly and the round ligament of the femur was removed. The surgical field of the acetabulum was then clear, and the chief surgeon used the acetabular file to grind the acetabulum to the preoperatively planned size.
## Placement of prosthesis
The models were used before prosthesis implantation. The appropriate acetabular component was placed after management of the acetabulum. The femoral medullary canal was reamed using cylindrical reamers and rasps so that the maximal stem could be inserted and the initial stability was ensured. After placement of a suitable femoral component and reset, the stability of the test molds was verified, and the size of femoral head, the range of motion and the soft tissue balance were assessed. Subsequently, the corresponding prosthesis substituted the molds, and the same test was performed again.
## Reconstruction
In the final stage of the surgery, the posterior hip capsule and the piriformis were repaired to provide enhanced postoperative muscle strength and to lower the dislocation rate. Tranexamic acid was infiltrated around the soft tissue to reduce postoperative blood loss. Whether a drainage tube was used depended on the local blood loss before closure.
## Perioperative management
The perioperative management of the two groups was based on the theory of FTS [bib_ref] Fast track in total hip and knee arthroplasty-experiences from Hvidovre University Hospital,..., Husted [/bib_ref]. In the preoperative period, patients in each group received the same education. Nerve blocks and local infiltration anesthesia were not used. Blood, pain, and sleep management were unremarkable in all patients postoperatively. The patients were mobilized on the day after the operation under supervision of a physiotherapist, and protected weight-bearing on the operated leg was initiated for 4 weeks. Anteroposterior and lateral radiographs were taken on the day of discharge to ensure initial stability.
## Intervention and follow up
One researcher was in charge of maintaining the connection with all subjects after discharge by phone. Different targets for postoperative rehabilitation were given in each group by the chief researcher: 2000 meters 0-2 months postoperation and 4000 m 3-6 months postoperation in the HA group and1000 m 0-2 months postoperation and 2000 m 3-6 months postoperation in the LA group. Daily walking distance was recorded using a pedometer and patients were followed up by phone interview. At 6 months after surgery, patients came back to our outpatient service to complete their clinical and radiological evaluation.
## Outcomes
The basic preoperative characteristics were classified into demographic and clinical factors. Demographic variables included gender, age, BMI, and ASA physical status. The clinical factors were ESR, hypersensitive CRP, duration of surgery, intraoperative bleeding, and HHS.
## Primary outcomes
## Radiographic evaluation
Stress shielding and prosthetic stability were evaluated using anteroposterior and lateral radiographs at 6 months postoperatively, focusing on the femoral prosthesis. The incidence and degree of these indicators were collected. According to the Engh scale, stress shielding could be divided into five levels, from 0 (none) to 4 (4th degree), based on X-ray, with a higher degree of stress shielding representing more severe bone resorption [bib_ref] Porous-coated hip replacement. The factors governing bone ingrowth, stress shielding, and clinical..., Engh [/bib_ref]. Prosthetic stability was judged using the criteria of Engh et al.; that is, no subsidence and radioopaque lines could be found along the implant in consecutive examinations [bib_ref] Roentgenographic assessment of the biologic fixation of porous-surfaced femoral components, Engh [/bib_ref].
## Bone mineral density
Bone mineral density examination was conducted at 6 months postoperatively. A DEXA (DPX-L; Lunar, Madison, WI, USA) using specific software for measuring BMD was applied in the coronal plane. The femoral side that was operated on was scanned, and BMD values were analyzed in seven Gruen zones. Then the unoperated side was superimposed with a prosthesis mask, which was stimulated from the opposite by the software at the same level. The values were expressed as real BMD (g/cm 2 ). The ratio between the operated side and the unoperated side was calculated in each zone.
## Gruen zone
The Gruen classification is a system for evaluating periprosthetic bone changes around the femoral stem [bib_ref] Modes of failure" of cemented stemtype femoral components: a radiographic analysis of..., Gruen [/bib_ref]. There are seven areas in this classification system: Zone 1, greater trochanter; Zone 2, proximal lateral; Zone 3, distal lateral; Zone 4, tip; Zone 5, distal medial; Zone 6, proximal medial; and Zone 7, calcar region. With the help of specialized software, DEXA can be used to analyze BMD changes in these zones automatically.
## Secondary outcomes
Secondary outcomes were day 1 ESR, day 1 CRP, LOS, and the HHS. ESR and CRP were collected at the first postoperative day. HHS was evaluated by a chief doctor on the day of discharge and at 2 months and 6 months postoperatively.
## Harris hip score
The HHS is an instrument for evaluating the functional ability in THA. The HHS contains four items: pain, function, degree of deformity, and range of motion of the hip joint [bib_ref] Comparison of the responsiveness of the Harris hip score with generic measures..., Hoeksma [/bib_ref]. Final scores range from 0 to 100. A score <70 is considered poor, while 70-80 is fair, 80-90 is good, and 90-100 is excellent. Flow diagram according to CONSORT. HA, high activity group, LA group, low activity group.
# Statistical analysis
Referring to previous research [bib_ref] Total hip arthroplasty for osteonecrosis: matched-pair analysis of 188 hips with long-term..., Ortiguera [/bib_ref] [bib_ref] Fundamentals and pitfalls of bone densitometry using dual-energy X-ray absorptiometry (DXA), Watts [/bib_ref] [bib_ref] Seven to 10 years followup of an anatomic hip prosthesis: an international..., Herrera [/bib_ref] and data from our hospital, we presumed that stress shielding in the ER group would be more severe than in the LR group. Thus, we set a primary end point (incidence rate of stress shielding) of 75% in the ER group and 15% in the LR group for an exploratory study. A total of 20 cases (10 cases per group) were thought to be essential to reveal a difference, with a type I error rate of 5% and a statistical power of 80%. We decided to enroll 22 subjects, anticipating a 10% dropout rate. Analysis in this study followed the intention-to-treat (ITT) principle.
All tests were two-sided, with a significance level of 5% (α value). Numerical data were described as mean AE standard deviation, including ESR, CRP, HSS, and LOS. Degree of stress shielding was compared between the two groups using the rank-sum test. Fisher's exact test was used for the rate comparison for stress shielding. The percentage changes in BMD were expressed by the median values (25th-75th percentiles). The Wilcoxon signed-rank test (paired observations) was used to compare differences in sides, while the Mann-Whitney U-test (unpaired observations) was used for intergroup comparison. We used SPSS software version 22.0 (IBM SPSS, Armonk, New York, USA) to analyze the collected data. Statistical significance was confirmed if P-values < 0.05.
# Results
## Study flow and patient characteristics
A total of 51 patients met the criteria; 27 patients were excluded based on the inclusion criteria and two patients refused to participate. Ultimately, a total of 22 subjects were enrolled in this study. All subjects were followed up and data collection was complete. The follow-up period was 6 months after surgery. A flow diagram is presented in . There were nine men and two women in the HA group, with an average age of 53.18 years (range, 26-70 years). There were five men and six women in the LA group, with an average respectively. The above demographic and clinical factors at baseline are shown in [fig_ref] TABLE 1: Demographics and clinical factors [/fig_ref]. The baseline balance was assured because of complete randomization in this randomized controlled trial. The target daily walking distance (interventions) was accomplished in each group (
## Primary outcomes
## Radiographic evaluation
At the endpoint, all prostheses were assessed as stable with bone ingrowth (with spot welds, absence of subsidence, and radiolucent lines). The distribution of the stress shielding scale was different between groups: four with no stress shielding, four with first-degree stress shielding, and three with second-degree stress shielding in the HA group, while nine with no stress shielding and two with first-degree stress shielding in the LA group (P < 0.05; [fig_ref] TABLE 3: Primary outcomes [/fig_ref]. Although the HA group had a higher incidence of stress shielding, no statistical significanve was found compared with the LA group (63.64% vs 18.18%, P > 0.05; [fig_ref] TABLE 3: Primary outcomes [/fig_ref]. The relative risk (RR) was 3.5 (confidence inteval 0.9-13.2). Typical cases are displayed in [fig_ref] Figure 3 X: -ray of three patients with osteonecrosis of the femoral head [/fig_ref].
## Bone mineral density
Compared with the contralateral side, a significant reduction of BMD was found at Zone 1 and Zone 7 in both groups. In comparing the operated side and the unoperated side, the BMD change was −25% for Zone 1 and −24% for Zone 7 in the HA group, while it was −13% for Zone 1 and −12% for Zone 7 in the LA group (P < 0.05;. Furthermore, the BMD was significantly increased at Zone 4 in the HA group (+6%, P < 0.05;. However, when comparing the BMD change of the operated side between HA and LA groups, no significant difference was found at all zones.
## Secondary outcomes
Comparing the HA and LA groups, day 1 ESR was 24.00 AE 23.56 mm/1 h and 24.55 AE 21.33 mm/1 h, day 1 CRP was 49.10 AE 33.66 mg/L and 69.36 AE 46.71 mg/L, and LOS was 7.00 AE 2.53 days and 7.18 AE 2.71 days, respectively. No statistical significance was found for these data (P > 0.05). The HHS was higher in the HA group than the LA group on the day of discharge (60.73 AE 5.37 vs 51.18 AE 8.05, P < 0.05); it was not different at follow up at 2 months and 6 months postoperatively [fig_ref] TABLE 7: Secondary outcomes [/fig_ref].
## Adverse events
Patients were observed for signs of fracture, dislocation, severe pain, and lack of mobility. No adverse events occurred during the study.
# Discussion
# Summary of results
To our knowledge, the present study was the first to investigate the relationship between early rehabilitation and the periprosthetic bone environment after primary THA, and the primary finding was that patients with high activity levels had a different distribution on the stress shielding scale (more severe), and the risk of appearance of stress shielding was 3.5 times higher in patients with high activity levels compared with those with low activity levels. Furthermore, an enhanced process of periprosthetic bone remodeling could be confirmed by considering BMD changes together. Based on secondary outcomes, the functional recovery of the HA group was better that that of the LA group on the day of discharge, which was revealed by HHS; however, at follow up, the results for the two groups no longer differed.
## Radiographic evaluation: stress shielding
Although few studies have focused on the relationship between rehabilitation and the periprosthetic environment, there are some previous related published studies that are useful for reference. First several demographic and surgical factors, including gender, age, BMI, cortical index, and surgeon's technique, have been suggested to affect stress shielding [bib_ref] Clinical outcome in total hip arthroplasty using a cemented titanium femoral prosthesis, Jergesen [/bib_ref] [bib_ref] Treatment of periprosthetic femoral fractures following total hip arthroplasty with femoral component..., Springer [/bib_ref] [bib_ref] Monitoring of periprosthetic BMD after uncemented total hip arthroplasty with dual-energy X-ray..., Venesmaa [/bib_ref]. Second, based on finite element analysis (FEA), optimal load transfer (good stress shielding) was thought to be achieved in short stems. However, Rietbergen et al. showed that reducing the stem length provided no benefit in reducing stress shielding [bib_ref] Load transfer and stress shielding of the hydroxyapatite-ABG hip: a study of..., Van Rietbergen [/bib_ref]. In addition, a trade-off between stress shielding and initial stability was found with a reduction in stem length in an in vitro biomechanical study [bib_ref] Trade-off between stress shielding and initial stability on an anatomical cementless stem..., Yamako [/bib_ref]. Results can be contrary in theory and in practice. showed that reduction of femoral bone dissolution was minimal in a patient 3 years after THA, while it was calculated to be high based on FEA [bib_ref] Finite element model of a novel short stemmed total hip arthroplasty implant..., Lerch [/bib_ref]. In clinical trials, bone loss areas were observed to be different in various uncemented stems and femoral canal shapes [bib_ref] Difference in postoperative periprosthetic bone mineral density changes between 3 major designs..., Inaba [/bib_ref] [bib_ref] Effect of femoral canal shape on mechanical stress distribution and adaptive bone..., Oba [/bib_ref]. One explanation was that loading conditions were changed by each matching situation and this effect was magnified over time.
In the same way, after controlling the above factors, we believed that activity, as an important part of rehabilitation, will influence the periprosthetic bone environment by creating substantial daily pressure, which could lead to an earlier and higher degree of stress shielding by enhancing load transfer. In the present study, the HA group had a higher incidence (although not statistically different) and a more severe degree of stress shielding than the LA group, which could be confirmed by comparison of BMD. These results demonstrated that the speed of bone remodeling was faster in the HA group, which was supported by the changes in BMD.
## Bone mineral density
For BMD, the results showing the bone remodeling procedure, reflected by self-comparision, were in accordance with those of Bodén et al., who showed lateral proximal and medial proximal bone loss (Zone 1 and Zone 7) and distal cortical hypertrophy (Zone 4) in hydroxyapatite coated Bi-Metric modular femoral stems [bib_ref] Unstable versus stable uncemented femoral stems: a radiological study of periprosthetic bone..., Bodén [/bib_ref]. Although the variation of BMD was more substantial in the HA group than in the LA group in all zones, particularly Zone 1 (−25% vs −13%) and Zone 7 (−24% vs −12%), there were no statistical differences between the groups in this study. However, comparing the absence of statistical differences between operated and unoperated sides in the LA group in Zone 4 (+ 6%[+32% to 0%]; P < 0.05), the opposite result in the HA group (+ 6% [+20% to −2%]; P > 0.05) indicated an enhanced process of bone remodeling. Therefore, we assumed that the acceleration of bone remodeling with intense activity may be found in a larger sample size and/or with a greater stimulus factor (amount of activity). Future studies in which the design is focused on BMD are needed to understand this effect. Considering the high degree of stress shielding in the HA group, the significant distal BMD change (Zone 4) in HA group and unremarkable medial BMD change (Zone 1 and Zone 7) in both groups, we concluded that the difference in HA group was a consequence of the earlier achievement of bone remodeling.
## Clinical outcomes
The aim of monitoring secondary outcomes was to observe potential influences on prosthesis and patients' motivation to participate in activity. ESR and CRP were used to reflect postoperative physiological status, while LOS and HHS were used to indicate patients' motivation to participate in activity. The non-significance of day 1 ESR and CRP indicated that patients in both groups experienced a similar pathophysiology process after surgery. Early rehabilitation has been proven to have a positive impact on functional recovery, but the duration of this effect may only last for 6-8 weeks [bib_ref] The first 6 weeks of recovery after primary total hip arthroplasty with..., Klapwijk [/bib_ref] [bib_ref] Early progressive strength training to enhance recovery after fast-track total knee arthroplasty:..., Jakobsen [/bib_ref]. In the present study, only HSS on discharge day was different between HA and LA groups (60.73 AE 5.37 vs 51.18 AE 8.05); this was no longer significant at 2 months and 6 months, postoperatively, which was consistent with other studies. Consequently, high activity levels after THA had a limited impact on functional recovery. Considering that there were no differences in LOS, functional outcomes contributed little in terms of patients' willingness to stay. There was no occurrence of adverse events. Therefore, any change in the bone environment after surgery should be attributed to rehabilitation.
## Clinical significance
Despite activity being encouraged for patients, there is no standard criteria for the recommended quantity of activity (walking distance) per day after THA. A range of 500-3500 steps after THA was recorded during hospitalization (first week approximately) and gender difference was found. The activity level in THA or TKA patients could be increased to 4000-7000 steps per day at 6 weeks after surgery [bib_ref] Feedback from activity trackers improves daily step count after knee and hip..., Van Der [/bib_ref]. In the present study, activity level was set as high and low postoperatively in two groups, with a range of set walking distances as the sole variable, with changes analyzed based on X-rays and BMD. Consequently, as the RR showed for the primary outcomes, although the integral mechanism of bone remodeling around the prosthesis was unclear, according to the present study, activity did play a role in changing the progress of bone remodeling. Therefore, intense rehabilitation (high activity level) after primary THA should be considered as a stimulus factor enhancing the process of periprosthetic bone remodeling.
# Limitations
As an initial exploratory trial to investigate the connection between rehabilitation and the periprosthetic environment, some limitations were inevitable. First, defining whether a daily walking distance was high or low was difficult. A contrary conclusion is possible in future studies if the variable is changed between groups. Thus, plenty of work is needed to find a critical value for producing earlier stress shielding to provide a guideline for postoperative home rehabilitation after primary THA. Furthermore, even if the conclusion was subsequently confirmed, whether an enhanced bone remodeling procedure would lead to related complications, such as periprosthetic fractures, remains unknown. Ultimately, overestimation of the influence of activity on the periprosthetic environment was possible prior to this trial, which could have led to an insufficient sample size. Therefore, a multicentric, consecutive, long-term follow-up study on this topic would be worthwhile.
# Conclusion
High activity levels during early rehabilitation after primary THA accelerate the process of bone remodeling and aggravate stress shielding, with no significant benefits for functional recovery.
[fig] Figure 1: The stem used in this study: An Echo Bi-Metric RPP stem, which was a collarless 3 bi-planer tapered stem made of titanium alloy. The neck angle of the stem was 135 . The proximal part was porous-coated with a plasma-sprayed high activity (HA) layer, and the distal part was polished and bullet-shaped. [/fig]
[fig] Figure 3 X: -ray of three patients with osteonecrosis of the femoral head (ONFH). Femoral collapse and acetabular arthritis could be found on preoperative X-ray (A1, B1, and C1).Stress shielding levels of 0, 1, and 2 were apparent on X-ray 6 months postoperatively (A2, B2, and C2). [/fig]
[table] TABLE 1: Demographics and clinical factors: Age, BMI, duration of surgery, intra-operative bleeding, preoperative ESR, preoperative CRP, and preoperative Harris hip score as mean AE standard deviation [/table]
[table] TABLE 2: Completion of daily walking distance (m) in each group: Walking distance was recorded daily using a pedometer and followed up by phone interview [/table]
[table] TABLE 3: Primary outcomes: Incidence and degree of stress shielding at 6 months postoperatively [/table]
[table] TABLE 7: Secondary outcomes: Day 1 means the first postoperative day * Statistically significant. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate. [/table]
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Night-time sedating H1-antihistamine increases daytime somnolence but not treatment efficacy in chronic spontaneous urticaria: a randomized controlled trial
## What does this study add?
- This study compared 5-day treatment of CSU with the second-generation H 1 -antihistamine, levocetirizine (20 mg daily), with levocetirizine (15 mg daily) plus hydroxyzine (50 mg nightly).
- The treatments were equally effective in decreasing symptoms and night-time sleep disturbances and increasing quality of life, but the addition of night-time hydroxyzine significantly increased daytime somnolence.
- The belief that addition of a night-time sedating H 1 -antihistamine is of benefit in the treatment of CSU is unfounded.
Chronic spontaneous urticaria (CSU) is a relatively common condition, with 0Á5-1Á0% of the population suffering from it at any single time. [bib_ref] Unmet clinical needs in chronic spontaneous urticaria. A GA 2 LEN task..., Maurer [/bib_ref] Of the symptoms of this condition, pruritus is the most bothersome, particularly at night when it causes sleep disturbances. [bib_ref] Unmet clinical needs in chronic spontaneous urticaria. A GA 2 LEN task..., Maurer [/bib_ref] [bib_ref] The impact of chronic urticaria on the quality of life, O'donnell [/bib_ref] These disturbances in sleep lead to chronic fatigue, with a direct impact on quality of life (QoL) and physical and emotional well-being, which may be assessed for chronic urticaria using specifically designed questionnaires such as the Chronic Urticaria Quality of Life Questionnaire (CU-Q 2 oL). [bib_ref] A new tool to evaluate the impact of chronic urticaria on quality..., Baiardini [/bib_ref] In addition, it has been reported that pruritic skin diseases impair workplace productivity, classroom productivity and daily activity by 39%, 45% and 42%, respectively. [bib_ref] Effects of nonsedative antihistamines on productivity of patients with pruritic skin diseases, Murota [/bib_ref] Because chronic urticaria has such a profound impact on QoL, effective treatment is required. The European Academy of Allergology and Clinical Immunology/Global Allergy and Asthma European Network/European Dermatology Forum/ World Allergy Organization guideline for management of urticaria 5 recommends second-generation 'nonsedating' H 1 -antihistamines as first-line treatment. It also states that if standard dosing is not effective, increasing the dosage up to fourfold is recommended. This recommendation is reiterated in other, newer international guidelines. [bib_ref] Consensus statement on the management of urticaria, Godse [/bib_ref] [bib_ref] Management of chronic urticaria in Asia: 2010 AADV consensus guidelines, Chow [/bib_ref] These guidelines also recommend against the use of older, sedating first-generation H 1 -antihistamines in patients with urticaria unless there is a special indication. [bib_ref] EAACI/GA 2 LEN/ EDF/WAO guideline: management of urticaria, Zuberbier [/bib_ref] [bib_ref] Consensus statement on the management of urticaria, Godse [/bib_ref] [bib_ref] Management of chronic urticaria in Asia: 2010 AADV consensus guidelines, Chow [/bib_ref] This is because first-generation H 1 -antihistamines have pronounced unwanted effects, including anticholinergic effects and sedative actions on the central nervous system. At night, first-generation H 1antihistamines increase the latency to the onset of rapid eye movement (REM) sleep and reduce the duration of REM sleep. Furthermore, residual effects, or hangover, are still present the next morning. Such effects include impairment in divided attention, vigilance, working memory and sensory-motor performance. [bib_ref] Risk of first-generation H 1 -antihistamines: a GA 2 LEN position paper, Church [/bib_ref] For commonly used drugs, the incidence of subjectively reported somnolence has been documented to vary from 40% with chlorpheniramine or brompheniramine to 80% with hydroxyzine. [bib_ref] loratadine, and placebo in allergic rhinitis: a placebo-controlled comparative clinical trial, Druce [/bib_ref] Disturbingly, lack of subjective drowsiness does not mean that an individual is able to drive a vehicle without impairment because subjective somnolence and impairment of the ability to perform tasks are not necessarily correlated. [bib_ref] Individual differences in central nervous system response to antihistamines (H 1 -receptor..., Simons [/bib_ref] Despite the potential of first-generation H 1 -antihistamines to cause unwanted effects, many physicians believe that the most effective treatment for chronic urticaria is a nonsedating second-generation H 1 -antihistamine in the morning and a sedating first-generation H 1 -antihistamine, usually hydroxyzine or chlorpheniramine, at night to reduce night-time itch and enhance sleep. The rationale for this is that the sedative component makes people sleep better in spite of the pruritus. This is supported by some guidelines, particularly older ones. [bib_ref] British Association of Dermatologists Therapy Group and Audit Subcommittee. Guidelines for evaluation..., Grattan [/bib_ref] [bib_ref] BSACI guidelines for the management of chronic urticaria and angio-oedema, Powell [/bib_ref] But is this belief well founded? To date, there are no backto-back comparisons of therapy of CSU with a nonsedating H 1 -antihistamine during the day plus a sedating first-generation sedating H 1 -antihistamine at night vs. monotherapy with a nonsedating H 1 -antihistamine. Consequently, we have compared the effectiveness of therapy and the prevalence of unwanted effects when treating patients with severe CSU with levocetirizine 15 mg daily plus hydroxyzine 50 mg at night (levocetirizine plus hydroxyzine) vs. levocetirizine 20 mg daily (levocetirizine monotherapy).
# Patients and methods
This was a prospective, randomized, double-blind, cross-over study in patients with CSU in whom the efficacy and adverse effects of treatment with levocetirizine 20 mg daily were compared with those of levocetirizine 15 mg daily plus hydroxyzine 50 mg at night before sleep. In total, 25 patients with a minimum of 6 weeks' documented history of chronic urticaria treated with systemic steroids were recruited from the Clinic of Allergy and Asthma in Sofia. One woman withdrew from the study for personal reasons during the in-hospital assessment period. The demographics of the 24 patients who completed the study are shown in [fig_ref] Table 1: Demographic characteristics of patients [/fig_ref]. The group size was estimated from a previous study of ours [bib_ref] The effectiveness of levocetirizine and desloratadine in up to 4 times conventional..., Staevska [/bib_ref] using a power of 80% (t-test) with a two-sided significance level of 5% and a medium effect of 1Á2 SD.
The study was approved by the ethics committee of the Medical University in Sofia (approval no. 3443/18.10.10), and was conducted in accordance with the general principles of Good Clinical Practice and the Declaration of Helsinki as amended in Edinburgh in 2000; its clinicaltrials.gov identifier number is NCT 01250652. Recruitment began in December 2010 and the study was completed in December 2012. All participants gave signed informed consent at the beginning of the study.
## Patient selection
At the screening visit, information about concomitant disease and previous medication use was collected, and the patients were subjected to a general physical examination, laboratory blood analyses and an electrocardiogram. The primary inclusion criterion was a minimum of 6 weeks' documented history of chronic spontaneous urticaria, with or without concomitant inducible urticaria or angio-oedema, treated with systemic corticosteroids. The exclusion criteria were a documented or suspected history of allergic disease; any acute or chronic disease; symptoms of a clinically significant illness, especially liver or kidney disease; a history of hypersensitivity to the study drug(s) or formulation ingredients; epilepsy or other seizure conditions; hereditary galactose intolerance, lactase deficiency or glucose-galactose malabsorption; drug abuse or excessive use of alcohol or tobacco. Pregnant or nursing women were also excluded. Oral H 1 -and H 2 -antihistamines, antidepressants, antipsychotics, corticosteroids, aluminium-and magnesium-containing antacids, ketoconazole and erythromycin, as well as topically applied H 1 antihistamines, corticosteroids or mast-cell stabilizers, were forbidden for 2 weeks prior to testing.
## Outcome measures
## Quality of life
Patients completed a self-administered urticaria-specific QoL questionnaire (CU-Q 2 oL). The original Italian CU-Q 2 oL, supplied to us by Professor Canonica (University of Genoa, Italy), was translated into Bulgarian, back-translated into Italian and validated by administration to patients with urticaria of differing severity.
The questionnaire consisted of 23 questions structured into three areas: symptoms (four questions), activities (six questions) and social aspects (13 questions). [bib_ref] A new tool to evaluate the impact of chronic urticaria on quality..., Baiardini [/bib_ref] Each question had five possible grades, from 0 = 'none' to 4 = 'maximal impact'. Global summary scores were computed (maximum score = 92 indicating worst possible impact on QoL), reflecting the overall impact of the disease on the health-related QoL of patients. While the accepted recall period for CU-Q 2 oL in clinical practice is 2 weeks, a period of only 5 days was used in this study.
## Urticaria activity score
The Urticaria Activity Score (UAS) was calculated using a standard operating procedure as recommended by the 2009 guidelines on the diagnosis of urticaria. [bib_ref] EAACI/GA 2 LEN/ EDF/WAO guideline: definition, classification and diagnosis of urticaria, Zuberbier [/bib_ref] The investigators, who were blinded to the treatment groups, calculated weal scores as follows: 0, none; 1, mild (< 20 weals); 2, moderate (20-50 weals) or 3, intense (> 50 weals or large confluent areas of weals). The severity of pruritus was recorded as 0, absent; 1, mild (present but not annoying or troublesome); 2, moderate (troublesome but does not interfere with normal daily activity or sleep) or 3, intense (severe pruritus, which is sufficiently troublesome to interfere with normal daily activity or sleep).
## Night-time sleep disturbance
Sleep disturbance during the previous night was judged by patients marking a 100-mm visual analogue scale (VAS), marked 'none' on one end and 'worst possible' on the other. The distance in millimetres between the 'none' end and the patient's mark was used further in the analyses.
## Daytime sedation
Daytime somnolence was also recorded by patients in the morning using a similar VAS scale to that above. All patient assessments were made by patients between 07Á00 h and 10Á00 h. CU-Q 2 oL and UAS scores referred to the previous 24 h, sleep disturbance to the previous night and daytime somnolence to the present time.
## Study design
Day 0
After signing informed consent, the patients' demographics were documented and they were subjected to thorough clinical evaluation by the physician in charge. Baseline assessments of CU-Q 2 oL (5 days), UAS, night-time sleep disturbance and daytime somnolence were also made.
## Days 1-6
Patients were randomized alternately into one or other of the two treatment groups depending on the sequence of their recruitment. Randomization was made pairwise at a specialized website, http://www.randomizer.org/, to ensure a balanced number of cases in the two treatment arms. Patients underwent an in-hospital assessment of the effectiveness and tolerability of levocetirizine 10 and 20 mg vs. hydroxyzine 100 and 200 mg. This was done in a double-blind fashion on alternate-day regimens [fig_ref] Fig 1: Study design [/fig_ref]. Medication was given morning and evening in opaque gelatine capsules that were prepared by a technician who was not aware of the clinical work.
Patients swallowed the capsules in front of the caregiver with 50 mL plain water. Physician assessments were carried out about noon the same day, and UAS was taken on the mornings after.
## Days 7-16
Day 7 marked the beginning of the main outpatient part of the study, which involved two 5-day periods comparing treatment with levocetirizine plus hydroxyzine (levocetirizine 15 mg daily, plus hydroxyzine 50 mg at night before sleep) and levocetirizine monotherapy (levocetirizine 20 mg daily). Patients started with their initial medication on day 7 and were crossed over to the opposite medication on day 12. There was no washout period between treatments. Patients were instructed to take two different opaque gelatine capsules each day: a grey one containing two 5-mg levocetirizine tablets in the morning and a blue one containing either two 5mg levocetirizine tablets, or one 5-mg levocetirizine tablet and two 25-mg hydroxyzine tablets, in the evening before going to sleep. All capsules were placed in coded boxes in accordance with the study period and the treatment arm. The coding was generated by a technical assistant who did not have contact with study participants. Assessments of CU-Q 2 oL (5 day), UAS, sleep disturbance and daytime somnolence were made on day 12 and day 16.
## Statistics
Analysis of results by the Kolmogorov-Smirnov test for normality showed that elements of all outcome measures were not distributed normally. Consequently, all results are expressed as medians with 25% and 75% limits, and differences between groups were tested using Wilcoxon's nonparametric test for paired data. The minimum level of statistical significance was accepted to be P < 0Á05.
# Results
## Prestudy assessment
In the in-hospital assessment of study drug effectiveness and tolerability, the UAS was reduced from a baseline of 5Á5 (3Á75-6Á00; median with 25% and 75% limits) to 3Á5 (1Á75-5Á00) or 2 (0Á75-5Á25) following 1 day's dosage with levocetirizine 10 mg or hydroxyzine 100 mg, respectively. Following 2 days of therapy with levocetirizine 20 mg or hydroxyzine 200 mg, the UAS was 2 (0Á75-4Á00) or 2 (0Á75-4Á00), respectively. No patient reported adverse responses that prevented their taking part in the subsequent part of the study. During this period one woman withdrew from the study for personal reasons. She was the only dropout in the whole study.
Main study of levocetirizine plus hydroxyzine vs. levocetirizine monotherapy
## Quality of life
The effect of 5 days' treatment of patients with levocetirizine 15 mg daily plus hydroxyzine 50 mg at night before sleep (levocetirizine plus hydroxyzine) vs. levocetirizine alone 20 mg daily (levocetirizine monotherapy) is shown in [fig_ref] Fig 2: Chronic Urticaria Quality of Life Questionnaire [/fig_ref]. The median CU-Q 2 oL scores (25-75% limits) were reduced from a baseline of 41 (28Á25-52Á00) to 17Á5 (9Á75-27Á75) (P < 0Á001) and 13Á5 (6Á75-33Á25) (P < 0Á001) with levocetirizine plus hydroxyzine, and levocetirizine monotherapy, respectively. There was no significant difference between the two treatments (P = 0Á426).
## Urticaria activity score
The individual UASs are shown in [fig_ref] Fig 3: Fig 3 [/fig_ref]. The median scores were reduced from a baseline of 5Á5 (3Á75-6Á00) to 2 (0-3) (P = 0Á002) or 1 (0-4) (P < 0Á001) following 5 days' treatment with levocetirizine plus hydroxyzine or levocetirizine monotherapy, respectively. There was no significant difference between the two treatments (P = 0Á182). As the UAS combines objective and subjective elements, i.e. weals and pruritus, these were also analysed separately. Weal Urticaria Activity Scores at the start of the study (baseline) and after 5 days of treatment with levocetirizine 15 mg daily + hydroxyzine 50 mg at night (Levo + Hydroxy) or levocetirizine 20 mg daily alone (Levo Monotherapy). Horizontal bars indicate median values. Significant differences between treatments were calculated by Wilcoxon's nonparametric test for paired data. NS, not significant. scores were significantly reduced by levocetirizine plus hydroxyzine (P = 0Á005) and by levocetirizine monotherapy (P = 0Á003). There was no significant difference between treatments (P = 0Á314). Pruritus scores were also significantly reduced by levocetirizine plus hydroxyzine (P = 0Á001) and by levocetirizine monotherapy (P < 0Á001). Again, there was no significant difference between treatments (P = 0Á141).
## Night-time sleep disturbance
There were highly significant reductions in night-time sleep disturbances [fig_ref] Fig 4: Visual analogue scale [/fig_ref] , with the median baseline VAS of 71 (21Á75-78Á5) being reduced to 10 (0-25Á25) (P = 0Á001) by levocetirizine plus hydroxyzine and 17 (0-24Á75) (P = 0Á002) by levocetirizine monotherapy. There was no significant difference between treatments (P = 0Á868).
## Daytime sedation
Daytime sedation [fig_ref] Fig 4: Visual analogue scale [/fig_ref] was significantly (P = 0Á007) reduced by levocetirizine monotherapy from a median baseline VAS of 14 to 0 (0-11). In contrast, the median sedation VAS following levocetirizine plus hydroxyzine of 5 (0-23Á5) was not significantly different from baseline (P = 0Á218). Furthermore, patients treated with levocetirizine monotherapy were significantly less sedated than those receiving levocetirizine plus hydroxyzine (P = 0Á030).
# Discussion
The primary result of this study was that both levocetirizine monotherapy and therapy with levocetirizine plus hydroxyzine were similarly effective in reducing urticarial symptoms, reducing night-time sleep disturbances and improving QoL. In contrast, daytime sedation was significantly less with levocetirizine monotherapy in comparison with levocetirizine plus hydroxyzine.
The effectiveness of levocetirizine in relieving the symptoms of CSU and improving QoL confirms previous findings. [bib_ref] The effectiveness of levocetirizine and desloratadine in up to 4 times conventional..., Staevska [/bib_ref] [bib_ref] Cost effectiveness of levocetirizine in chronic idiopathic urticaria: a pooled analysis of..., Kapp [/bib_ref] [bib_ref] Levocetirizine: from scientific evidence to a potent modern-day treatment of today's allergic..., Klimek [/bib_ref] [bib_ref] H 1 -antihistamines and urticaria: how can we predict the best drug..., Church [/bib_ref] It is of particular relevance in this study that the subjective element of the UAS, the severity of pruritus, was similarly reduced by both treatment regimens, suggesting that the central effects of hydroxyzine do not contribute to the overall antipruritic effect, which is agreement with the hypothesis that histamine is involved primarily in the peripheral genesis of itch. [bib_ref] Basic mechanisms of itch, Potenzieri [/bib_ref] Furthermore, both treatment regimens having a similar effect in reducing night-time sleep disturbances again indicates that it is the peripheral antipruritic effect of the nonsedating second-generation H 1 -antihistamine that is of prime importance rather than any central sedative effects.
What is very clear from this study is that patients receiving levocetirizine monotherapy were significantly less sedated during the day than they were before treatment at the beginning of the study, confirming the findings of Staevska et al. [bib_ref] The effectiveness of levocetirizine and desloratadine in up to 4 times conventional..., Staevska [/bib_ref] In contrast, when treated with levocetirizine plus hydroxyzine, patients experienced a similar level of sedation to those who were not treated. Furthermore, patients receiving levocetirizine monotherapy experienced less daytime sedation than those receiving levocetirizine plus hydroxyzine. This strongly suggests that the detrimental sedative effects of hydroxyzine were due to its prolonged central nervous system effects. With a terminal half life of 20-25 h, [bib_ref] The pharmacokinetics and antihistaminic of the H1 receptor antagonist hydroxyzine, Simons [/bib_ref] it is not surprising that hydroxyzine has hangover effects into the next day.
We believe that this study has two weaknesses. Firstly, we did not perform objective assessments of alertness or productivity. Even so, our study would support the conclusion of Murota et al. [bib_ref] Impact of sedative and nonsedative antihistamines on the impaired productivity and quality..., Murota [/bib_ref] that pruritic diseases negatively impact on daily activity and impair productivity, and that this is improved in patients taking second-generation nonsedative, but not firstgeneration sedative, H 1 -antihistamines, and that the improve- ments correlated with the alleviation of itch and improved QoL. The second weakness is that the length of the study, 10 days, is less than that of many similar studies in urticaria. This was because the study was performed at a tertiary referral clinic with patients from all over Bulgaria and we wanted to ensure a sustainable follow-up of patients, particularly given compliance issues.
In conclusion, the results of this study do not support the widely held belief that the most effective treatment for chronic urticaria is a nonsedating second-generation H 1 -antihistamine in the morning and a sedating first-generation H 1 -antihistamine, usually hydroxyzine, at night to enhance sleep. In view of the potential detrimental effects of residual daytime sedation on school performance and study, impaired productivity at work and, possibly more importantly, driving motor vehicles, [bib_ref] Risk of first-generation H 1 -antihistamines: a GA 2 LEN position paper, Church [/bib_ref] it is clear that it is better not to offer a sedating antihistamine at night for the treatment of CSU.
[fig] Fig 1: Study design. The study consisted of two separate double-blind phases. Phase one was an in-hospital assessment of the effectiveness and tolerability of levocetirizine and hydroxyzine at two doses. Phase two was a comparison of levocetirizine monotherapy vs. levocetirizine plus hydroxyzine at night. Complete data were collected at baseline and at the end of each study period. Broken lines indicate collection of urticaria activity scores during the assessment period. [/fig]
[fig] Fig 2: Chronic Urticaria Quality of Life Questionnaire (CU-Q 2 oL) scores at the start of the study (baseline) and after 5 days of treatment with levocetirizine 15 mg daily + hydroxyzine 50 mg at night (Levo + Hydroxy) or levocetirizine 20 mg daily alone (Levo Monotherapy). The maximum possible score for CU-Q 2 o Lis 92. Horizontal bars indicate median values. Significance of differences between treatments was calculated by Wilcoxon's nonparametric test for paired data. NS, not significant. [/fig]
[fig] Fig 3: Fig 3. Urticaria Activity Scores at the start of the study (baseline) and after 5 days of treatment with levocetirizine 15 mg daily + hydroxyzine 50 mg at night (Levo + Hydroxy) or levocetirizine 20 mg daily alone (Levo Monotherapy). Horizontal bars indicate median values. Significant differences between treatments were calculated by Wilcoxon's nonparametric test for paired data. NS, not significant. [/fig]
[fig] Fig 4: Visual analogue scale (VAS) scores for (a) night-time sleep disturbance and (b) daytime sedation at the start of the study (baseline) and after 5 days of treatment with levocetirizine 15 mg daily + hydroxyzine 50 mg at night (Levo + Hydroxy) or levocetirizine daily 20 mg alone (Levo Monotherapy). Horizontal bars indicate median values. Significant differences between treatments were calculated by Wilcoxon's nonparametric test for paired data. NS, not significant. [/fig]
[table] Table 1: Demographic characteristics of patients [/table]
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VOSviewer and Bibliometrix
# Introduction
As the materials on a given topic increases, researchers and librarians find it more difficult to grasp the big picture of a field. Outlining the state of the art, the relationships, opportunities and main players of a given community of practitioners and scholars calls for a map that connects research information, venues, themes, as well as relationships among authors and institutions. The understanding that may emerge from such a big picture is necessary to formulate research, publication, institutional or career strategies. Authors such as Beerepoot et al [bib_ref] Competition in Science: Links Between Publication Pressure, Grant Pressure and the Academic..., Waaijer [/bib_ref] and Waaijer et al [bib_ref] Competition in online job marketplaces: towards a global labour market for outsourcing..., Beerepoot [/bib_ref] argue that this need has increased with the reduction of the psychological distance of the world, a phenomenon that accelerated during the Covid-19 pandemic. By furthering remote work, many more connections became possible on a worldwide scale. This has led to a change in expectations, that increasingly takes international collaboration and multi-country data for granted, enlarging the requirements of what is to be considered "doing good research".
## Domain visualization
Domain visualization based on citation analysis represents the relationships between sources in a two-dimensional space. This diagram offers a map of the dynamics of the literature and the paths that connect it. This network comprises nodes and edges and admit to customization in order to support a given analysis. Nodes may represent individual pieces of publication, journals, researchers, institutions or keywords. Edges represent the existence or type of relationship between pairs of nodes [bib_ref] bibliometrix: An R-tool for comprehensive science mapping analysis, Aria [/bib_ref]. This allows the expression of any of a number of possible big pictures of the state of field. It may serve to enlarge the understanding of an individual researcher about the shape and directions of a given subject, it can support the definition of the breadth and reach of the plan for a Systematic Review and avoid biases during the process of source selection. This article will review two of the most popular and promising domain visualization software packages available: VOSviewer and Bibliometrix.
## Vosviewer
VOSviewer was released in 2010 by Nees Jan van Eck and Ludo Waltman (Leiden University) [bib_ref] Software survey: VOSviewer, a computer program for bibliometric mapping, Van Eck [/bib_ref]. VOSviewer is a software tool for creating and exploring maps based on network data. While intended primarily for analyzing academic records, it can be used on any type of network data (social networks, e.g.). VOSviewer explores co-authorship, co-occurrence, citation, bibliographic coupling, and co-citation links in one of three possible representations: network, overlay, or density visualization.
## Bibliometrix
Bibliometrix was launched in 2017 by Dr. Massimo Aria (University of Naples Federico II) and Dr. Corrado Cuccurullo (University of Campania Luigi Vanvitelli) [bib_ref] bibliometrix: An R-tool for comprehensive science mapping analysis, Aria [/bib_ref]. It is a package that must be used within the R software environment. R is both a programming language and a free environment for statistical computing, supported by the R Core Team and the R Foundation for Statistical Computing. Bibliometrix is a comprehensive mapping analysis tool that supports three phases of the bibliometric analysis process: (i) data import and conversion to R format; (ii) bibliometric analysis of a dataset and (iii) the construction of matrices. Matrices are customizable and allow mapping in great resolution, being input data for performing network analysis, multiple correspondence analysis, and many other data reduction techniques including domain visualization.
## Main purpose
For the purposes of this article, the main concerns of domain visualization correspond to the representation of the attributes and connections of:
[formula] - Authors - Institutions or nations - Publications - Sources - Relationships - Keywords [/formula]
## Software comparison
VOSviewer and Bibliometrix can be compared succinctly in terms of their installation and usage requirements and of their functionalities [fig_ref] Table 1: Comparison of VOSviewer and Bibliometrix [/fig_ref]. VOSviewer is a button-and-window-interface software, confined to its preprogrammed functions and possibilities.
Bibliometrix is a coding terminal that requires knowledge of the R programming language and allows complete customization.
It is important to note that Bibliometrix's developers also created Biblioshiny, a user interface that requires no coding knowledge to simplify usage. However, Biblioshiny does not allow file import of multiple files for the same analysis, imposing the task of merging different files that must be from the same database. This excludes it from consideration in this article.
## Features
## Software requirements
Both Bibliometrix and VOSviewer can be used in the main operating systems (Windows, Linux, and MacOS). VOSviewer was developed in Java, which leads to platform portability. Bibliometrix is a package in the R programming language, which leads to operating system flexibility, but requires an IDE for R, like R Studio.
## Need for programming knowledge
VOSviewer does not require any programming knowledge.
Only Bibliometrix requires the user to have knowledge of programming in R language.
## Ease of use
VOSviewer is immediately accessible with a standard graphical interface.
Bibliometrix requires a customized interface to be programmed, if desired.
## Loading multiple files
The main reference databases limit the export of metadata in a single file. Web of Science allows users to export the metadata of 500 references; Scopus allows 2000. Users have to create more than one file and import them one at a time whenever the number of desired references exceeds the limit.
Both VOSviewer and Bibliometrix allow loading metadata from multiple files.
## Data analysis from multiple sources
VOSviewer accepts files from different databases, but only one source can be used at a time. Depending on user's requirement, this may be a significant issue, because any one database does not hold the whole literature of any given field: users must combine metadata from different sources.
Only Bibliometrix allows users to analyze data from multiple sources concurrently.
## Flexibility and responsiveness to the user
Flexibility is linked to this diversity of possible manipulation of parameters (colors, lines, labels). Responsiveness to the user is the possibility of seeing changes as they are made without the need to run the program again.
VOSviewer is both more flexible and more responsive than Bibliometrix.
## Api support
Both VOSviewer and Bibliometrix allow using an API to automate the communication with the indexing databases (Web of Science, PubMed etc.), keeping the database updated automatically.
## Analysis that only one of the solutions delivers
## Vosviewer
## Spreadsheet export
This functionality allows the data used to create bibliometric maps to be exported to spreadsheets, easing the usual clutter of domain visualization to identify relationships and nodes that might otherwise be missed.
## Creation of thesaurus
Due to the diversity of keywords in the documents, it is common to have to deal with the occurrence of different words that have the same meaning, as simply as the use of singular and plural in keywords. A thesaurus makes it possible to deal with this problem, allowing references with keywords that are synonymous to be addressed jointly.
## Temporal data visualization
The visualization of temporal data can color code network nodes to the year of publication allowing the identification of trajectories and trends in a given field [fig_ref] Figure 1: Overlay map of keyword co-occurrence over time for papers published at the... [/fig_ref] , for keywords, for example).
## Bibliometrix
## H-index, g-index, m-index
The impact indexes of sources and authors (H-index, G-index, M-index)
## Production over time
A map of authors' production over the time.
## Total numbers
The total of authors, sources and publications from the database used.
# Conclusion
VOSviewer and Bibliometrix are fit for purpose in providing domain visualization, yet each offers a distinctive set of capabilities and requirements.
VOSviewer is the more userfriendly offering simplicity, flexibility and responsiveness to user demands as well as greater graphic quality at the price of bounding alternatives to its preprogrammed functions and requiring repeating analysis due to its inability to combine data from different sources.
Bibliometrix is the more robust and versatile, being capable of greater customization by users and of (a) performing analyses using files from multiple databases, (b) accepting files from the Cochrane Database of Systematic Reviews (CDSR) and from RISmed, (c) offering exclusive analyzes at the price of a steeper learning curve that includes programming.
It can be argued that each fills a particular niche. VOSviewer may be all that a given user or set of users need. However, as a particular capability is required, it may turn out to be a first step before deciding on the extra effort that Bibliometrix will demand.
[fig] Figure 1: Overlay map of keyword co-occurrence over time for papers published at the Journal of the Medical Library Association (JMLA). Note: Metadata includes all 1826 articles in the Journal of the Medical Library Association (JMA) (references), retrieved from Web of Science [September 7th, 2021]. [/fig]
[table] Table 1: Comparison of VOSviewer and Bibliometrix. [/table]
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Monitored anesthesia care in and outside the operating room
The American Society of Anesthesiologists (ASA) defines monitored anesthesia care (MAC) as a planned procedure during which the patient undergoes local anesthesia together with sedation, and analgesia is provided by an anesthesiologist[1]. A responsible anesthesiologist with a broad understanding of drug application performs the MAC since the degree of sedation and analgesia is dependent on the characteristics of the procedures and patients. Sedation during MAC may be considered safer than that of general anesthesia in that less drugs are typically administered. However, the application of sedatives and analgesics should be titrated to avoid central respiratory depression and airway obstruction, since the airway of the patient is not secured during MAC. Preoperative evaluation, perioperative management, monitoring, and postoperative recovery care of MAC is similar to those of general or regional anesthesia. Moreover, the attending anesthesiologist should be aware of the possibility of airway obstruction, desaturation, or even aspiration due to the possibility of deep sedation after infusion of a combination of two or more drugs.The use of MAC as the technique of choice for a variety of diagnostic and therapeutic procedures in and outside of the operating room is increasing due to rapid postoperative recovery with relatively small amounts of sedatives and analgesics compared to general anesthesia. Additionally, investigations on the effect of MAC on postoperative outcomes have been increasing. Therefore, the responsibility and roles of the anesthesiologistReview ArticleMonitored anesthesia care (MAC) is an anesthesia technique combining local anesthesia with parenteral drugs for sedation and analgesia. The use of MAC is increasing for a variety of diagnostic and therapeutic procedures in and outside of the operating room due to the rapid postoperative recovery with the use of relatively small amounts of sedatives and analgesics compared to general anesthesia. The purposes of MAC are providing patients with safe sedation, comfort, pain control and satisfaction. Preoperative evaluation for patients with MAC is similar to those of general or regional anesthesia in that patients should be comprehensively assessed. Additionally, patient cooperation with comprehension of the procedure is an essential component during MAC. In addition to local anesthesia by operators or anesthesiologists, systemic sedatives and analgesics are administered to provide patients with comfort during procedures performed with MAC. The discretion and judgment of an experienced anesthesiologist are required for the safety and efficacy profiles because the airway of the patients is not secured. The infusion of sedatives and analgesics should be individualized during MAC. Many procedures in and outside of the operating room, including eye surgery, otolaryngologic surgery, cardiovascular procedures, pain procedures, and endoscopy are performed with MAC to increase patient and operator satisfaction.
VOL. 69, Monitored anesthesia care should be considered in terms of the safety and effectiveness of MAC. In this review, MAC in and outside of the operating room was described in terms of preoperative patient evaluation, monitoring, and intraoperative care with sedatives and analgesics during a variety of procedures and potentially serious complications.
## The definition and purpose
According to the ASA, a MAC is a planned procedure during which the patient undergoes local anesthesia together with sedation and analgesia [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. During MAC, systemic sedation and analgesia is provided by an anesthesiologist, and local anesthesia, including local infiltration or field block, is mainly performed by a surgeon. MAC should be distinguished from simple sedation/analgesia in that sedation/analgesia is performed by a nonanesthesiologist, whereas sedation and analgesia during MAC is provided by an anesthesia care team. Therefore, preoperative evaluation, intraoperative monitoring, and postoperative care for patients with MAC are the same as those with general or regional anesthesia.
The purposes of sedation during a MAC are to provide patients with safe sedation, comfort, pain control, and satisfaction [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. Safe sedation is achieved due to the wide spectrum of knowledge on sedatives and analgesics available to the attending anesthesiologist; the degree of sedation should be individualized according to the medical condition of the patients. In addition, each diagnostic and therapeutic procedure requires a different degree of sedation and analgesia, and the amount of drug should be carefully titrated. The administration of the proper dosage of anxiolytics as a premedication can provide patient amnesia and comfort without compromising cardiovascular function. Adequate control of pain with analgesia during and after the procedure can contribute to satisfactory MAC, allowing patients to be discharged as quickly as possible [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref].
## Preoperative evaluation
Preoperative evaluation of patients with MAC is not different from that of patients given general or regional anesthesia; in all cases, patients should be comprehensively assessed. In addition, patient cooperation is essential during MAC [bib_ref] Remifentanil as an analgesic adjunct in local/regional anesthesia and in monitored anesthesia..., Servin [/bib_ref]. Patients undergoing MAC are able to respond to orders; therefore, whether they are ready for the procedure should be evaluated. If the patients cannot cooperate, general anesthesia may be a better alternative. On the other hand, there are no specific exclusion criteria for a MAC, and this anesthetic technique can be performed even for the elderly or high perioperative risk patients.
Communication between patients and the anesthesia care team can be used as a tool for monitoring sedation levels and to offer verbal assurance; it can also improve patient cooperation [bib_ref] The changing role of monitored anesthesia care in the ambulatory setting, Sá Rêgo [/bib_ref]. MAC may be chosen due to patient cardiovascular and respiratory instability, but persistent cough or movement during microscopic procedures may limit the use of MAC. Therefore, preoperative evaluation of whether patients can cooperate without movement during the procedure should be determined.
Preoperative visits are helpful for the patient-anesthesiologist relationship, in that patients can be informed about safe and comfortable analgesia, as well as provided with an explanation regarding the MAC procedure. Communication between patients and anesthesiologists is essential for evaluation of the level of consciousness during infusion of sedative and analgesics for MAC. The patient's physical status, including cardiovascular and respiratory capacity, may determine or affect their sensitivity to sedatives and analgesics. Assessment of co-morbidity, past history, drug reactions, and postoperative anesthetic complications are required during the preoperative visit. The number of ambulatory surgeries performed without allowing sufficient time for preoperative evaluation and patient informed consent has increased recently, although an adequate explanation of anesthesia can be given in preoperative patient clinics.
## Intraoperative monitoring
The ASA has established a basic level of patient monitoring during MAC. Intraoperative monitoring should be effective, applicable, noninvasive, and economical [bib_ref] The changing role of monitored anesthesia care in the ambulatory setting, Sá Rêgo [/bib_ref]. The presence of a qualified anesthesiologist is essential and patient oxygenation, ventilation, circulation, and temperature must be monitored continuously [bib_ref] The changing role of monitored anesthesia care in the ambulatory setting, Sá Rêgo [/bib_ref].
Prevention of respiratory depression through continuous respiratory monitoring is essential during the infusion of sedatives and analgesics for MAC. Pulse oximetry is useful for monitoring patient oxygenation, but its use is limited due to the delayed detection of hypoxemia during sedation. Real time detection of hypoxemia with capnography is required immediately, and precordial or esophageal stethoscopes can be used for continuous monitoring of ventilation and inspired oxygen [bib_ref] Capnography accurately detects apnea during monitored anesthesia care, Soto [/bib_ref]. In addition, the attending anesthesiologist should observe clinical signs continuously through detection of arterial pulses and observation of chest movement and the surgical field.
## Monitoring the level of consciousness
In addition to standard monitoring, evaluation of the sedation level is of extreme importance during MAC, in terms of the safety and efficacy of the sedatives [bib_ref] The changing role of monitored anesthesia care in the ambulatory setting, Sá Rêgo [/bib_ref]. For this purpose, clinical or electroencephalographic methods can be used.
For clinical evaluation, several scales pertaining to the sedation level during MAC have been used to reduce the subjectiv-
## Korean j anesthesiol
## Sohn and ryu
ity of anesthesiologist judgments. The Observer Assessment of Alertness/Sedation scale (OAA/S scale) is a well-established instrument for evaluating the level of consciousness. The OAA/ S scale was developed in 1990 to measure the level of consciousness in patients sedated with midazolam [bib_ref] Validity and reliability of the Observer's Assessment of Alertness/ Sedation Scale: study..., Chernik [/bib_ref] ; the scale is sensitive to the level of midazolam administered. A score of 3-4 on the OAA/S scale represents a moderate level of sedation-analgesia and a score of 1-2 represents unconsciousness [fig_ref] Table 1: Observer Assessment of Alertness/Sedation Scale [/fig_ref]. Therefore, a score of more than 3 on the OAAS/S scale is required for MAC. However, some degree of patient stimulation, such as calling, prodding, or shaking, is required during the operation to evaluate the patient's level of sedation with the OAAS/S scale, and these assessments may affect sedation status. In addition, the results of the evaluation may be dependent on the assessor and fail to account for the changes in sedation level that may occur between assessments.
The Ramsay Sedation Scale (RSS) was introduced in 1974 as a subjective tool to evaluate the level of consciousness during titration of sedative medications in the intensive care unit [bib_ref] Controlled sedation with alphaxalone-alphadolone, Ramsay [/bib_ref]. The RSS assesses the level of consciousness, agitation, and anxiety through observation of behavior, and response to voice, a loud auditory stimulus or a light glabellar tap .
Real time and continuous monitoring of the sedative level can be performed with electroencephalographic (EEG) methods such as the Bispectral Index (BIS). The BIS is an instrument used specifically for easy analysis of EEG variations, which correspond to different levels of sedation. Sedatives and analgesics specifically change the frequency and amplitude of EEG waves, which are analyzed and correlated with a numerical index by using the BIS [bib_ref] Update on bispectral index monitoring, Johansen [/bib_ref]. In a study with the BIS and propofol sedation, the BIS reliably assessed the sedation depth achieved with propofol; a decrease in the BIS was associated with the incidence of amnesia during the operation [bib_ref] Electroencephalographic bispectral index correlates with intraoperative recall and depth of propofol-induced sedation, Liu [/bib_ref].
## Patient satisfaction score (iowa satisfaction with anesthesia scale)
The Iowa Satisfaction with Anesthesia Scale (ISAS) is a tool for measuring patient satisfaction during anesthetic experiences including MAC [bib_ref] Systematic review of questionnaires measuring patient satisfaction in ambulatory anesthesia, Chanthong [/bib_ref]. The ISAS is a self-administered, written questionnaire on which patients respond to 11 items on a sixchoice, vertical answer column [bib_ref] Systematic review of questionnaires measuring patient satisfaction in ambulatory anesthesia, Chanthong [/bib_ref].
## Systemic sedatives and analgesics
In addition to the use of local anesthetic drugs, systemic sedatives and analgesics are required to provide patients with comfort during surgical procedures performed with MAC. A pharmacological approach is required since there is a synergistic effect between sedation and analgesia. Continuous infusion of rapid elimination drugs helps the anesthesiologist achieve target drug concentrations at the effect-site, as well as maintain safety.
Several infusion regimens, including bolus injection, continuous infusion, target controlled infusion (TCI), and patient controlled sedation (PCS) can be used during MAC [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. During continuous infusion, the velocity of the drug infusion can be determined by the attending anesthesiologist according to the patient's level of consciousness and clinical signs [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. TCI calculates the infusion velocity to obtain and maintain specific plasma level or effect site concentration targets, based on the patient's pharmacokinetic parameters [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. PCS allows patients to administer intravenous drugs and achieve a specific level of consciousness according to the requirement for more sedation and analgesia [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. PCS was programmed by the anesthesiologist with a bolus injection and a lock out time to avoid over-sedation since there is wide variability in drug effects among patients.
The judgment and discretion of an experienced anesthesiologist are required for safety because the airway of the patient is Monitored anesthesia care not secured; the infusion of sedatives and analgesics should be individualized during MAC. Monitoring devices are especially required for MAC outside of the operating room. According to previous reports, the severity of injury in claims after MAC was comparable to those pertaining to general anesthesia [bib_ref] Injury and liability associated with monitored anesthesia care: a closed claims analysis, Bhananker [/bib_ref]. Respiratory compromise resulting from over-sedation was the most common cause of injury, and 41% of those claims were related to death or permanent brain damage [bib_ref] Injury and liability associated with monitored anesthesia care: a closed claims analysis, Bhananker [/bib_ref]. Sedation related respiratory depression can be preventable with better monitoring, vigilance (of the attending anesthesiologist), and early resuscitation [bib_ref] Injury and liability associated with monitored anesthesia care: a closed claims analysis, Bhananker [/bib_ref]. Geriatric anesthesia is increasing and MAC is an attractive option for many procedures to minimize the physiological stress of elderly patients. However, elderly patients show reduced functional reserve, including loss of normal compensation against the stress of cardiovascular and respiratory compromise, and altered mental status [bib_ref] Monitored anaesthesia care in the elderly: guidelines and recommendations, Ekstein [/bib_ref]. In addition, geriatric patients show different and variable pharmacokinetic responses to sedatives and analgesics due to changes in body composition and functions. Therefore, the attending anesthesiologist should administer the drugs while monitoring the consciousness level and hemodynamics [bib_ref] Monitored anaesthesia care in the elderly: guidelines and recommendations, Ekstein [/bib_ref].
Common causes of complications during infusion of sedatives and analgesics for MAC include airway obstruction, hypoxia, and cardiovascular collapse. In addition to local anesthesia administered by surgeons, sedatives and analgesics are co-infused and their synergistic effect results in complications. Inhibition of the airway reflex after infusion of sedatives and analgesics may lead to respiratory compromise, upper airway obstruction, and aspiration. Careful and continuous infusion of sedatives instead of intermittent bolus injection may minimize the risk of respiratory depression. In addition, oxygen delivery [bib_ref] Supplying sub-100% oxygen gas mixtures during monitored anesthesia care: respiratory monitoring and..., Loeb [/bib_ref] , as well as monitoring through capnography or thoracic impedance [bib_ref] Capnography accurately detects apnea during monitored anesthesia care, Soto [/bib_ref] , are recommended to prevent and detect airway complications during drug administration.
## Sedatives
Patients typically suffer from discomfort and anxiety during the procedure and sedatives are used for amnesia and anxiety relief. Proper concentrations of sedatives are required since light sedation may cause patient anxiety while heavy sedation leads to airway obstruction and compromise. The level of sedation can be monitored through patient communication and hemodynamic variables. Patients are cooperative without airway compromise during proper sedation. Using PCS, the infusion rate of sedatives can be individualized according to the patient's needs. Ideal characteristics of sedatives during MAC are rapid onset and recovery, easy titration, and minimal respiratory and cardiovascular depression. In addition, each procedure requires a different level of sedation, which should be decided according to the type of surgery.
Benzodiazepines provide patient comfort and amnesia during the procedure; midazolam is the most commonly used benzodiazepine. Midazolam (starting dose: 0.03 mg/kg, infusion rate: 0.6-6.0 mg/kg/h) is typically co-injected with propofol [bib_ref] Monitored anesthesia care, Ghisi [/bib_ref]. It shows maximal CNS effects within 2-3 min; repeated or continuous injection within a relatively short period may lead to heavy sedation, which can be reversed with flumazenil.
Propofol remains the mainstay drug of MAC because of its favorable pharmacodynamic and pharmacokinetic profile. Compared with midazolam, cognitive function recovery is faster and the degree of postoperative sedation, dizziness, amnesia, and postoperative nausea and vomiting (PONV) are lower after propofol sedation [bib_ref] Clinical assessment of target-controlled infusion of propofol during monitored anesthesia care, Casati [/bib_ref]. However, propofol does not have an analgesic effect, and other opioid analgesics are often required during painful procedures [bib_ref] Clinical assessment of target-controlled infusion of propofol during monitored anesthesia care, Casati [/bib_ref].
Dexmedetomidine and clonidine are α2 agonists that inhibit endogenous catecholamine release in the locus ceruleus, which results in a sedative-analgesic effect without respiratory depression [bib_ref] Monitored anesthesia care with dexmedetomidine: a prospective, randomized, double-blind, multicenter trial, Candiotti [/bib_ref]. Dexmedetomidine is eight times more selective for the α2 receptor than clonidine. Due to its sedative properties, intravenous dexmedetomidine (1 µg/kg) relieves patient anxiety. In addition to its sedative effect, dexmedetomidine shows analgesic properties and reduces opioid requirements [bib_ref] Monitored anesthesia care with dexmedetomidine: a prospective, randomized, double-blind, multicenter trial, Candiotti [/bib_ref]. Since dexmedetomidine reduces the occurrence of respiratory depression, it is a useful sedative and analgesic during MAC. However, dexmedetomidine leads to hypotension and bradycardia due to the inhibition of catecholamine release [bib_ref] Clinical use of dexmedetomidine in monitored anesthesia care, Lee [/bib_ref]. Therefore, these adverse events should be considered for elderly patients with cardiovascular disease. The onset and offset of dexmedetomidine is slower than midazolam and the administration of dexmedetomidine to ambulatory patients should be individualized considering delayed recovery.
## Analgesics
Analgesics are used during MAC to relieve the discomfort and pain associated with procedures. Fentanyl is one of the most commonly used analgesics during MAC, and fentanyl 50-100 μg has an onset of 3-5 min and duration of 45-60 min [bib_ref] The comparative effectiveness of fentanyl and its newer analogs during extracorporeal shock..., Gesztesi [/bib_ref]. Even small amounts (25-50 μg) of fentanyl may cause respiratory depression if co-infused with other sedatives. Alfentanil can be injected intermittently to relieve procedure-associated discomfort and pain [bib_ref] Median effective infusion dose (ED50) of alfentanil for monitored anesthesia care of..., Sesay [/bib_ref]. Remifentanil, an ultra-short-acting opioid with a rapid onset time (1 min) and short duration of action (3-10 min), is an ideal opioid for continuous infusion, and for managing pain related to surgical stimulation [bib_ref] Remifentanil as an analgesic adjunct in local/regional anesthesia and in monitored anesthesia..., Servin [/bib_ref]. However, an anesthesia care provider should be cautious during remifentanil infusion since remifentanil often causes respiratory depression [bib_ref] Remifentanil as an analgesic adjunct in local/regional anesthesia and in monitored anesthesia..., Servin [/bib_ref].
## Korean j anesthesiol
## Sohn and ryu
Ketamine is an N-methyl-o-aspartate receptor antagonist that has profound analgesic, sedative, and amnestic characteristics. Ketamine is a particularly valuable analgesic during MAC because it does not cause clinically significant respiratory depression or PONV. Low dose ketamine (0.25-0.50 mg/kg) with propofol has been used during ambulatory MAC for plastic surgery [bib_ref] The use of a ketamine-propofol combination during monitored anesthesia care, Badrinath [/bib_ref]. Ketamine-dexmedetomidine combination is known to be effective in sedation and analgesia for pediatric patients during magnetic resonance imaging [bib_ref] Monitored anesthesia care with a combination of ketamine and dexmedetomidine during magnetic..., Luscri [/bib_ref].
Non-steroidal anti-inflammatory drugs (NSAIDs) such as ketorolac have analgesic properties. Compared with opioids, NSAIDs cause less adverse effects such as pruritus and PONV. Ketorolac has been used as an adjunct analgesic during local anesthesia or propofol infusion because it has lower analgesic effects than opioids [bib_ref] Ketorolac or fentanyl to supplement local anesthesia?, Bosek [/bib_ref].
## Procedures performed under mac
## Eye surgery
Cataract surgery is typically performed under topical anesthesia [bib_ref] Monitored anaesthesia care (MAC) and ophthalmic surgery, Bhattarai [/bib_ref]. Topical anesthesia is simple to perform and avoids the potential risk of retrobulbar or peribulbar nerve block. However, topical anesthesia may not provide complete analgesia and may cause discomfort and anxiety. For this reason, MAC with topical anesthesia and infusion of intravenous sedatives and analgesics can provide anxiety relief and patient comfort during cataract surgery [bib_ref] Monitored anaesthesia care (MAC) and ophthalmic surgery, Bhattarai [/bib_ref].
Sedation with midazolam has been commonly used with topical anesthesia for cataract surgery. In a randomized, doubleblind trial in patients with cataract surgery, dexmedetomidine was associated with slightly better patient satisfaction compared with midazolam [bib_ref] Dexmedetomidine vs midazolam for monitored anaesthesia care during cataract surgery, Alhashemi [/bib_ref]. However, dexmedetomidine group patients showed relative cardiovascular depression and delayed recovery [bib_ref] Dexmedetomidine vs midazolam for monitored anaesthesia care during cataract surgery, Alhashemi [/bib_ref]. Cardiovascular depression including bradycardia and hypotension should be considered during MAC for cataract surgery since patients with cataract surgery typically have comorbidities such as hypertension and diabetes mellitus. Another study compared dexmedetomidine with remifentanil during cataract surgery in patients under topical anesthesia; the result showed that surgeon satisfaction was lower for the dexmedetomidine group than for the remifentanil group because of poor cooperation and deep sedation of the patients [bib_ref] Remifentanil or dexmedetomidine for monitored anesthesia care during cataract surgery under topical..., Park [/bib_ref].
In previous studies on cataract surgery-related adverse events, cataract surgery performed under topical anesthesia with MAC required anesthesiologist intervention in 21.6% of cases [bib_ref] Systemic adverse events during 2005 phacoemulsifications under monitored anesthesia care: a prospective..., Basta [/bib_ref]. Agitation was more common in younger patients with neurological or psychiatric diseases, while hypertension was more common in older patients with higher ASA scores [bib_ref] Systemic adverse events during 2005 phacoemulsifications under monitored anesthesia care: a prospective..., Basta [/bib_ref].
## Otolaryngologic surgery
Surgeries that had previously been performed with only local anesthesia have since been performed with MAC due to it its advantages for patient safety and satisfaction. During tympanoplasty, surgeons provide local anesthesia on the surgical spot and the anesthesiologist infuses sedatives and analgesics [bib_ref] A prospective randomized double-blind study comparing dexmedetomidine vs. combination of midazolam-fentanyl for..., Parikh [/bib_ref]. Recently, dexmedetomidine has been investigated and compared with midazolam-fentanyl combination during tympanoplasty; the dexmedetomidine group had higher surgeon satisfaction scores than the midazolam-fentanyl group [bib_ref] A prospective randomized double-blind study comparing dexmedetomidine vs. combination of midazolam-fentanyl for..., Parikh [/bib_ref]. Percutaneous dilatational tracheostomy can be performed under local anesthesia, but MAC with midazolam, propofol, and alfentanil increased patient comfort and satisfaction [bib_ref] The clinical application of monitored anesthesia care in percutaneous dilatational tracheostomy, Dong [/bib_ref].
## Inguinal herniorrhaphy
Inguinal herniorrhaphy is one of the most common surgical procedures performed under MAC in an ambulatory setting. A previous study reported that MAC with local anesthesia plus intravenous sedatives and analgesics resulted in more rapid recovery and lower medical costs than general anesthesia [bib_ref] Eighty cases of monitored anesthesia care (MAC) for inguinal hernia repairs using..., Adachi [/bib_ref]. Spinal anesthesia and MAC with ilioinguinal-hypogastric nerve block plus remifentanil infusion were compared for inguinal herniorrhaphy: MAC was associated with hemodynamic stability, fewer side effects, and higher satisfaction than spinal anesthesia [bib_ref] Comparison between monitored anesthesia care with remifentanil under ilioinguinal hypogastric nerve block..., Bang [/bib_ref].
## Cardiovascular procedures
Endovascular aortic aneurysm repair (EVAR) can be performed under general anesthesia, regional anesthesia, or MAC [bib_ref] Results of endovascular aortic aneurysm repair with general, regional, and local/monitored anesthesia..., Edwards [/bib_ref] [bib_ref] Comparison of anesthesia technique on outcomes of endovascular repair of abdominal aortic..., Franz [/bib_ref]. However, the use of general anesthesia for EVAR was related with increased postoperative hospital stay and respiratory compromise compared with regional or MAC [bib_ref] Results of endovascular aortic aneurysm repair with general, regional, and local/monitored anesthesia..., Edwards [/bib_ref] [bib_ref] Comparison of anesthesia technique on outcomes of endovascular repair of abdominal aortic..., Franz [/bib_ref]. In the majority of patients with transcatheter-aortic valve replacement, MAC was associated with a shorter procedure time and hospital stay than general anesthesia [bib_ref] Transfemoral aortic valve implantation under sedation and monitored anaesthetic care--a feasibility study, Bergmann [/bib_ref]. In this case, sedation should be performed by an experienced anesthesiologist and immediate conversion to general anesthesia should be prepared [bib_ref] Transfemoral aortic valve implantation under sedation and monitored anaesthetic care--a feasibility study, Bergmann [/bib_ref].
## Pain procedures
MAC can be provided for patients with invasive percutaneous vertebroplasty or diagnostic imaging discography [bib_ref] Use of dexmedetomidine for monitored anesthesia care for diskography in adolescents, Furstein [/bib_ref] [bib_ref] Clinical evaluation of the Capnomask in the supine vs. prone position during..., Sesay [/bib_ref]. During percutaneous vertebroplasty, TCI with propofol provided satisfactory sedation and high operator satisfaction [bib_ref] Median effective infusion dose (ED50) of alfentanil for monitored anesthesia care of..., Sesay [/bib_ref]. In a preliminary trial of dexmedetomidine for analgesia and sedation during diagnostic discography, dexmedetomidine was reported to be an adequate sedative and analgesic [bib_ref] Use of dexmedetomidine for monitored anesthesia care for diskography in adolescents, Furstein [/bib_ref]. Monitored anesthesia care Gastroendoscopic procedure MAC for gastro-endoscopic procedures is increasingly applied outside the operating room for operator and patient satisfaction. MAC for Gastro-endoscopic procedures should allow for safe sedation and complete examination, as well as rapid recovery. Especially, endoscopic retrograde cholangiopancreatography (ERCP) is a painful and long procedure in the lateral decubitus position, and MAC for ERCP procedures requires rather deep sedation and complete analgesia while allowing for respiratory and cardiovascular stability [bib_ref] Target-controlled infusion during monitored anesthesia care in patients undergoing EUS: propofol alone..., Fanti [/bib_ref]. Conventionally, the combination of midazolam and meperidine or fentanyl has been used for MAC in ERCP because of the relative lack of respiratory depression; different drug combinations have been recently investigated and compared [bib_ref] Target-controlled propofol infusion during monitored anesthesia in patients undergoing ERCP, Fanti [/bib_ref] [bib_ref] Midazolam with meperidine and dexmedetomidine vs. midazolam with meperidine for sedation during..., Lee [/bib_ref]. The addition of propofol or dexmedetomidine with this conventional regimen decreased the requirement for additional sedatives and increased operator satisfaction [bib_ref] Target-controlled propofol infusion during monitored anesthesia in patients undergoing ERCP, Fanti [/bib_ref] [bib_ref] Midazolam with meperidine and dexmedetomidine vs. midazolam with meperidine for sedation during..., Lee [/bib_ref]. During MAC for ERCP, relative deep sedation is required and cardiovascular or respiratory complications. including arterial hypotension, desaturation, bradycardia, arterial hypertension, arrhythmia, and aspiration may occur [bib_ref] Adverse events during monitored anesthesia care for GI endoscopy: an 8-year experience, Agostoni [/bib_ref]. For that reason, monitoring of the level of sedation, and the presence of a qualified anesthesiologist, are required for MAC in ERCP [bib_ref] Adverse events during monitored anesthesia care for GI endoscopy: an 8-year experience, Agostoni [/bib_ref].
## Flexible bronchoscopy
Flexible bronchoscopy is a diagnostic and therapeutic procedure typically performed under MAC. The ideal characteristics of sedatives during MAC, for flexible bronchoscopy, should allow the patient to maintain spontaneous ventilation and protect their own airway, while blunting sympathetic responses during bronchoscope insertion. The major safety concern with MAC for flexible bronchoscopy is excessive sedation depth and duration of hypoxemia. In addition, other indicators of cardiovascular instability, such as hypotension, hypertension, bradycardia, and tachycardia, should be considered during MAC for flexible bronchoscopy.
The combination of midazolam and hydrocodone was shown to reduce cough during flexible bronchoscopy without causing significant desaturation [bib_ref] Cough suppression during flexible bronchoscopy using combined sedation with midazolam and hydrocodone:..., Stolz [/bib_ref]. The combination of propofol and alfentanil during flexible bronchoscopy resulted in greater respiratory depression than propofol alone [bib_ref] Comparison of propofol and the combination of propofol and alfentanil during bronchoscopy:..., Yoon [/bib_ref]. The major advantage of ketamine use is preservation of airway patency and respira-tory function. Ketamine was superior to alfentanil when used in combination with propofol because of the high patient satisfaction and amnesia [bib_ref] Comparison of alfetanil and ketamine in combination with propofol for patient-controlled sedation..., Hwang [/bib_ref]. The safety profiles and efficacies of remifentanil and dexmedetomidine for sedation during flexible bronchoscopy have been investigated, with the results suggesting that dexmedetomidine was associated with a lower incidence of oxygen desaturation and reduced need for oral cavity suction than remifentanil during MAC for flexible bronchoscopy [bib_ref] Randomized double-blind study of remifentanil and dexmedetomidine for flexible bronchoscopy, Ryu [/bib_ref]. However, dexmedetomidine seemed to be less effective than remifentanil in that the dexmedetomidine group showed lower bronchoscopist satisfaction scores and more frequent need for topical anesthesia than the remifentanil group [bib_ref] Randomized double-blind study of remifentanil and dexmedetomidine for flexible bronchoscopy, Ryu [/bib_ref].
## Neurosurgery
Patients should be awake and cooperative under MAC to monitor neurologic functions during awake craniotomy [bib_ref] Monitored anesthesia care in awake craniotomy for brain tumor surgery, Berkenstadt [/bib_ref]. In such patients, adequate analgesia and sedation without respiratory and hemodynamic depression are required during application of the head frame, skin incision, and craniotomy, and the patient should be awake and cooperative for evaluation of neurologic functions during brain mapping and tumor resection [bib_ref] Monitored anesthesia care using remifentanil and propofol for awake craniotomy, Berkenstadt [/bib_ref]. In addition to scalp blocks, short half-life drugs such as propofol and remifentanil should be titrated, according to the surgical procedure step, by experienced anesthesiologists during awake craniotomy performed under MAC [bib_ref] Monitored anesthesia care in awake craniotomy for brain tumor surgery, Berkenstadt [/bib_ref] [bib_ref] Monitored anesthesia care using remifentanil and propofol for awake craniotomy, Berkenstadt [/bib_ref].
# Conclusions
Diagnostic or therapeutic procedures in and outside of the operating room are increasing and MAC is preferred to general or regional anesthesia due to its cost-effectiveness and rapid recovery. Systemic sedatives and analgesics should be infused carefully after monitoring the patient's level of consciousness and hemodynamic variables, since heavy sedation may lead to central respiratory depression or airway obstruction. MAC should be chosen as a proper anesthetic alternative after patient co-morbidities and preferences, and the type of procedure being performed, have been considered. The choice of sedative and analgesic is based on the required depth of sedation and analgesia of each procedure. The presence of a sufficiently experienced anesthesiologist, as well as oxygen supply, monitoring devices and emergency equipment, are required during MAC both in and outside the operating room.
[table] Table 1: Observer Assessment of Alertness/Sedation Scale (OAA/S)[6] Lethargic responses to name spoken in normal tone Mild slowing or thickening [/table]
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The Effect of Parenting Quality on Child Development at 36–48 Months in China’s Urban Area: Evidence from a Birth Cohort Study
Environmental exposures, especially parenting quality, are critical for later child development. This study aimed to determine the status of parenting quality and suspected development delay of preschool children in China's urban area and explore the associations between these two factors. The research was based on a birth cohort study conducted in Changsha, Hunan province, China. We used the Parenting Assessment Tool and Ages and Stages Questionnaires, Third Edition (ASQ-3), to measure parenting quality and child development status, respectively. Other data were collected from maternal health manuals and self-administered questionnaires during the follow-up period. The generalized estimating equation was used to examine whether parenting quality was significantly associated with child development outcomes. In the study, good parenting quality was 33.6% measured at 18 months, and suspected development delay was below 10% at 36-48 months among urban China; we observed negative associations between parenting quality scores and child development scores; poor parenting quality had a negative association with suspected development delay [OR and 95% CI: 2.74 (1.17, 6.40)], girls [OR and 95% CI: 0.33 (0.16, 0.69)] and maternal education years (>12 years) [OR and 95% CI: 0.27 (0.12, 0.64)] were protective factors for suspected development delay. Our findings highlighted the importance of good parenting quality among children in urban areas of China through a birth cohort study and may be used to reduce the children at high risk of developmental delay as a future intervention program. early childhood increase the risk of low social, cognitive, and health outcomes, and these outcomes are increasingly challenging to reverse beyond early childhood[10].According to the Integrated Maternal and Child Health Development (IMCHD) project in China[11], the prevalence of suspected developmental delay in children younger than three years in China poverty-stricken areas was 39.7%. It also emphasized that parenting played an essential role in the suspected development delay. According to a randomized comparison trial, the children who received encouraging parenting behaviors had higher development scores than the control group[12]. Disadvantaged experiences in early childhood have lasting effects on cognitive outcomes [13], but higher parenting quality predicted better performance after children suffered from brain injuries in early childhood[14]. Most of the current studies focusing on child development are cross-sectional studies and lack longitudinal study evidence[15,16]and could not infer causal and temporal associations. Due to the regional economic development disparities, most studies were conducted in poor rural areas in China and less in urban areas[11,17]. The effect of family rearing quality on cognitive development differs in diverse economic conditions[18]. A longitudinal birth cohort study showed a stronger association of child cognitive development and socioeconomic status compared to measures of other home environmental determinants (parenting) in low-and middle-income areas[19]. We need to conduct surveys in China's urban areas to find an association between parenting quality and child development.To address these research gaps, we conducted a cohort study in urban areas of Hunan province. This study aimed to determine the status of parenting quality and suspected development delay of the children at 36, 42, 48 months, explore the associations between these two factors and provide evidence for future intervention to reduce suspected development delay.
# Introduction
Early childhood, especially the first two years in early life, is a critical period for rapid growth and cognitive development. indicated that 250 million children (43%) younger than 5 years in low-income and middle-income countries are at risk of not reaching their developmental potential, and the number is as high as 17.43 million in China. Cognitive development in early childhood is attributed to genes and the environment. Genetic influences on cognitive development occur through a transactional process, in which genetic predispositions lead children to evoke cognitively stimulating experiences from their environments. In low-income and middle-income countries, individual differences in environments have a greater influence on IQ and genetics less so. Child cognitive development depends on multiple factors, which interact with each other and can be mutually reinforcing the process of development. Disadvantaged exposures and experiences in We collected the development data at , and 48 months, parenting quality data at 18 months, and the baseline data at 1 month.describes the number of participants at each time point. The study was approved by the Independent Ethics Committee Institute of Clinical Pharmacology, Central South University, Changsha, China. (Project number: CTXY-130041-3-2).
## Measures
## Children development outcomes
ASQ-3 was used to measure preschool children's development outcomes at 36, 42, 48 months. ASQ-3 is a parent-completed questionnaire used as a general developmental screening tool to identify children's potential developmental problems from 1 to 59 months. It has excellent psychometric properties, with high reliability and validity in China. The ASQ-3 is a developmental screening instrument consisting of 21 intervals, different child's age group has a corresponding one. Each questionnaire includes 30 items in five domains: communication (CM), gross motor (GM), fine motor (FM), problem-solving (CG), and personal social (PS). Each "yes" item is scored 10 points, "sometimes" is scored 5 points, and "not yet" is scored 0 points, and the sum score of each domain is 60. We compared the sum scores of every domain with the national normative cutoff point of China, then regarded children whose scores were lower than the cutoff point of the national norm in any field as suspected developmental delay.
## Parenting quality
We used the parenting assessment tool (PAT) to assess the parenting quality and skill at 18 months. PAT is a reliable and marginally valid measurement tool for determining Chinese urban parents. Moreover, PAT referred to the content, structure, and scoring method of Home Observation for Measurement of the Environment (HOME). The questions were designed to be broad, to elicit the general parental attitudes about parenting. PAT includes 8 factors: parenting concepts, acceptance, parent-child relationship, learning material, language stimulation, outside activity, feed, and safety. Each element has 4-5 items; the answer of each item as "yes" is scored 1 point, "no" is scored 2 points, and "not clear" is scored 3 points. The higher the PAT score was, the fewer parents knew about parenting. When all items' answers were "yes," the parenting quality was considered good parenting quality and vice versa.
## Confounding factors
Based on previous research, we covered multiple potentially confounding factors in our analysis, including child gender, child order, family socioeconomic factor, delivery mode, and paternal smoking status during pregnancy. Child order was classified into 2 groups: less than 2 children, more than or equal to 2 children. Family socioeconomic factors contained family income and parental education. Family income was measured by 4 groups: less than 2000 Chinese Yuan (CNY), 2000-4999 CNY, 5000-9999 CNY, and more than 10,000 CNY. Parental education was divided into 2 categories: less than or equal to 12 years and greater than 12 years. Delivery modes included vaginal and cesarean delivery. The paternal smoking status was classified as smoking and not smoking during pregnancy.
# Statistical analysis
Cohort characteristics for participant demographics were presented as mean (SD) for numerical variables n (%) for categorical variables. A generalized estimating equation (GEE) model was applied to find the correlation between parenting quality and developmental outcomes at different follow-up times from the same child. We constructed two kinds of GEE models to describe the associations. In the first model, the scores in different domains of ASQ-3 and the scores in 8 factors of PAT as dependent and independent variables were quantitative variables. In the second model, dependent and independent variables were qualitative variables. Suspected development delay was the dependent variable, and independent variables were parenting quality and other factors. The potential factors in both analyses were child gender, family income, parental education, delivery mode, child order, and paternal smoking during pregnancy. The above analyses were performed in SPSS version 22 (IBM, New York, NY, USA).
The number of respondents varied across waves. Most of the missing data were due to wave no-response (move and exit follow-up). Of the 976 respondents, 246 did not respond to the variables of interest (ASQ-3) in this study, so we removed these respondents. Besides, 538 respondents provided data on all three waves, 102 on two waves, and 90 on one wave. Altogether, 730 respondents provided data on ASQ at least once. There was no statistical difference in the general characteristics of the included and excluded subjects. The analysis was based on these 730 respondents. We used the "MissMech" function to test missing data; the result indicated that missing data were non-systematic [p = 0.730], which supported the multiple imputations. We completed quantitative and qualitative variables by "mice" package; the method was a random forest model. Missing data analysis was calculated on the R 3.5.1 version.summarizes participant demographics among the included 730 children. We got all the information of gender, and boys were 373 while girls were 357. Most families in the study were single-child families (71.9%). About a quarter of the families (27.8%) had 2 children or more. According to the delivery method, 59.1% were vaginal delivery, and 40.65% were cesarean delivery. The constituent ratios of family income categories were 3.4%, 51.5%, 37.8%, and 4.2%. A total of 617 (84.5%) mothers had more than 12 years of education; the number of fathers was 622 (85.2%). About 52.7% of fathers smoked during pregnancy. A third of families (224) provided good parenting quality, while 443 children had poor parenting quality.was a descriptive analysis of ASQ at 36 months, 42 months, 48 months. As shown in the figure, each domain's mean scores were higher than 50, and the FM domain had the lowest scores in three check times. The prevalence of suspected development delay was 6.8% at 36 months, 1.4% at 42 months, and 3.7% at 48 months.describes parenting quality at 18 months of children, including PAT scores and the proportion of good parenting quality in each factor. The proportion of good parenting quality in each factor was from 54% to 89.7%; those in most factors were higher than 80%. The lowest proportion of good parenting was in the parent-child relationship (54%); the highest proportion was in language stimulation (89.7%).presents the associations between parenting qualities and children's cognitive development. The results showed us that if parents knew more about parenting, children's ASQ scores would be higher, and different PAT factors affected the ASQ domains. As shown in, children's CM scores would be higher if parents knew more about parenting concepts, parent-child relationships, language stimulation, and children's safety. Poor parenting qualities in attention, communication, learning material, and language stimulation had adverse effects on children's GM scores. FM scores were higher when scores of parenting concepts and learning material were closer to 5 points. Parental feeding concepts were negatively correlated with CG. We found a negative correlation between attention, parent-child relationship, the outside activity of PAT, and PS of ASQ.
# Results
# Discussion
In the study, we found that the proportion of good parenting quality in urban China was 33.6% measured at 18 months. The prevalence of suspected development delay was 6.8% at 36 months, 1.4%
# Discussion
In the study, we found that the proportion of good parenting quality in urban China was 33.6% measured at 18 months. The prevalence of suspected development delay was 6.8% at 36 months, 1.4% at 42 months, 3.7% at 48 months. We also observed the negative association between parenting quality and communication, gross motor, fine motor, problem-solving, and personal-social of children; poor parenting quality increased the risk of suspected development delay; girls and maternal education years (>12 years) were the protective factors for suspected development delay.
Our study revealed that the prevalence of suspected development delay was below 10% at 36-48 months among urban China. According to a survey about suspected development delay, the prevalence of suspected developmental delay among children aged 6-35 months was 35.7% in poor rural areas of China, 17% in Senegal, and 24% in Brazil. Other studies' higher prevalence may reflect the environmental and nutritional factors, including socioeconomic status, contributing to disparities in child development. The scores of FM were the lowest among five domains of ASQ, which suggested the need for further intervention of fine motor in an early lifetime. In our study, good parenting quality was 33.6%, and poor parenting quality was 66.4% at 18 months. According to the IMCHD project in rural China, children's good care quality was about 25%, poor and medium parenting quality was about 75% at 12-23 months. The different proportions were likely due to positive associations between socioeconomic classifications and the availability of children's books and playthings.
We observed negative associations between parenting quality scores and child development scores, similar to the previous study. A longitudinal birth cohort in Canada has revealed the protective effects of parent-child interactions and language stimulation on child language development. An adequate home environment, which represents good parenting quality, is associated with better child motor development in southern Brazil. Some randomized controlled trials had highlighted that interventions on maternal play and parenting skills have also improved young children's social, emotional, communication, language, and cognitive competence. Our multivariable analysis confirms these findings, and improving the quality of different parenting behaviors is a feasible and effective way to enhance child development in primary community health services.
In public health, the development of effective intervention strategies requires an understanding of risk factors. We used the GEE model to testify parenting quality and confounders affecting suspected development delay. Poor parenting quality was a risk factor of suspected development delay, which was consistent with previous studies. A pregnancy cohort has highlighted that nondaily parent-child interaction increased the risk of delay. Child gender, as an essential demographic characteristic, may play a role in child development. For example, boys were statistically significant predictors of low cognitive development, according to an American birth cohort study. In our study, we found that gender impacted child development; girls got better outcomes than boys. A meta-analysis of parent-child language interactions showed that mothers talk more to their daughters than their sons. Thus, more maternal language interaction preference for girls than boys could explain this result. We found that maternal education affected suspected development delay, and family income had no effect on child development among socioeconomic variables. Mothers with higher education levels may have more knowledge of parenting, pay more attention to children's cognitive development, and improve their cognitive development through scientific methods. The result of family income may be that the role of socioeconomic status on children's development gradually decreases with the development of social income. We observed that paternal education was not statistically associated with suspected development delay. One possible explanation may be that limited father participation was insufficient to show positive child development outcomes. The result suggested that intervention strategies should target different child gender and focus on parenting quality to avoid adverse developmental outcomes.
To our knowledge, this is one of the first longitudinal studies investigating the impact of parenting quality on child development in urban China. The study population was representative of the urban population in Hunan. We randomly selected three communities of the Kaifu District as our study sites. However, the present study was subject to certain limitations. First, there exists evidence that parenting quality would change over time; one explanation may be the bidirectional nature of parenting behavior and child development. Further evidence from cohort studies and interventional studies is needed to consider the change in parenting quality. The second limitation is that the self-administered questionnaire was not assessed for validity and might result in some inaccurate information. Another limitation was that ASQ is only a screen tool for suspected developmental delay. The potential bias caused by misclassification error should be considered when interpreting the findings.
# Conclusions
The prevalence of child development in China's urban areas is lower than in China's poor rural areas, and parenting quality is higher than in rural areas. Diverse parenting factors influence different development outcomes, and the parenting quality is associated with suspected development delay. For a single development domain, we could have interventions individually to prevent children from suspected development delay. Strategies targeting parenting quality among children in China's urban areas should be addressed for a future intervention program to reduce the children at high risk of developmental delay. |
Lightning Strike Presenting as Fatal Lung Contusion: A Case Report
# Introduction
Lightning is a life-threatening natural disaster with an electrical energy content of 100-300 million volts and produces heat at a temperature of about 3000 °C. Prior studies estimate that there are 0.09 to 0.12 lightning strikes per 100,000 individuals worldwide [bib_ref] Lightning caused injuries in humans, Cooray [/bib_ref]. Around 0.2-1.7 deaths per million people are reportedly caused by lightning each year globally [bib_ref] Exadaktylos AK: Injuries, sequelae, and treatment of lightning-induced injuries: 10 years of..., Pfortmueller [/bib_ref]. In India, lightning strikes are thought to be responsible for over 2500 fatalities every year [bib_ref] Delayed esophageal perforation following lightning strike: a case report and review of..., Figgis [/bib_ref].
Many lightning-related fatalities cause sudden cardiac death by either ventricular fibrillation or asystole. Among the survivors, neurological manifestations like encephalopathy, intracranial hemorrhage, and neuropathy, cardiac dysrhythmias, other forms of injuries like burns, tympanic and ocular impairment are common. Solid organ damage can occasionally result from lightning's blasting action [bib_ref] Findings in fatal lightning strike cases, Turan [/bib_ref]. The spleen, liver, lungs, and bowels can be harmed by blunt trauma from shock waves, falling, or being struck with some object.
In this study, we describe the case of a 62-year-old man who presented to the emergency room with recurrent laryngeal nerve palsy and pulmonary contusion after being struck by lightning on his farm.
## Case presentation
After being struck by lightning, a 62-year-old man lost consciousness and was taken to the emergency room. he had been working on a farm with his family members when the incident occurred. He regained consciousness after half an hour in the ambulance. At the time of arrival at the hospital, he complained of severe pain all over the body, hoarseness of voice, coughing, and regurgitation. The patient also complained of shortness of breath at rest, which was sudden in onset. He denied any history of hemoptysis, tuberculosis, smoking, asthma, and past hospitalization with similar complaints.
On admission, he was irritable, opened his eyes when hearing any voice, and was able to move all four limbs, with a power grade of 5/5. His respiratory rate was 34 breaths per minute, his pulse was 120 beats per minute, and his blood pressure was 90/60 mmHg. Saturation was 88% on room air. Respiratory examination revealed bilateral crepitations in the infra-axillary, infra-scapular, and mammary regions. A cardiovascular examination revealed no abnormalities. Per abdomen examination was normal. His ear examination revealed a rupture of the tympanic membrane on the right side, but the left ear showed no abnormality.
His physical examination revealed an entrance wound of 1 × 3 cm in diameter, on the right side of the forehead [fig_ref] FIGURE 1: Entry wound of 2 x 2 cm in diameter on the right... [/fig_ref].
## Video 1: video laryngoscopy showing unilateral vocal cord palsy
View video here: https://youtu.be/O49g3_yS2Ms
All the routine investigations were done, and the findings are summarized in .
## 1: routine investigation findings in our patient
Creatinine kinase levels were monitored on a daily basis, as summarized in [fig_ref] TABLE 2: Creatinine phosphokinase levels in our patientDaily creatinine phosphokinase levels [/fig_ref]. Hydration with IV fluids and nasogastric tube feeding was started, and injectable tramadol and fentanyl were given for pain relief. Starting on hospital day one, the patient was kept on oxygen support for a period of seven days, after which the patient's respiratory distress increased, and hence he was put on non-invasive ventilatory support. From day three of admission, the patient developed weakness in all four limbs and his power grade declined from 5/5 to 2/5 due to rhabdomyolysis-induced myositis. Electromyography was also done for confirmation, which was suggestive of a decreased duration of motor unit potentials.
## Creatinine kinase levels
For myopathy, steroid methylprednisolone 1 g was started and given for five days. After five days on steroids, his power improved to grade 4, and other supportive medications were started. During the second week, his oxygen saturation was maintained on non-invasive ventilation (NIV). However, during the third week of admission, arterial blood gas (ABG) monitoring revealed hypoxia with pO 2 of 70%, pH of 7.202, CO 2 of 44%, and base deficit of -10; hence, the patient was intubated in light of hypoxia, tachypnoea, and tachycardia and was put on a mechanical ventilator with volume control mode with FiO 2 of 100%, peep of 6, vital capacity of 350, and RR of 20/min. Consecutive ABG monitoring was suggestive of pO 2 of 95%, pH of 7.351, and CO 2 of 40%.
In view of the long-standing mechanical ventilator support, a tracheostomy was planned on the seventh day of intubation. Cultures were sent of ET tube secretion; sputum culture and blood and urine cultures were suggestive of no growth after 48 hours of incubation. A concurrent chest radiogram suggested no new changes with regard to ventilator-acquired pneumonia. Despite aggressive medical management, the patient's condition kept deteriorating and he eventually succumbed to respiratory failure on the 39th day of admission.
# Discussion
Lightening causes injury by way of five different mechanisms: thermal, electrical, explosive, magnetic field, and blunt damage from falls; however, the precise mechanism behind lightning injury remains unknown. The most serious clinical manifestation is most likely to result from direct hits [bib_ref] Injuries and deaths from lightning, Blumenthal [/bib_ref]. The second mechanism produces a blast wave as a result of abrupt and significant changes in temperature. The blastic injury effect of a lightning strike can cause myocardial injury, lung contusion, lung parenchymal tear, major vascular rupture, intestine rupture, tympanic membrane rupture, and ocular damage [bib_ref] Lung contusion: a clinico-pathological entity with unpredictable clinical course, Ganie [/bib_ref]. In our case, lightning's electrical, thermal, and explosive effects all contributed to skin burns.
A pulmonary contusion is defined as a lung injury caused by a blunt explosive event without any accompanying chest wall injuries [bib_ref] Pulmonary contusion, Rendeki [/bib_ref]. The lung tissue is not ripped or torn in pulmonary contusion cases, but blood and other fluids do pool there because of injury to the alveolar capillaries. Clinical indicators might vary significantly in terms of their severity and timing, which is unusual. Hypoxia and hypercarbia-related respiratory distress frequently manifests gradually and reaches their peak in around 72 hours [bib_ref] Blast injury of the lungs due to lightning, Moulson [/bib_ref] [bib_ref] Lightning injury as a blast injury of skull, brain, and visceral lesions:..., Ohashi [/bib_ref]. When lightning hits, symptoms are often classified into immediate symptoms and symptoms that appear after a period of time, as in our case.
Moulson [bib_ref] Blast injury of the lungs due to lightning, Moulson [/bib_ref] described the first case of pulmonary contusion resulting from blastic injury in a lightning strike case without any burn injuries. According to Ohashi et al. [bib_ref] Lightning injury caused by discharges accompanying flashovers: a clinical and experimental study..., Ohashi [/bib_ref] , injuries such as cerebral hemorrhage, pulmonary hemorrhage, and solid organ rupture can be caused by falls or the lightning strike's current effects. The concussive action of rapidly expanding steam created by superheating water on the body surface of a wet person is what causes these injuries. Rapid surface flashover decreases internal energy dissipation and increases the likelihood of survival. We believe that in our case, the pulmonary contusion and hemorrhage were caused by the blastic effect of lightning that resulted from a quick surface flashover.
Tolunay et al. have reported the case of a 15-year-old male patient who was admitted to the emergency department due to a lightning strike in open terrain; even though the patient initially did not have any complaints, he developed breathlessness on the fourth day and was managed with oxygen therapy, and was discharged on 10th day with advice to follow up.
Uzel Şener et al. have described a case of a 19-year-old male patient who presented with a history of a lightning strike and developed cardiomyopathy with low ejection fraction; HRCT revealed bilateral pleural effusion and there were patchy consolidations in the upper lobes and areas of ground glass opacities around it, which were treated with oxygen therapy, steroids, antibiotics, and supportive measures. The patient was discharged with instructions to follow up after 10 days [bib_ref] Lightning-strike-induced acute lung injury: a case report, Şener [/bib_ref].
Schleich et al. have reported a case of a 23-year-old male patient who presented with severe third-degree burns on the chest who developed pulmonary embolism confirmed on CT angiography and was treated with heparin, antibiotics, and supportive care with ventilator support and systemic steroids. The patient recovered after a hospital stay of two months and was subsequently discharged [bib_ref] Survival after severe intrathoracic electrical injury, Schleich [/bib_ref]. The details of the studies mentioned are summarized in [fig_ref] TABLE 3: Summary of previous studies on lung contusion due to lightningHRCT [/fig_ref].
## Age/sex
# Conclusions
Our case report presented a unique combination of complications caused by a lightning strike. Rhabdomyolysis is the most common complication, with laryngeal nerve palsy and lung contusion causing respiratory failure. Through a variety of causes, including blunt trauma, lightning strikes pose a serious hazard to human life. These patients must be closely monitored and should undergo a thorough respiratory evaluation on admission for early identification and management of any potential lung injury.
# Additional information disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
[fig] FIGURE 1: Entry wound of 2 x 2 cm in diameter on the right side of the forehead: a linear-shaped first-degree burn The exit wound was 2 x 3 cm in diameter, at the left iliac region, a linear-shaped first-degree burn showing ruptured bullae with a reddish base (Figure 2). [/fig]
[fig] FIGURE 2: Exit wound of 2 x 3 cm in diameter, at the left iliac region: a linear-shaped first-degree burn showing ruptured bullae with a reddish base Video laryngoscopy was done in light of the hoarseness of voice and regurgitation, which revealed right vocal cord palsy with absent gag reflex (Video 1). [/fig]
[fig] FIGURE 3: HRCT thorax revealing multifocal areas of ground glass appearance in both the upper lobe, left lingular lobe, and left lower lobe suggestive of possible pulmonary contusion HRCT: high-resolution computed tomographyFIGURE 4: HRCT thorax revealing multifocal areas of ground glass appearance on both sides and pulmonary contusion on the left side HRCT: high-resolution computed tomography [/fig]
[table] TABLE 2: Creatinine phosphokinase levels in our patientDaily creatinine phosphokinase levels; reference range: 0.6-1.25 mg/dl [/table]
[table] TABLE 3: Summary of previous studies on lung contusion due to lightningHRCT: high-resolution chest computed tomography [/table]
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Multifunctional Templates for Minimized Osteotomy, Implantation, and Palatal Distraction with a Mini-Screw-Assisted Expander in Schizodontism and Maxillary Deficit
Purpose. Schizodontism is complete separation of a dental germ. It results in a twin tooth and supernumerary teeth. The treatment of transverse constriction in combination with supernumerary dental germs and impacted central incisors can pose a challenge, especially in young patients, when the number of permanent teeth is not adequate to ensure secure anchorage. The use of navigation templates based on three-dimensional X-ray images allows for precise insertion of temporary mini-implants for the acquisition of palatal distractors. In addition, templates allow for minimally invasive biopsies and osteotomies. Methods. The treatment of schizodontism, dentitio tarda, and transverse constriction is to be assessed as an interdisciplinary method by using mini-screw-assisted devices. Minimized osteotomy of impacted supernumerary teeth or dental implantation can be carried out in a one-step-procedure based on digital preplanning and prefabrication of orthodontic devices. Results. Multifunctional templates allow for early planning, preoperative fabrication, and intraoral fixation of orthodontic appliances. In the case of an adolescent patient, a sustainable, interdisciplinary treatment concept could be demonstrated that shows age-appropriate gnathological development and stable growth conditions over a follow-up period of 10 years. Conclusion. One can likely assume that multifunctional templates allow for minimally invasive one-step surgeries as an interdisciplinary tool between orofacial surgery and modern orthodontics.
# Introduction
Schizodontism, germination, and twinning are uncommon developmental anomalies of the hard dental tissue. These aberrations are manifested either as anomalous teeth or supernumerary teeth. The prevalence rate is variable in individual reports, and the overall prevalence appears to be approximately 0.5% in the deciduous teeth and 0.1% in the permanent dentition. It is more prevalent in the anterior maxillary region affecting incisors and canines. Bilateral cases are seen less frequently, with a prevalence of 0.02% in both dentitions. Cone beam computed tomography (CB-CT) is a three-dimensional (3D) imaging technique that allows proper localization of supernumerary teeth, measurement of palatal constriction, and treatment planning prior to orthodontic or surgical intervention. The diagnostic workflow and treatment concept by using a mini-screwassisted device for guided osteotomy, palatal distraction, and orthodontic treatment will be presented and critically discussed.
Schizodontism, intermaxillary incongruences, and transverse constriction will be discovered early in the context of child or adolescent medical check-ups. This means that patients are given dental and orthodontic treatment at an early stage. A precise and carefully considered strategy of treatment is therefore important. Intervention should be as gentle as possible to reduce comorbidities. In addition, predictability and sustainability should be mandatory. Orofacial backward-planning enables surgical interventions to be carried out in a minimally invasive manner, bundled with orthodontic treatment, and could be-if necessary-reduced to a minimum.
## Case presentation
A 12-year-old girl visited the dental clinic because of delayed tooth eruption and deciduous tooth persistence. The first tooth breakthrough for deciduous incisors was observed at the age of 14 months. The family dental history was inconspicuous. Pediatric check-ups were performed regularly and a percentile conformal normal growth was found.
## Clinical findings
The clinical findings showed a brachyfacial facial type corresponding to the chronological age. Intraoral eruption of the six-year molars all lower incisors. In addition, the lateral upper incisors were broken through, rotated, and had a gap to the canines. There was persistence of all deciduous molars and deciduous canines. Owing to the absence of the anterior tooth breakthrough, a functional switching gap resulted between the central maxillary incisors. The anterior and posterior arch width of the maxilla was reduced.
## Radiological findings
Two supernumerary dorsocaudopalatal displaced microforms of central incisors (schizodonts: 11b, 21b; Figures 1-3) and nasocranial-positioned normal forms of incisors were seen on radiographics (orthopantomography (OPG); Orthophos XG, Sirona Dental Systems, Bensheim, Germany), digital volume tomography (CB-CT; KaVo 3D eXam ConeBeam XG; KaVo Dental GmbH, Biberach/Riss, Germany), and lateral cephalometric view (Orthophos XG, Sirona Dental Systems, Bensheim, Germany). The software used for CB-CT was eXam-VisionQ®, Version 1.9.3.13 (Kaltenbach & Vogt Dental GmbH, Biberach/Riss, Germany). The X-ray images corresponded to the status of a mixed dentition at the end of the first dentition with persistence of 55, 54, 53, 63, 64, and 65 and retention of all permanent tooth germs, with narrow germination of 13 and 23.
## Diagnosis
(i) Schizodontism of 11 and 21 with palatal dislocated schizodonts (microforms)(ii) Persistence of deciduous teeth(iii) Transverse constriction of the upper jaw; narrow germination of 13 and 23
6. Therapy 2 Case Reports in Dentistry (a) Guided insertion of mini-implants with immediate loading: as an anchorage-tool, mini-implants were planned, placed, and loaded immediately with a palatal distractor (b) Minimally invasive biopsy: templates were used for the targeted, minimally invasive removal of palatal displaced schizodonts. The horizontally displaced incisors were provided with vestibular brackets and aligned by traction (c) Implant-guided palatal distraction (IGPD): to obtain space for the horizontally displaced incisors , transverse distraction was necessary. To align the exposed teeth, the distractor was extended anteriorly to hold the elastics or wire ligatures
## Prefabrication of the individual implant-guided palatal distractor (igpd)
A special model with implant analogues (Nobel Biocare Ser-vices®, Klothen, Switzerland) was prefabricatedby using the template. Then, 8°conical abutments were placed on each implant-analogue. A mesiostructure with 2°milled bars and two anterior extensions was added to each extension to hold the ligature wire. We rigidly fixed this device with frictional fit over the bars and began to activate transverse expanding by use of a Memory Palatal Split Screw (No. 167 M1529, Forestadent® Pforzheim; Germany).
Owing to divergence of the mini-implants, the superstructure had to be fabricated in the following 3 parts: part 1-8°cone construction; part 2-attached bar in two parts; and part 3-connector with a memory screw.
## Surgical procedure
At first, the NobelGuide® template was positioned on the maxillary teeth. For osseous fixation, four diverging anchor pins (Nobel Biocare Services®, Zurich, Switzerland; diameter: 1.5 mm) were inserted from the palatal side. Guided punching of the mucosa (Soft Tissue Punch®, Nobel Biocare Services®, Zurich, Switzerland; diameter: 5.2 mm) was added. Osteotomy of the palatal compacta uncovered the schizodonts for extraction. The harvested gingival punch was replaced palatal to the mucosa. We removed the template and anchor pins (1:5 mm x 10 mm) and replaced them with primary stable immediate implants (2:8 mm x 13:0 mm) by using the implant holes. The cone connectors of the mesiostructure were cemented precisely to the patrixes of the immediate implants. The anterior branches were located at the level of the palatal gingiva of 12 and 22. Then, the distractor was attached to the mesiostructure. The distal ligature ends were fixed to the anterior branches of the distractor. The two permanent anterior teeth were exposed from the vestibular side. Brackets were fixed adhesively using the acid etching technique and 11 and 21 were attached by ligaturesto the branches of the mesiostructure.
## Postoperative course
Over a period of 18 days, discontinuous palatal distraction of the posterior dental arch (7 mm) was performed. Finally, the transverse screw was blocked to remain in position. The follow-up after 12 weeks showed a stable gap widening along the entire Raphe median plane up to the anterior region. The planned lowering and ventral alignment of the central incisors also became evidentCase Reports in Dentistry
It resulted in complete alignment of the central anterior teeth. The vitality test of all permanent teeth after breakthrough was positive. The 6-year radiological follow-up showed that the central incisors were in the correct position according to the standards. The initially protruded front teeth to OK1 SN: 138°could be retruded to an inclination of OK1 to SN: 108°. A normal interincisal angle of 133°was achieved by compensatory protrusion of the lower incisors. The previously retruded lip profile was compensated. The lateral cephalometric radiographs showed development from the brachyfacial-to the mesiofacial-type during the treatment. For abbreviations, see.
## 5
Case Reports in Dentistry 11. Discussion 11.1. The Navigated Implant and Biopsy Planning. The NobelGuide planning is easily linkable with intraoral scanners. This technology has registered a constantly increasing use in many fields of dentistry, such as restorative dentistry, prosthodontics, orthodontics, and implantology. It allows for a completely digital workflow, from impres-sion to final framework, with clinical reliability (precision of the virtual occlusal record)and good patient feedback.
In conventional NobelGuide planning, the anchor pins are used to fix the position of the template in the edentulous jaw and the implants are placed in the dental arch in a planned and navigated manner. In our modified case, the IGPD fixed the template to the deciduous teeth; they Case Reports in Dentistry serve as the dental support. The actual implant planning involves guided biopsy of the palatal schizodonts. At best, it is planned with the largest diameter (x > 5 mm).
The anchor pins serve to position and fix the template precisely during the biopsy. In addition, the position of the anchor pins can be used as a pilot hole for the mini- impl. diameter, 2.8 mm). Owing to the exact drilling in the palatal D1-D2 maxilla bone, a high insertion torque of x > 35 N cm can be achieved. This allows for immediate loading of the implants. Furthermore, single-stage insertion of the IGPD can be implemented with early loading. This makes the performance of intraoperative impressions and a second surgical intervention unnecessary, which is particularly important in younger or anxious patients. The CB-CT Case Reports in Dentistry recording technique offers the best accuracy for the described navigation-supported intervention in the first dentition, because there are usually no artifacts due to metallic restorations. Despite an inaccuracy of 0.3 mm due to the system, this technique is also successful in CB-CT-assisted surgery; however, there is higher radiation intensity. In cases of palatal implant insertion with divergent implant axes, it is essential to pay attention to the roots of the permanent teeth. Algorithms for hard tissue usually allow a good differentiation of the teeth from the bone substance in the CB-CT analysis. This allows IGPDs to be planned and clinically implemented with a high degree of accuracy. The alternative of a mere implant at insertion along the raphe median plane would be associated with a lower morbidity with respect to the tooth germs, but it does not allow for the use of mini-implants for palatal extension. Systems that are only dental supported (e.g., the hyrax screw) often lead to tooth displacement or accelerated resorption of deciduous teeth without any real transverse increase.
## Tooth-borne apparatus versus bone-borne apparatus.
Owing to the generally delayed tooth penetration, our patient did not have enough permanent teeth available for dental anchorage of the palatal expansion appliance and the looping of the first incisors. Even after transverse expansion with a conventional removable expansion plate and pulling of the attached upper first incisors against it, the plate would not have provided support due to the beginning of tooth change in the second mixed dentition. Mini-screw-assisted palatal expanders are supported with titanium, stainless steel, or orthodontic miniscrews, like the used bone-borne apparatus (IGPD). In addition, anchoring by implants offers the advantage that neither the setting of the incisors nor the palatal expansion is dependent on the compliance of the young patient. Owing to stable connection of the IGPD with the upper jaw, the forces can be applied in a targeted, dosed manner and 24 hours a day. Owing to the 3D representation on the CB-CT, the IGPD could be constructed in such a way that an optimal pulling direction for the upper first incisors could be realized. However, a possibly necessary change in the pulling direction would have been difficult. The inserted palatal implants transfer the forces of the expansion screw directly to the upper jaw, thus avoiding the load and potential overload of the palatal expansion appliance on the anchor teeth. This eliminates the typical risks of forced transverse expansion (bite opening due to buccal tilting of the first molars and root resorption of the anchor teeth). The lowering of the nasal floor leads to an improvement in the nasal passage (nasal airflow) and thus often leads to a spontaneous change from oral to nasal breathing. If the patient's midface hypoplasia would have been more pronounced, the IGPD could have also served as an anchor for a Delaire mask. In the present case, however, the case was finished with moderate dental compensation for a slightly mesial bite, even if the jaws-see lateral view-were not in maximum intercuspation during the course of the treatment. The bone-borne apparatus requires good oral hygiene in the patient. Otherwise, peri-implant inflammation could occur. Implant loosening is a rare complication with orthodontic mini-implants. Miniscrews inserted in midpalatal locations have shown a failure rate of 1.3%.
Owing to soldering of the IGPD construction, explantation of all implants had to be performed simultaneously. However, in the present case, this was not a disadvantage, because the setting of the incisors coincided approximately with the end of the retention time for the transverse expansion. The IGPD was relatively flat; thus, speech was hardly affected. Moderate impressions on the back of the tongue occurred only in the initial period after insertion of the appliance. In summary, IGPD or other mini-screw-supported devices present good clinical reliabilityand excellent mechanical properties even with small diameters. The presented patient case shows that CB-CT-based planning and the preoperative appliance (customized distractor) improved the predictability of the surgical result, and it achieved a minimally invasive guided biopsy and a shortened duration of surgery. In addition, the subsequent orthodontic therapy could be conducted effectively and without any anchorage loss due to the inserted mini-implants. However, the mini-screw-supported technique is more invasive and Supernumerary teeth often cause palatal constriction and are a therapeutic challenge for orthodontists and oral surgeons. Therefore, interdisciplinary therapeutic concepts are needed for the benefit of the patient.
Treatment planning depends on various factors, such as the time of diagnosis, the age of the patient, the position of the supernumerary tooth, and possible complications. This case report presents only one possible treatment option for a multidisciplinary approach in the supernumerary permanent dentition.10 Case Reports in Dentistry: Lateral cephalometric radiograph with evaluation 48 months postoperative (end of treatment); for abbreviations, see also11 Case Reports in Dentistry |
Pre-hospital care time intervals among victims of road traffic injuries in Iran. A cross-sectional study
Background: Road traffic injuries (RTIs) are a major public health problem, requiring concerted efforts both for their prevention and a reduction of their consequences. Timely arrival of the Emergency Medical Service (EMS) at the crash scene followed by speedy victim transportation by trained personnel may reduce the RTIs' consequences. The first 60 minutes after injury occurrence -referred to as the "golden hour"-are vital for the saving of lives. The present study was designed to estimate the average of various time intervals occurring during the pre-hospital care process and to examine the differences between these time intervals as regards RTIs on urban and interurban roads. Method: A retrospective cross-sectional study was designed and various time intervals in relation to pre-hospital care of RTIs identified in the ambulance dispatch centre in Urmia, Iran from 20 March 2005 to 20 March 2007. All cases which resulted in ambulance dispatches were reviewed and those that had complete data on time intervals were analyzed. Results: In total, the cases of 2027 RTI victims were analysed. Of these, 61.5 % of the subjects were injured in city areas. The mean response time for city locations was 5.0 minutes, compared with 10.6 minutes for interurban road locations. The mean on-scene time on the interurban roads was longer than on city roads (9.2 vs. 6.1 minutes, p < 0.001). Mean transport times from the scene to the hospital were also significantly longer for interurban incidents (17.1 vs. 6.3 minutes, p < 0.001). The mean of total pre-hospital time was 37.2 (+/-17.2) minutes with a median of 32.0. Overall, 72.5% of the response interval time was less than eight minutes. Conclusion: The response, transport and total time intervals among EMS responding to RTI incidents were longer for interurban roads, compared to the city areas. More research should take place on needs-to and access-for EMS on city and interurban roads. The notification interval seems to be a hidden part of the post-crash events and indirectly affects the "golden hour" for victim management and it needs to be measured through the establishment of the surveillance systems.
# Background
Road traffic injuries (RTIs) are a major public health problem, requiring concerted efforts for prevention. The best strategy for RTI control is crash prevention, however total prevention is obviously impossible and crashes can occur at any time. However, it is often possible to minimize crash consequences by promptly providing effective pre-hospital services [bib_ref] Community-based trauma system development: key barriers and facilitating factors, Bazzoli [/bib_ref]. Each year, many of the 1.2 million lives lost globally could be saved and much of the ensuing disability suffered by the 50 million injured could be prevented if rapid and competent pre-hospital services were available at the crash scene [bib_ref] Prehospital emergency care and the global road safety crisis, Von Elm [/bib_ref].
In most low-and middle-income countries (LMICs), transport of road traffic victims, is usually provided by relatives, taxi drivers, truck drivers, police officers and other motorists; who are usually untrained [bib_ref] Emergency medical systems in low-and middle-income countries: recommendations for action, Kobusingye [/bib_ref] [bib_ref] Improvements in prehospital trauma care in an African country with no formal..., Mock [/bib_ref]. Ambulances, if available, usually exist only in urban areas [bib_ref] Prehospital emergency care and the global road safety crisis, Von Elm [/bib_ref]. Significant numbers of neurological injuries appear to be a result of the extrication process or victim transportation without adequate immobilization [bib_ref] Efficacy of cervical spine immobilization methods, Podolsky [/bib_ref] [bib_ref] Acute cervical spine injuries, Cloward [/bib_ref] , generally by untrained people [bib_ref] Vehicle entrapment rescue and pre-hospital trauma care, Wilmink [/bib_ref]. Studies have shown that the inadequacy of public health infrastructure and poor access to health services are important reasons for the high burden of RTIs and/or their severity. Many LMICs have insufficient pre-hospital emergency medical services including rapid services and effective management of RTI victims and their transportation [bib_ref] Trauma mortality patterns in three nations at different economic levels: implications for..., Mock [/bib_ref] and therefore their improvement and system evaluation is crucial [bib_ref] Traffic injury deaths in West Azarbaijan province of Iran: a crosssectional interview-based..., Khorasani-Zavareh [/bib_ref] [bib_ref] The requirements and challenges in preventing of road traffic injury in Iran...., Khorasani-Zavreh [/bib_ref].
Pre-hospital care is unsatisfactory in many countries, especially in LMICs [bib_ref] Emergency medical systems in low-and middle-income countries: recommendations for action, Kobusingye [/bib_ref] [bib_ref] Improvements in prehospital trauma care in an African country with no formal..., Mock [/bib_ref] [bib_ref] Efficacy of cervical spine immobilization methods, Podolsky [/bib_ref] , where the majority of trauma deaths occur in the pre-hospital phase. Rapid arrival of the EMS at the crash scene and proper victim transportation by trained personnel may reduce injury severity and reduce the number of preventable deaths. It is important to note that many trauma experts consider that the first 60 minutes after injury occurrencereferred to as the "golden hour"-are the most effective for saving lives [bib_ref] Branas CC: A meta-analysis of prehospital care times for trauma, Carr [/bib_ref]. After this period, the risk of death or injury severity rises significantly [bib_ref] Branas CC: A meta-analysis of prehospital care times for trauma, Carr [/bib_ref]. This "golden hour" consists of various time intervals, e.g. notification interval, activation interval, response interval, on-scene interval, and transport interval (see .
Rapid responses are believed to be one of the most important criteria for the quality of care provided to trauma patients [bib_ref] Branas CC: A meta-analysis of prehospital care times for trauma, Carr [/bib_ref]. Measuring these various time intervals can be an important step towards the evaluation of the EMS function. However, to our knowledge there is a lack of information about the various pre-hospital time intervals of road traffic injuries and the differences between these for city and interurban roads area in Iran. The present study therefore was designed to estimate the average timings of various time intervals of RTI at the pre-hospital phase by EMS to trauma centres in the capital city of West Azarbaijan Province of Iran.
# Method
This is a retrospective cross-sectional study on time intervals of RTIs that were identified in the centre for ambulance dispatch sites from 20 March 2005 to 20 March 2007 in the Urmia city of Iran. The pre-hospital data of all RTIs were reviewed and the average of the different interval times was analyzed.
## Study area and study population
This study was undertaken in Urmia, the capital city of the West Azarbaijan Province of Iran, which is located in the centre of the province. West Azarbaijan province shares a common border with Iraq, Turkey and Russia Azerbaijan. For feasibility reasons Urmia was chosen for the study. The population was about 887 318 in 2006. The rate of fatal RTIs in this province was estimated at 34 per 100 000 in 2005 [bib_ref] Estimating road traffic mortality more accurately: Use of the capturerecapture method in..., Zavareh [/bib_ref].
## Data collection instrument
A standard questionnaire designed by the Ministry of Health and Medical Education in Iran, was used in this study. The questionnaire has demographic information on patients or victims including name, sex and age, disease or external cause of injury and information about time of services including time of emergency call, time of ambulance departure to scene, time of arrival at scene, time of patients' transportation, time of arrival at hospital, time of leaving the hospital and time of returning to ambulance depot, location of crash scene (in cities or on interurban roads), as well as distance covered by the ambulance from the departure to return to depot. In this study, time intervals including time of emergency call, time of ambulance departure to scene, time of arrival at scene, time for patients' transportation and time of arrival at the hospital were used. [fig_ref] Figure 1: Specific intervals and points in time for road traffic injury victims [/fig_ref] illustrates these different time intervals.
## Data source and case selection
Trained EMS ambulance personnel record information about patients/or victims of RTIs. Ambulance personnel are responsible for death and injury registration. For each patient there is a questionnaire to be filled out (see also data collection). After data collection by technicians, these data will be fed into a central computer located at the ambulance site dispatch centre by a trained technician. Initially, all calls to the ambulance centre were reviewed. In total, there were 22 182 registered calls to the EMS in Urmia that resulted in EMS activity. Among all calls during the study period, 2 210 were related to RTIs. The inclusion criteria were, if they qualified as RTI victims and resulted in a ground ambulance dispatch; and received service from them, e.g. they were transported from the scene by one of the region's EMS. Incident locations were defined as "city" and "interurban roads" if they occurred in the Urmia geographic area, according to the Statistical Centre of Iran definition. The cases lacking in complete information about time intervals were excluded (183 cases). In total, 2027 cases were analyzed in this study.
## Data treatment
Descriptive analysis on various pre-hospital time intervals including: activation interval, response interval, onscene interval, transportation interval and the total prehospital intervals were investigated, using mean, median, mode, maximum, minimum and 95% confidence intervals. Moreover, bivariate analyses were conducted for time intervals (five categories) and crash location (two categories) using t-tests and Chi 2 test, to detect significant association and differences (P < 0.05) in distribution between categorical and continuous variables, respectively. Moreover, the distribution of time of injury occurrence (four categories for time of injury; seven categories for date of injury; and four categories for season of injury) was considered for city and interurban roads, using Chi 2 test. In order to test the difference between weekdays and seasons of injury occurrence in relation to crash location, the t-test used again. The SPSS version 13.00 (SPSS Inc, Chicago, IL, USA) was used for data analysis.
The study was approved by the Iranian National Ethics Committee at the Ministry of Health and Medical Education of Iran. Permission was also obtained from Urmia University of Medical Sciences.
# Results
From all ambulance dispatches by EMS, 61.5% of RTIs victims were injured within the city compared to 38.5% cases that occurred on interurban roads. Among them, 27.7% deaths occurred in the city and the rest on interurban roads. Overall, 1.8% of all subjects died after EMS arrival at crash scene or en route to hospitals.
## Time intervals of ems activities
The mean values for the different time intervals of the activities are summarized in [fig_ref] Table 1: Mean, median, mode, minimum and maximum of the time intervals of road... [/fig_ref]. The mean of the response interval and on-scene interval were approximately the same. The transport interval was slightly longer than response intervals. The variation of the total time interval was long with a minimum and maximum of 14-114 minutes, respectively. [fig_ref] Table 2: Pre-hospital time intervals of road traffic injury stratified by crash location in... [/fig_ref] presents time intervals by measurements of the central tendency in the EMS, stratified by city and interurban roads. The mean response time on interurban roads was longer than within the city (10.6 minutes vs. 5.0 minutes, p < 0.001). Moreover, the on-scene interval was longer on interurban roads compare to the city (9.2 vs. 6.1 minutes, p < 0.001). The transport time interval for both city and interurban roads was slightly longer than the response time interval. The mean of total pre-hospital time was almost twice for interurban roads compared to the city (p < 0.001).
As [fig_ref] Table 3: Proportion of the different time intervals of road traffic injury stratified by... [/fig_ref] shows, there was no significant association between activation time for RTIs on city and interurban roads. However, there was significant association for response time in the city compared to on interurban roads (p < 0.001). Focusing on total pre-hospital time intervals, around eight out of ten victims in the city were transported in less than 30 minutes, while for victims on interurban roads; one out of four had been transported in less than 30 minutes.
Response times varied significantly between the city compared to interurban roads (P < 0.001). Close to 90% of response time within the city were less than 8.0 minutes, compared to 45.7% on interurban roads. There was significant association between transportation interval time and crash site (P < 0.001). In general, 99.5% of victims of RTIs in the city had arrived at hospital within 20 minutes of departure from the crash scene. Close to half of the cases on interurban roads reached the hospital more than 45 minutes after departure (P < 0.001).
Asshows, seven out of ten of the injuries occurred between 8.00 am and 8.00 pm. There was an association between time of injury occurrence and crash site (P < 0.001); of which close to half of the injuries on interurban roads occurred between 14:01-20:00, which at the same time this is different from the injury occurrence in the city area. There was an association between the victims' crash site and the days of the week (P = 0.02).
The effect of the date and season of injury on the mean time intervals (response time, transport time and total time interval) of the ambulance dispatches was also explored in this analysis. As [fig_ref] Table 5: Mean of the time intervals on city and interurban roads stratified by... [/fig_ref] shows, there was an association between the mean of the above time intervals during weekdays at crash location (P < 0.05). However, none of the time intervals varied significantly between city and interurban roads areas, when the time intervals were stratified according to season (P < 0.05).
# Discussion
This study estimated the various time intervals for ambulance services and the differences between them for city against interurban roads. The time intervals in this study can be important indicators for EMS performance evaluation in terms of resource planning and maybe also useful for assessing quality of patient care. Most of the time intervals were lower than similar studies in Iran. The time interval between the RTI occurrence and the onset of care at a designated trauma centre has been thought to be an important predictor of victim survival [bib_ref] Systems of trauma care. A study of two counties, West [/bib_ref] [bib_ref] Preventable trauma deaths: Dade County, Florida, Kreis [/bib_ref]. The average response interval in this study was lower than the findings from the capital city of Iran (7.1 vs. 14.9 minutes). The reasons for this shorter interval compared to Tehran, are mainly related to the infrastructure of the study area [bib_ref] Estimating road traffic mortality more accurately: Use of the capturerecapture method in..., Zavareh [/bib_ref]. Urmia is a small city compared to Tehran, which is a very big city and usually suffers from traffic congestion that can result in delays in response time. Focusing on the location of the injury occurrence, the response interval for interurban roads was longer than in the city. Other studies have revealed that rapid responses are believed to have a major effect on the quality of care provided to trauma patients. If time to the trauma centre is a critical variable in the prediction of trauma outcome; then planners of emergency medical services on interurban road areas may be faced with the difficult task of providing services to victims in this geographic location in terms of both prehospital care and hospital-based care, that is in line with line with previous recommendation in other studies [bib_ref] Urbanrural differences in prehospital care of major trauma, Grossman [/bib_ref] [bib_ref] Highway fatal accidents and accessibility of emergency medical services, Brodsky [/bib_ref].
The on-scene interval in this study was shorter than findings in Tehran, 7.4 vs. 18.0 minutes [bib_ref] Prehospital trauma care in Tehran: potential areas for improvement, Modaghegh [/bib_ref]. This time interval mainly can be affected by the skill of EMS team members as well as the involvement of lay people at the crash scene. In recent years many activities have been carried out in the study area [bib_ref] Post-crash management of road traffic injury victims in Iran. Stakeholders' views on..., Khorasani-Zavareh [/bib_ref] aimed at enhancing EMS personnel skills, which has resulted in their better performance during victim rescue. The other reason for such a short interval could also be related to the involvement of members of the public, initially as first responder and then their sense of urgency regarding victim transportation before ambulance arrival. A study in Iran has indicated that, untrained laypeople feel that removing victims from the crash scene and taking them to hospital quickly is the best course of action for their survival [bib_ref] Post-crash management of road traffic injury victims in Iran. Stakeholders' views on..., Khorasani-Zavareh [/bib_ref]. This can result in over-rapid extrication of the casualties from the trapped vehicles before the arrival of the ambulance crew. It is also important to note that, since motor vehicle crashes on interurban road areas often involve high speeds resulting in more serious crashes and injuries and they may require longer extrication times, a result which is line with Grossman et al. [bib_ref] Urbanrural differences in prehospital care of major trauma, Grossman [/bib_ref].
The transport interval in this study was shorter than at other settings in Iran, 10.5 minutes compare to in Tehran [bib_ref] Time intervals for emergency children in Tehran (in Persian), Panahi [/bib_ref]. The reason for this again may mainly be related to the infrastructure of the city and the availability of different hospitals combined with low traffic congestion; factors which can provide better conditions for victim transportation. It is important to note that the response time interval on interurban roads was significantly longer than that within the city. However, compared to the transport interval, this variation was lower. This may imply, on the one hand, that the location of the ambulance dispatch sites allows ambulances to arrive at crash scenes more quickly and hence provide better ambulance access to victims, but, on the other hand, that it takes longer for them to transport victims to hospital. As a result of this comparison, information regarding the effect of time on outcomes may be helpful in decisions on the geographic location of transport and first response teams as well as the sites for ambulance dispatch.
In general, the total pre-hospital time interval in this study was 37.2 minutes. Focusing on this time interval, when it is stratified by crash occurrence in the city and on interurban roads, it was 29.2 and 45.0 minutes, respectively. Compared to a study in Tehran, our finding was low, 37.2 vs. 45.0 minutes. As explained earlier regarding the response time interval, the same reason could be considered for this difference. Moreover, in recent years, the EMS have dramatically improved in the country as a whole [bib_ref] Pattern of motorcycle-related injuries in Tehran, Zargar [/bib_ref] , including an increase in the number of ambulances and ambulance dispatch sites, improvements in the equipment, and educational plans for emergency team staff [bib_ref] Post-crash management of road traffic injury victims in Iran. Stakeholders' views on..., Khorasani-Zavareh [/bib_ref]. All these factors can affect the shorter response as well as total pre-hospital time intervals.
As Calland (2005) noted that " the term Golden Hour, i.e. the first 60 minutes after crash occurrence, was first introduced in 1961, but due to misinterpretation as to what period this actually referred to, a second concept, the "Platinum Ten Minutes" was proposed as the time taken to move a victim to the ambulance. To achieve this rapid removal, the ambulance and medical personnel must work in harmony with the police and fire service to secure the scene and remove the RTI victims safely without causing injury either to the casualty or other personnel on the scene".
Currently there is no available information about the notification interval in this study, however as [fig_ref] Figure 1: Specific intervals and points in time for road traffic injury victims [/fig_ref] shows, this time interval is a hidden part of the total interval and it is crucial to the eventual outcome of each dispatch. This interval may be affected by many factors other than improvement in the pre-hospital care. A study in Tehran revealed that the mean time between the crash occurrence and arrival at the hospital was 170 minutes, while the total time interval from notification to arrival hospital was 120 minutes [bib_ref] Prehospital trauma care in Tehran: potential areas for improvement, Modaghegh [/bib_ref]. This difference between injury occurrence and notification time can argue for the importance of evaluation of the notification interval in Iranian settings through establishment of injury surveillance [bib_ref] Post-crash management of road traffic injury victims in Iran. Stakeholders' views on..., Khorasani-Zavareh [/bib_ref] , which currently is underway.
## Limitation and strength of the study
Accuracy when filling in the data on the questionnaire is one issue. However, data are collected by the trained personnel in the EMS and authors recommend quality control on the data collection process. The future limitation of the study concerns focusing on time intervals and not on quality of care. From the data at hand we could focus on that part of data rather than the severity of the injury and a lack of analysis of potential correlations between injury type and severity. However, they were not within the scope of this study. Another limitation concerns the removal of 187 cases from the list, because of their incomplete data for analysis. We critically tested using the available information at hand and there was no skew and significant variation between this data with that presented in the tables. A strength of this study was that, to our knowledge, it was the first in the country that compared city and interurban roads and the associations and differences for various time intervals, using all cases for a period of two years.
# Conclusion
All time intervals among accident victims on interurban road areas are longer than those for city areas. The short response interval and transport interval compared to other settings may indicate improvement in the prehospital services in the study area. It is important for more attention and care to be paid to the notification interval which should be measured by means of the establishment of a surveillance system at the EMS. A public education campaign to increase cooperation is also an important recommendation of this study. Finally, this study implies the need for investigation of the need-for and access-to emergency medical services [bib_ref] Branas CC: A meta-analysis of prehospital care times for trauma, Carr [/bib_ref] [bib_ref] When is the helicopter faster? A comparison of helicopter and ground ambulance..., Diaz [/bib_ref].
[fig] Figure 1: Specific intervals and points in time for road traffic injury victims. Adapted with some modification from references[15,25]. [/fig]
[table] Table 1: Mean, median, mode, minimum and maximum of the time intervals of road traffic injury in the Urmia from 20 March 2005 to 20 March 2007 [/table]
[table] Table 2: Pre-hospital time intervals of road traffic injury stratified by crash location in the Urmia from 20 March 2005 to 20 March 2007 [/table]
[table] Table 3: Proportion of the different time intervals of road traffic injury stratified by crash location in the Urmia from 20 March 2005 to 20 March 2007 [/table]
[table] Table 5: Mean of the time intervals on city and interurban roads stratified by date, season and crash occurrence in the Urmia from 20 March 2005 to 20 March 2007 [/table]
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Total knee replacement: A comparison of the subvastus and medial parapatellar approaches
Objective: The goal of this study is to compare the subvastus method to the usual medial parapatellar technique for total knee replacement in patients with osteoarthritis who present to a tertiary care centre, based on quadriceps function recovery in days after surgery. Materials and methods: We retrospectively reviewed case notes of 76 patients with osteoarthritis who had total knee replacements in a tertiary care hospital over the course of a year from August 2019 to August 2020. We divided them into two groups: group A received TKR via the subvastus approach, and group B received TKR via the medial parapatellar approach. Preoperative quadriceps strength, BMI, and baseline demographics were all recorded from their initial pre-operative workup case notes. Starting on the first postoperative day, patients recorded first unassisted straight leg raise (SLR) was kept as the main determinant for muscle function. The data were evaluated to determine the quadriceps muscle function post TKR. Results: When compared to the medial parapatellar approach, the quadriceps muscle function returns sooner with the subvastus technique. Patients in their sixties showed the highest improvement. Preoperative quadriceps strength has a major impact on muscle recovery after surgery. Conclusion: The subvastus method to total knee replacement is linked to a faster recovery of quadriceps muscle strength, resulting in a shorter hospital stay and postoperative therapy.
# Introduction
Knee osteoarthritis (OA) is a major public health concern and a primary cause of functional disability and reduced quality of life in the elderly [bib_ref] Women and arthritis: burden, impact and prevention programs, Hootman [/bib_ref]. It is the most common chronic condition in the United States, afflicting 59% of those aged 65 and up. Women, on average, have a higher prevalence of OA than men. The diagnosis of OA of the hip or knee is the same for both men and women. Significant joint pain, unpleasant and limited ambulation, the discomfort that is exacerbated by weight-bearing activities, and noticeable joint space narrowing and osteophyte growth are common symptoms in patients with severe hip or knee OA. Total knee arthroplasty [bib_ref] Health-related quality of life in total hip and total knee arthroplasty. A..., Ethgen [/bib_ref] is a successful operation for treating knee joint deterioration. Over the last three decades, this procedure has grown in popularity. In the United States, the age and gender-adjusted incidence per 100,000 people per year climbed from twenty-nine in , showing a >400% rise in total knee arthroplasty. From 51% of total knee arthroplasties in , the proportion of total knee arthroplasties performed for the treatment of osteoarthritis has increased significantly. The number of primary total knee arthroplasties in the United States is expected to rise from 450,400 in 2008 to 3.48 million by 2030 [bib_ref] What's new in adult reconstructive knee surgery, Deirmengian [/bib_ref].
An anterior midline incision with medial parapatellar arthrotomy is the most common skin incision for primary total knee arthroplasty. To gain access to the knee joint with patellar subluxation, a portion of the quadriceps tendon is incorporated in the arthrotomy.
Alternative ways of exposure have been described to prevent patellofemoral problems and speed up the restoration of quadriceps function after surgery. In terms of subluxing the extensor mechanism laterally for knee exposure, the subvastus ("Southern") technique varies from the medial parapatellar technique [bib_ref] Subvastus (Southern) approach for primary total knee arthroplasty, Hofmann [/bib_ref]. Leave the extensor mechanism intact, according to proponents, results in faster restoration of quadriceps strength, retains greater vascularity in the patella, increases patient satisfaction while reducing postoperative pain, and reduces the need for lateral release. The exposure may be reduced compared to the medial parapatellar approach, especially in obese individuals and those who have had previous knee procedures.
Other approaches have been documented in the literature, with the majority, if not all, aiming to retain the quadriceps mechanism in order to have fast postoperative recovery and quadriceps function.
The midvastus technique, which varies from the subvastus technique in that the vastus medialis muscle is split in line with its fibres rather than being subluxated laterally in its whole [bib_ref] A midvastus muscle-splitting approach for total knee arthroplasty, Engh [/bib_ref] , is another strategy to access the joint. The quadriceps tendon and the superior genicular artery to the patella are preserved with this technique.
The goal of this study is to compare the subvastus technique to the usual medial parapatellar technique for total knee replacement in patients with osteoarthritis who present to a tertiary care centre based on quadriceps function recovery in days.
## Operational definitions
## Quadriceps function
The first unassisted straight leg raise (SLR) was assessed objectively with time in days following total knee replacement and indicated that the muscle had recovered.
## Straight leg raise (slr)
Quadriceps muscle function is recovered when the hip joint is flexed and the knee joint is extended above ground level.
# Materials and methods
This is a retrospective study carried out in a UK tertiary care institution registered under Zaud no 12216. Data from patients who had complete knee replacements from August 2019 to August 2020 was collected after clearance from the clinical governance department and registration as a quality improvement initiative. The study comprised patients of either gender between the ages of 50 and 70, with a BMI between 18.5 and 40 [measured as weight (kg)/height2 (m2)], who were diagnosed with osteoarthritis and required a total knee replacement as a result. Patients with ischemic heart disease with an ejection fraction of <30% were excluded from the study. The sample size after applying the necessary criteria was down to a comparison of 76 patients keeping an equal number of patients for each approach. After collection, the patients were divided into 2 groups based on the approach for surgery. Group A consisted of patients with subvastus approach and Group B included patients with medial parapatellar approach.
The SPSS 16.0 statistical programme was used to analyse the data. The results were provided as mean sd for straight leg raise in days and gender frequency/%. Using the t-test to compare means, a statistical comparison of quadriceps function in days will be done between the two groups. A P-value of less than 0.05 was considered significant. Stratification would be done in regards to age, gender, quadriceps strength and BMI to see the effect of these on outcomes.
# Results
A total of 76 patients' data were examined. The subvastus route was used in 38 individuals, whereas the medial parapatellar route was used in 38 patients. The majority of the patients in our research were women (78%) [fig_ref] Table 1: Demographics. [/fig_ref]. The average BMI was 31.98 ( ±8.858). The preoperative quadriceps was rated as MRC 4 and 5 on the Medical and Research Council (MRC) scale with only a small fraction of the studies patients (29%) having an MRC of 4 and the majority (71%) having an MRC 5 [fig_ref] Table 1: Demographics. [/fig_ref]. Out of the studied patients (72%) had no co-morbidities [fig_ref] Table 1: Demographics. [/fig_ref].
The data clearly show that patients in group A (subvastus approach) had faster recovery of SLR, indicating that the surgery spared the quadriceps. Those in Group A recovered 0.45 days faster in SLR than patients in Group B. With a p-value of 0.008, this difference was likewise statistically significant. [fig_ref] Table 2: Straight Leg Raise [/fig_ref]. BMI did not affect the post-operative recovery of patients' quadriceps strength, and no significant difference (p = 0.104) was detected between patients with a BMI of >30 and those with a BMI of 30 [fig_ref] Table 3: Affect of BMI on straight leg raise [/fig_ref]. Side of surgery again did not show significant relevance (p = 0.833) to the mean number of days for the first postoperative SLR . Gender of the studied patients didn't show any statistical importance (p = 0.182) on SLR in our analysis of collected patient data [fig_ref] Table 5: Stratification according to gender [/fig_ref] (see .
In contrast to the above-mentioned results in and [fig_ref] Table 5: Stratification according to gender [/fig_ref] , when the preoperative quadriceps strength of two groups was compared, it was discovered that there is a statistically significant relationship (p = 0.000) between preoperative quadriceps strength and postoperative muscle function recovery, with patients with preoperative MRC V strength recovering faster than those with MRC IV strength.
It's worth noting that there was a statistically significant association between the ages of the two groups. When the mean age of patients in the medial parapatellar group was 62.74 (5.598) and the mean age of patients in the subvastus group was 58.55 (4.919), we discovered a significant result with p = 0.001. Further investigation of the data revealed that patients in their sixth decade had a statistically significant difference in postoperative SLR between the two groups, as indicated in [fig_ref] Table 7: Stratification according to age [/fig_ref].
# Discussion
TKA procedures that are less invasive are rapidly becoming available. For uni-compartmental knee arthroplasty, small knee capsular incisions were originally documented [bib_ref] Minimally invasive surgical technique for unicondylar knee arthroplasty, Repicci [/bib_ref]. The need for a faster recovery and less time in the hospital following TKA has led to the development of minimal-incision TKA procedures. To present, the literature on minimally invasive procedures for TKA is restricted to anecdotal reports based on one surgeon's experience, with only a few prospective, single-cohort or matched-control comparative studies available [bib_ref] Mini-subvastus versus medial parapatellar approach in total knee arthroplasty, Boerger [/bib_ref] [bib_ref] Minimally invasive total knee arthroplasty: a 10-feature evolutionary approach, Bonutti [/bib_ref] [bib_ref] Minimally invasive total knee replacement through a mini-midvastus incision: an outcome study, Laskin [/bib_ref] [bib_ref] Minimal-incision total knee arthroplasty: the early clinical experience, Tenholder [/bib_ref].
A previous observer blinded study done on 120 patients evaluated two nonrandomized groups of TKAs performed using either the usual medial parapatellar approach or a minimally invasive method using a mini-subvastus incision and followed up to 90 days [bib_ref] Mini-subvastus versus medial parapatellar approach in total knee arthroplasty, Boerger [/bib_ref]. When compared to the traditional technique, the minimally invasive method exhibited certain advantages in terms of functional recovery and postoperative pain. These benefits came at the cost of a heightened risk of problems. However, this was my first time, and there is a learning curve. Their findings matched those of a previous retrospective study done on 58 patients and followed up for 3 months that used minimally invasive techniques like the mini-midvastus or the "quadriceps-sparing" approach [bib_ref] Minimally invasive total knee replacement through a mini-midvastus incision: an outcome study, Laskin [/bib_ref]. A similar study done by the same authors on a 100 patients followed for 2 years gave similar conclusions [bib_ref] Minimally invasive total knee arthroplasty: the results justify its use, Laskin [/bib_ref]. Only recently has the subvastus technique to the knee acquired prominence in knee arthroplasty [bib_ref] Subvastus approach for total knee arthroplasty: a prospective, randomized, and observer-blinded trial, Roysam [/bib_ref] [bib_ref] The standard versus the subvastus apporach for total knee arthroplasty: a randomized..., Marie [/bib_ref] [bib_ref] Subvastus and medial parapatellar approaches in total knee arthroplasyt, Matsueda [/bib_ref] [bib_ref] Subvastus versus medial parapatellar apporahc in total knee arthroplasty, Cila [/bib_ref]. A randomized blinded trial done on 89 patients showed that the subvastas technique offered early advantages in gaining mobility for patients postop [bib_ref] Subvastus approach for total knee arthroplasty: a prospective, randomized, and observer-blinded trial, Roysam [/bib_ref].
This method offers several anatomic advantages that may help with knee arthroplasty function and survival. The supreme (descending) geniculate, superomedial and inferomedial geniculate, superolateral and inferolateral geniculate, and anterior tibial recurrent arteries all feed a parapatellar extraosseous ring of vessels that supplies blood to the patella [bib_ref] Comparison of the subvastus and paramedian surgical approaches in bilateral knee arthroplasty, Faure [/bib_ref].
The supreme, superomedial, and inferomedial geniculate arteries' contributions to the perivascular ring are severed in the medial parapatellar approach. The subvastus primarily preserves the contribution of the medial patellar blood flow, which may reduce the risk of patellar avascular necrosis in theory.
A comparison study of 20 patients undergoing one-stage bilateral knee arthroplasty found that patients prefer the subvastus method to the parapatellar technique. One knee was exposed using the parapatellar technique, while the second knee was exposed using the subvastus technique [bib_ref] Comparison of the subvastus and paramedian surgical approaches in bilateral knee arthroplasty, Faure [/bib_ref]. The objective and functional outcomes measured by the knee society rating scales did not differ substantially between the two groups in the same trial; however, the medial parapatellar approach group's higher incidence of patellar maltracking may have a late significant effect on knee function.
Another studydiscovered that subvastus appeared to provide an early benefit to knee arthroplasty patients in the postoperative period, as seen by significantly reduced pain medication dosages utilised in the subvastus approach group during the first 48 h. SLR might be completed in substantially less time by the Subvastus group. Patients who had the subvastus technique required 40% less postoperative pain medications and could straight leg lift on average 2.6-4 days sooner than those who had the traditional medial parapatellar technique, according to other research [bib_ref] Subvastus approach for total knee arthroplasty: a prospective, randomized, and observer-blinded trial, Roysam [/bib_ref] [bib_ref] The standard versus the subvastus apporach for total knee arthroplasty: a randomized..., Marie [/bib_ref].
Up to one month after surgery, the same patient who had the subvastus technique showed considerable improvement in quadriceps strength. However, by the third month after surgery, this early function advantage had faded and did not appear to confer any long-term benefit. These findings are consistent with other reports that patients with total knee arthroplasty usually regained quadriceps strength with little difference between the medial parapatellar approach and the subvastus approach, and that patients with total knee arthroplasty usually regained quadriceps strength with little difference between the medial parapatellar approach and the subvastus approach [bib_ref] Subvastus approach for total knee arthroplasty: a prospective, randomized, and observer-blinded trial, Roysam [/bib_ref] [bib_ref] The standard versus the subvastus apporach for total knee arthroplasty: a randomized..., Marie [/bib_ref] [bib_ref] Subvastus versus medial parapatellar apporahc in total knee arthroplasty, Cila [/bib_ref].
In line with prior studies [bib_ref] Subvastus approach for total knee arthroplasty: a prospective, randomized, and observer-blinded trial, Roysam [/bib_ref] [bib_ref] The standard versus the subvastus apporach for total knee arthroplasty: a randomized..., Marie [/bib_ref] , their findings revealed no significant changes in the postoperative range of knee motions offered by the subvastus approach at any time period, as well as no significant differences in operating time or postoperative blood loss.
A review by Bonutti et al. found that the least invasive and standard groups had equal mean Knee Society scores [bib_ref] Minimally invasive total knee arthroplasty, Bonutti [/bib_ref]. A randomized multi centre study done by Kolisek et al. on 80 knees found that at the three-month follow-up.these two approaches had identical scores [bib_ref] Clinical experience using a minimally invasive surgical approach for total knee arthroplasty:..., Kolisek [/bib_ref]. Seon et al. in their study found that patients who had been randomised to minimally invasive surgery had significantly better scores on a 10-point visual analogue pain scale on postoperative day 3 than patients who had a standard approach, but the scores obtained at two weeks indicated equivalence between the cohorts [bib_ref] Navigation-assisted less invasive total knee arthroplasty compared with conventional total knee arthroplasty:..., Seon [/bib_ref]. Some writers have taken a firm stance against the use of minimally invasive procedures, citing the lack of a demonstrated advantage in other similar studies [bib_ref] Clinical experience using a minimally invasive surgical approach for total knee arthroplasty:..., Kolisek [/bib_ref].
In this study, we looked at patients' quadriceps muscular strength recovery after surgery, as well as their probable implications on the outcome and early postoperative rehabilitation, which led to early discharges. In terms of faster recovery of straight leg raise (SLR) in the subvastus group, our findings are comparable with those found in the literature. It's also worth noting that this faster recovery was especially noticeable in people between the ages of 60 and 70. Preoperative quadriceps strength has a substantial impact on early postoperative SLR attainment. The current study, however, showed no evidence of a There are a few imitations to our study. Our study was a retrospective one and we had to work of data recorded earlier. Sample size for the study was a small one and we don't have long term follow up to see how patients in each group progressed on 3 and 6 month follow up. We plan on following up this study with a randomized control trial with a bigger cohort and following them up for at least a year to see how long term follow up in both approaches can be seen.
# Conclusion
In conclusion, complete knee replacement can be performed successfully using either of the above techniques; but, based on our findings, we may conclude that patients who underwent surgery using the subvastus approach have faster recovery of quadriceps strength. This supports our hypothesis that the subvastus technique is better anatomic and facilitates faster postoperative recovery.
[table] Table 1: Demographics. [/table]
[table] Table 2: Straight Leg Raise (SLR) in two approaches. [/table]
[table] Table 3: Affect of BMI on straight leg raise. Data stratification according to side of surgery. [/table]
[table] Table 5: Stratification according to gender. Stratification according to preoperative quadriceps strength. [/table]
[table] Table 7: Stratification according to age. [/table]
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Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation
[bib_ref] The changing preference of T and B cells for partners as T-dependent..., Maclennan [/bib_ref] [bib_ref] Clonal selection and learning in the antibody system, Rajewsky [/bib_ref] [bib_ref] Heavy-chain class switch does not terminate somatic mutation, Shan [/bib_ref] [bib_ref] Immunoglobulin class switch recombination, Harriman [/bib_ref] [bib_ref] Isolation of high-affi nity memory B cells: phycoerythrin as a probe for..., Hayakawa [/bib_ref] [bib_ref] The repertoire of somatic antibody mutants accumulating in the memory compartment after..., Weiss [/bib_ref] [bib_ref] Evidence for a large compartment of IgM-expressing memory B cells in humans, Klein [/bib_ref] [bib_ref] Identifi cation of functional human splenic memory B cells by expression of..., Tangye [/bib_ref] [bib_ref] B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B..., Agematsu [/bib_ref] [bib_ref] Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface..., Klein [/bib_ref] [bib_ref] B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B..., Agematsu [/bib_ref] [bib_ref] CD27 expression in the human splenic marginal zone: the infant marginal zone..., Zandvoort [/bib_ref] [bib_ref] Immunophenotypic analysis of B cells in PNH: insights into the generation of..., Richards [/bib_ref]
## Molecular footprints of a germinal center derivation of human igm + (igd + )cd27 + b cells and the dynamics of memory b cell generation marc seifert and ralf küppers
Institute of Cell Biology (Cancer Research), University of The origin of IgM + CD27 + B lymphocytes with mutated IgV genes, which account for ف
## 20% of human peripheral blood (pb) b cells, is controversially discussed. a generation in a primary diversifi cation pathway, in t cell-independent immune responses, or in t celldependent germinal center (gc) reactions has been proposed. we show here that igm + igd + cd27 + and igm + igd -/low cd27 + b cell subsets carry, like class-switched memory b cells, mutations in the bcl6 gene as a genetic trait of a gc experience. moreover, the identifi cation of pb igm + igd + cd27 + b cells clonally related to gc-derived igg + memory b cells with shared and distinct igv gene mutations demonstrates the gc origin also of the former subset. these fi ndings provide genetic evidence for a gc derivation of somatically mutated igm + b cells and indicate that adult humans harbor a large population of igm + igd + post-gc memory b cells. furthermore, the analysis revealed that a highly diverse and often very large population of memory b cells is generated from a given gc b cell clone, and that (preferentially igm) memory b cells are generated already early in the gc reaction. this provides novel insights into the dynamics of gc reactions and the generation of a memory b cell repertoire.
clonally related IgM + IgD + CD27 + and class-switched B cells in the PB, and these cells should carry shared and distinct somatic V gene mutations.
# Results
## Bcl6 mutations in human cd27 + b cell subsets
We analyzed the mutation status of the Bcl6 -MMC in FACSsorted B cells from three healthy adult blood donors by single-cell PCR. This approach allows the reliable detection of rare mutations, as PCR-introduced mutations are negligible in the direct sequence analysis of the amplifi cates from single cells. As it is relevant for the determination of the mutation frequency whether one or both alleles of a cell were amplifi ed, we analyzed donors who were prescreened to carry a known single-nucleotide polymorphism (SNP; rs3832246, 1 bp deletion) on one allele of Bcl6 , so that for unmutated sequences it could also be determined whether one or both alleles were amplifi ed. Overall, from ف 21% of the cells (range 14-25%) both alleles were amplifi ed [fig_ref] Table I: Bcl6 mutations in human PB B cells [/fig_ref]. The Bcl6 -MMC of all 60 PCR-positive naive B cells was unmutated, whereas 16 of 75 (21%) IgM + IgD + CD27 + B cells, 13 of 64 (20%) IgM-only, and 19 of 57 (33%) class-switched B cells carried mutations. In the CD27 + B cell subpopulations, 8-12 cells had a single mutation and 4-9 cells harbored 2-6 mutations per sequence. The mean mutation frequencies were 0.045% (range 0.036-0.084%) for IgM + IgD + CD27 + B cells, 0.050% (range 0.013-0.119%) for IgM-only B cells, and 0.085% (range 0.082-0.093%) for class-switched B cells. Thus, IgM + IgD + CD27 + and IgM-only B cells carry classswitched B cells in a fraction of cells Bcl6 mutations as a footprint of a GC passage of these B cells. The mutations were distributed throughout the whole MMC, and besides two 1-bp deletions and one 5-bp deletion, the mutations were single basepair substitutions with a bias for transitions in IgM + IgD + CD27 + , IgM-only, and class-switched B cells (63, 58, and 46%, respectively, as compared with an expected frequency of 33% for unbiased mutations). The overrepresentation of transitions over transversions was statistically signifi cant only for both IgM + B cell subsets (P < 0.001 each), but not in those of class-switched B cells (P < 0.09). The RGYW and WRCY motifs are known SHM hotspots [bib_ref] Somatic hypermutagenesis in immunoglobulin genes. II. Infl uence of neighbouring base sequences..., Rogozin [/bib_ref] , and each of the three CD27 + B cell subsets had an enrichment of mutations in RGYW/WRCY motifs. 28.6% of the mutations in IgM + IgD + CD27 + , 18.5% in IgM-only, and 40.5% in class-switched B cells were within the motifs, in comparison to an expected frequency of 16.7%, assuming unbiased targeting of these motifs. The diff erence between observed and randomly expected targeting of RGYW/WRCY motifs reached statistical signifi cance only for class-switched B cells (P < 0.001). However, in another study Bcl6 mutations in class-switched B cells from human tonsil also did not show signifi cant RGYW targeting [bib_ref] Diff erent patterns of bcl-6 and p53 gene mutations in tonsillar B..., Yavuz [/bib_ref]. The fact that the intrinsic SHM biases analyzed did not reach statistical signifi cance in all instances might be caused by the restricted number of mutation events analyzed and/or by the fact that some Bcl6 mutations infl uence Bcl6 cells upon appropriate stimulation [bib_ref] B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B..., Agematsu [/bib_ref] [bib_ref] Maintenance of serological memory by polyclonal activation of human memory B cells, Bernasconi [/bib_ref] [bib_ref] A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by..., Bernasconi [/bib_ref] [bib_ref] Functional analysis of human memory B-cell subpopulations: IgD+CD27+ B cells are crucial..., Shi [/bib_ref] , and respond in vitro to various stimuli more similar to class-switched memory cells than to naive B cells [bib_ref] Kinetics of human B cell behavior and amplifi cation of proliferative responses..., Good [/bib_ref] [bib_ref] Decreased expression of Kruppel-like factors in memory B cells induces the rapid..., Good [/bib_ref].
However, recent studies on human IgM + IgD + CD27 + B cells indicated that other mechanisms might be responsible for the generation and maintenance of these cells. Based on a reported dependency of these cells on the splenic environment, which is known to be important for T cell-independent (TI) type II immune responses, it was proposed that IgM + IgD + CD27 + (as well as IgM-only B cells, which were not distinguished in that work) are generated in TI responses [bib_ref] Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated..., Kruetzmann [/bib_ref]. In another scenario, it was proposed that the mutated IgM + IgD + CD27 + B cells underwent SHM in a primary, antigen-independent diversifi cation mechanism outside GC, as it is known to occur in sheep and rabbits [bib_ref] Rabbit IgH sequences in appendix germinal centers: VH diversifi cation by gene..., Weinstein [/bib_ref] [bib_ref] Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process, Reynaud [/bib_ref]. This idea was based on the observation that x-linked hyper-IgM patients apparently lack GC (caused by defective CD40Lsignaling) and class-switched, as well as IgM-only, B cells, but still have substantial numbers of mutated IgM + IgD + CD27 + B cells [bib_ref] CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversifi cation..., Weller [/bib_ref] [bib_ref] Human blood IgM "memory" B cells are circulating splenic marginal zone B..., Weller [/bib_ref]. Several fi ndings supported this scenario, including an analysis of CDRIII length diversity of the respective subsets, particularly the presence of rare mutated IgM + CD27 + B cells in fetal liver [bib_ref] T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+..., Scheeren [/bib_ref] and neonatal cord blood [bib_ref] CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversifi cation..., Weller [/bib_ref] , as well as the detection of transcripts of activation-induced cytidine deaminase, an enzyme that is essential for SHM and class switching, in human cord blood transitional type 1/immature B cells [bib_ref] Activation-induced cytidine deaminase expression and activity in the absence of germinal centers:..., Kuraoka [/bib_ref].
As IgM + IgD + CD27 + B cells and IgM-only B cells account for ف 15 and 5% of human peripheral blood B cells, respectively, and occur at similar frequencies also in secondary lymphoid tissues [bib_ref] Analysis of somatic mutation in fi ve B cell subsets of human..., Pascual [/bib_ref] [bib_ref] Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected..., Dunn-Walters [/bib_ref] , the cellular origin and functions of about a quarter of human mature B cells are currently controversially discussed.
To clarify whether mutated IgM + B cells are post-GC cells, we analyzed molecular footprints of GC reactions in human B cell subsets. It has been shown that Bcl6, the master transcriptional regulator in GC B cells [bib_ref] Germinal centres: role in B-cell physiology and malignancy, Klein [/bib_ref] , is aff ected by the SHM machinery in GC B cells, resulting in ف 30% of GC and class-switched memory B cells carrying Bcl6 mutations in the intronic major mutation cluster (MMC; [bib_ref] BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation..., Pasqualucci [/bib_ref] [bib_ref] Mutation of BCL-6 gene in normal B cells by the process of..., Shen [/bib_ref] [bib_ref] Nonimmunoglobulin gene hypermutation in germinal center B cells, Peng [/bib_ref]. As SHM of IgV and non-Ig genes is strictly dependent on high-level transcription of the target sequences [bib_ref] Somatic hypermutation in the heavy chain locus correlates with transcription, Fukita [/bib_ref] [bib_ref] Increased transcription levels induce higher mutation rates in a hypermutating cell line, Bachl [/bib_ref] [bib_ref] Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer..., Yang [/bib_ref] , Bcl6 should only be mutated in GC B cells that express high levels of this gene, but not in non-GC B cells that lack Bcl6 expression or show only low transcript levels. Thus, if the vast majority of IgM + CD27 + B cells acquired somatic IgV gene mutations in a GC-independent way, and hence in the absence of Bcl6 transcription, these cells should lack Bcl6 mutations. Second, if IgM + IgD + CD27 + and class-switched B cells derive from common GC B cell clones, one may fi nd and C /C ␦ primers [fig_ref] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences... [/fig_ref]. The C ␥ primer used in this and the experiment described further below is specifi c for the C ␥ 1 and C ␥ 3 genes, which are classically used by post-GC memory B cells, but does not amplify C ␥ 2 transcripts, which mainly derive from TI responses [bib_ref] Human antibodies to group A streptococcal carbohydrate. Ontogeny, subclass restriction, and clonal..., Shackelford [/bib_ref]. All C H primers were complementary to positions in equal distance from the fi rst coding nucleotide of the corresponding C H gene. Thus, an identical CDRIII length of putative clone members in IgM + IgD + CD27 + and IgG + CD27 + B cells would result in equal-size products in a CDRIII-spectratyping analysis, performed on neighboring lanes of a PAGE gel. Such bands of interest were cut out from the gel, reamplifi ed, and sequenced. Overall, 120 pairs of identical length were analyzed from sorted cells of the 10 donors. Most of these products (85%) were oligo-to polyclonal and not further followed up. From the monoclonal products in one instance, clonally related V H 1-18 -expressing IgM and IgG transcripts were obtained, and after cloning of the amplifi cates 7 distinct IgG + and 2 distinct IgM + members of the clone were identifi ed [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref]. The clone members contained between 3 and 9 V H gene mutations per sequence, of which 1 to 7 were shared by at least two clone members. Importantly, the genealogical tree depicting the most likely clonal evolution revealed that several IgG + clone members descended from a proposed intermediate with only a single point mutation, expression [bib_ref] Mutations of the BCL6 proto-oncogene disrupt its negative autoregulation in diff use..., Pasqualucci [/bib_ref] and hence may be selected for or against.
## Clonal relation of human igm + igd + cd27 + and classswitched memory b cells
If IgM + memory B cells and class-switched B cells both are descendants of common GC B cell clones, it should principally be possible to fi nd clonally related IgM + and IgG + B cells with shared and unique mutations in PB. We focused on IgM + IgD + CD27 + B cells because their origin is more controversial than that of IgM-only B cells [bib_ref] CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversifi cation..., Weller [/bib_ref] , and for our analysis it would have been diffi cult to obtain enough cells from the small IgM-only subset.
Our fi rst approach to identify such clonally related cells was based on a PAGE strategy and aimed at very large clones. Considering the number of cells that could be used per PCR reaction (30,000) and the total number of IgG + CD27 + and IgM + IgD + CD27 + B cells in the PB ( ف 6 × 10 8 per subset), we calculated that a putative clone should have at least 20,000 members in each of the two subsets to be detectable by this approach. Several aliquots of 30,000 IgG + CD27 + and IgM + IgD + CD27 + B cells each were sorted from PB of 10 healthy donors. Extracted RNA was separately reverse transcribed with gene-specifi c primers for C ␥ and C /C ␦ and amplifi ed with a V H 1 family-specifi c primer and the same C ␥ duced heterogeneity of the population. However, as V H 3-23 is one of the most frequently used V H gene segments [bib_ref] Analysis of the heavy chain repertoire of human peripheral B cells using..., Brezinschek [/bib_ref] , we aimed to clarify whether clonally related IgM + IgD + and IgG + B cells can also be found for such a gene segment. The amplifi ed V H 6 and V H 3-23 rearrangements of IgG + B cells were cloned and a number of sequences (8 to 30 per PB donor) were randomly chosen to design CDRIII-specifi c primers. These primers were mainly binding to the 3 Ј end of CDRIII (and in several instances parts of FRIV), so that enough sequence from the 5 Ј part of CDRIII was included in the resulting amplifi cates to allow an unequivocal determination of the clonal relationship of the amplifi ed rearrangements. The CDRIII primers were used together with the V H 6 or V H 3-23 primer to amplify clonally related sequences from the V H 6 -C /C ␦ -or V H 3-23 -C /C ␦ -amplifi ed cDNA from IgM + IgD + CD27 + B cells, respectively. From donors 1-6, we were able to amplify and directly sequence 21 monoclonal PCR products from V H 6 -expressing IgM + IgD + CD27 + B cells (range 2-5 per donor; oligo-or polyclonal amplifi cates caused by cross-hybridization of the "clone-specifi c" primers on other V H 6 gene rearrangements were not further considered). Of these 21 products, 11 V H 6 whereas the two IgM sequences shared 5 additional point mutations with the other IgG + sequences. We conclude that the IgM + B cells acquired most if not all of their mutations in the clonal evolution of the GC B cell clone that gave rise to these memory B cells.
As the gel-based approach was restricted by the relatively small number of cells that could be analyzed for a clonal relationship, we established an RT-PCR-based approach with clone-specifi c primers to screen larger cell samples [fig_ref] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences... [/fig_ref]. RNA from 10 6 to 1.5 × 10 6 sorted IgG + and IgM + IgD + CD27 + B cells isolated from 8 adult peripheral blood donors, and in two instances from naive B cells (donors 7 and 8), was extracted and reverse transcribed with C ␥ and C /C ␦ primers, respectively. The cDNAs from both subsets were amplifi ed with primers for V H 6 -FR1 (donors 1-6) or V H 3-23 leader peptide sequence (donors 7 and 8) in combination with nested constant region primers. The nesting eff ect of the second constant primer set prevents the amplifi cation of V H D H J Hrearrangements from potential cell sorting contaminations, i.e., IgM transcripts from an IgG + B cell sort and vice versa. By analyzing the single member V H 6 family, we aimed to enhance the chance to fi nd clonally related cells caused by a re- rearrangements from IgM + IgD + CD27 + B cells were clonally related to the respective V H 6 rearrangements from the IgG + B cells [fig_ref] Table I: Bcl6 mutations in human PB B cells [/fig_ref]. From donors 7 and 8, we obtained 9 and 7 monoclonal PCR products, respectively, from V H 3-23expressing IgM + IgD + CD27 + B cells. Of these 16 products, 3 V H 3-23 rearrangements from IgM + IgD + CD27 + B cells were clonally related to the respective V H 3-23 rearrangements from the IgG + B cells [fig_ref] Table I: Bcl6 mutations in human PB B cells [/fig_ref]. With the "clone-specifi c" primers for these three memory B cell clones, no clonally related sequences were obtained from the corresponding naive B cells, although 14-21 sequences from the cloned PCR amplifi cates were analyzed for the three clones. Instead, the sequences from the naive B cells consisted of 4-10 diff erent unmutated sequences with varying CDRIII length and composition [fig_ref] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences... [/fig_ref]. As one would not expect to fi nd naive B cell clone members among the isolated cells, the lack of CDRIII sequences shared by the naive and IgG + B cells validates the reliability of the approach to identify clonally related sequences among the mutated IgD + IgM + and IgG + B cells.
To study the intraclonal diversity of the clonally related V H -C and V H -C ␥ amplifi cates, for each of the 11 V H 6 and 3 V H 3-23 clones, PCR products obtained with the CDRIIIspecifi c primers from the IgG + and IgM + IgD + CD27 + B cells were cloned into plasmids and several of these were sequenced. Sequence alignments of fi ve selected sequences per clone, including the initial IgG sequence, are depicted in [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref].
For all 14 clones expressing V H 6 or V H 3-23 , multiple distinct members, diff ering from each other by single, few, or >24 point mutations were found [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref]. The overall mutation frequency of the IgG + CD27 + B cells (including clone A from the PAGE analysis) is 6.2% and of the IgM + IgD + CD27 + B cells 4.2%. Genealogical trees were generated to visualize the mutational evolution of the clones, and a heterogenous picture was obtained [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref]. Moreover, in 6 clones, IgM members are mainly early progeny of the clones and hence carry relatively few mutations [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] , clones H-M), whereas the IgG + post-GC B cells -with few exceptions -have acquired more shared and distinct mutations. In the other 9 clones, including clone A [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] , IgG + and IgM + clone members are found more intermingled. Notably, also in these trees, IgG sequences on average carry more mutations than IgM sequences. Unmutated IgM + sequences were detected in two clones [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] , and unmutated IgM and IgG sequences were detected in one clone [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref].
Considering the overall shapes of the trees depicting the clonal evolution of the B cell clones, a striking diversity is evident. Each clone consists of a high number of branches, and many distinct sequences were found. This shows that throughout the GC reaction, and starting early during clonal expansion and mutation accumulation, clone members are selected to leave the GC and become members of the memory B cell pool.
Assuming that all clone members in a sample were detected (certainly an underestimation), we estimated the total number of clone members in the PB of the donors. Counting repeatedly identifi ed sequences only once, we calculated a clone size of 190 B cells for the smallest clone [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] and 3,600 B cells for the largest clone [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] detected in our study (see Materials and methods).
# Discussion
Human IgM-only and IgM + IgD + CD27 + B cell subsets carry molecular footprints of a GC reaction To clarify the still unresolved origin of somatically mutated IgM + B cells in humans, we analyzed mutations in the Bcl6 gene as a genetic trait of a previous GC experience. These mutations are found in ف 30% of GC B cells and classswitched memory B cells and show key features of SHM [bib_ref] BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation..., Pasqualucci [/bib_ref] [bib_ref] Mutation of BCL-6 gene in normal B cells by the process of..., Shen [/bib_ref]. As the mutation frequency in Bcl6 is ف 50-100-fold lower than the one of IgV genes [bib_ref] BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation..., Pasqualucci [/bib_ref] , this explains why such mutations are not found in all class-switched memory B cells. Importantly, SHM is strictly dependent on transcription of its target genes [bib_ref] Somatic hypermutation in the heavy chain locus correlates with transcription, Fukita [/bib_ref] [bib_ref] Increased transcription levels induce higher mutation rates in a hypermutating cell line, Bachl [/bib_ref] [bib_ref] Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer..., Yang [/bib_ref]. On this basis, we reasoned that Bcl6 mutations should only be present in IgM + B cells with mutated IgV genes when these cells acquire their mutations in a GC reaction, i.e., when Bcl6 is strongly transcribed [bib_ref] Germinal centres: role in B-cell physiology and malignancy, Klein [/bib_ref]. Indeed, although there is a low basal level of Bcl6 transcription in mature resting naive human B cells [bib_ref] Alterations of a zinc fi nger-encoding gene, BCL-6, in diff use large-cell..., Ye [/bib_ref] , this is further reduced when the cells undergo TI immune responses outside GC [bib_ref] BCL-6 expression during B-cell activation, Allman [/bib_ref]. Thus, Bcl6 should not acquire mutations in case SHM would be active in TI responses. We cannot formally exclude that Bcl-6 is transcribed during the proposed primary V gene diversifi cation pathway for the generation of somatically mutated IgM + IgD + CD27 + B cells; however, this seems very unlikely considering that Bcl6 is the master regulator of the GC B cell diff erentiation program [bib_ref] Germinal centres: role in B-cell physiology and malignancy, Klein [/bib_ref].
As somatic mutations in IgV genes and Bcl6 are generated by the same SHM machinery [bib_ref] BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation..., Pasqualucci [/bib_ref] , one would expect that their levels are correlated, and this is exactly what we observed. Both IgM + CD27 + B cell subsets have ف 50-60% of the mutation load of class-switched memory B cells in their IgV genes and in Bcl6 . In conclusion, the detection of Bcl6 mutations in IgM + IgD + CD27 + and IgMonly B cells strongly argues that most if not all of these cells derive from a GC reaction.
## Derivation of igm + igd + cd27 + and igg + cd27 + b cells from common gc b cell clones
If one assumes that IgM + IgD + CD27 + and IgG + CD27 + B cells derive from common GC B cell clones, and hence both represent post-GC memory B cells, fi nding of clonally related IgM + and IgG + PB B cells would represent an important validation for this assumption. However, a clonal relationship as such does not discriminate between the three proposed origins of somatically mutated IgM + IgD + CD27 + B cells. If these cells acquire IgV gene mutations during an Gp denotes the IgG + B cell sequence that was used to design the clone-specifi c primer. Numbers besides the lines indicate the number of (additional) somatic mutations acquired from a given cell to its descendent. Each tree is rooted by the putative, unmutated V H rearrangement, marked as "gl" (the cells were generated in GC reactions. Nevertheless, the fact that we found clonally related IgM + IgD + CD27 + B cells for ف 40% of the informative IgG + memory B cells (in total, 14 of 37 C ␥ transcript sequences tested with monoclonal C or C ␦ amplifi cates) indicates that GC B cell clones regularly give rise to both IgM and IgG memory B cells. That we did not fi nd IgM + members for all clones may simply be caused by technical matters (e.g., mutations in IgM + clone members at primer binding sites) or a smaller size of many memory B cell clones, so that in the aliquot of PB that we analyzed no IgM members were present (note that we sampled ف 10% of PB B cells, but as only 2% of lymphocytes are present in PB [bib_ref] Lymphocyte numbers and subsets in the human blood. Do they mirror the..., Blum [/bib_ref] , overall we analyzed only an aliquot of 0.2% of B cells). Moreover, it is remarkable that in many of the clones the number of (distinct) members was similar for IgM + and IgG + B cells, which indicates that for at least half of the IgG + memory B cells a population of clonally related IgM + memory B cells of similar size exists. Considering that the frequency of IgG + memory B cells in the PB is similar to the frequency of IgM + CD27 + B cells, this, together with the results of the Bcl6 mutation analysis, suggests that indeed most if not all of the IgM + CD27 + B cells in human adults are post-GC B cells.
How do these fi ndings relate to the indications for a generation of somatic mutations in IgM + IgD + CD27 + (and IgMonly) B cells by a prediversifi cation mechanism or in the course of TI immune responses? Regarding an origin of IgM antigen-independent primary diversifi cation process, these cells may also enter GC reactions upon TD activation and give rise to IgG + memory B cells. Even if IgM + IgD + CD27 + B cells represent GC independent memory B cells from TI immune responses, such cells may, in particular circumstances, be driven into GC reactions and become IgG + memory B cells. It is only the mutation pattern within a clone that can discriminate between the diff erent scenarios. Several of the clones are indeed unhelpful in clarifying this issue. In three clones shown in [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] ( I, K, and L ), all IgG + clone members may potentially derive from one prediversifi ed IgM + B cell or a memory B cell from a TI response that was driven into a GC reaction. However, in two other clones [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] such a scenario is already less likely, as here one would have to postulate that two diff erent members of a prediversifi ed clone were driven into GC reactions. Importantly, the pattern of mutation accumulation in the other 10 clones [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] , A-G and M-O ) is incompatible with a non-GC dependent scenario. In these clones, IgG and IgM members are intermingled in the genealogical trees, and some of the IgG cells can be found at earlier branches in the trees than IgM members, despite a number of shared mutations. Thus, in these clones IgG + and IgM + members coevolved from a common GC B cell clone.
The selective search for clonally related IgG + CD27 + and IgM + IgD + CD27 + B cells does not allow one to determine whether all or only a small fraction of IgM + IgD + CD27 + B roots of clones E, G, and L also include unmutated members). The trees were constructed to maximize the number of shared and minimize the number of unique mutations. In rare instances, a reversion of a formerly shared mutation was assumed to ensure consistency of the dendrogram. Importantly, the overall shape of the respective tree was never affected. In two instances (clones H and M), the development of IgG clones members could principally be explained by two independent prediversifi ed IgM clone members (marked with an "x") entering GC reactions. ticular immune reactions associated with diminished V gene diversifi cation and/or hampered T cell help. Moreover, the detection of unmutated members of memory B cell clones might indicate that the few unmutated B cells that were described in previous V gene studies of CD27 + B cells are (mostly) memory B cells and not cell sorting contaminants. Also class switching is initiated very early during the clonal expansion, as multiple IgG + clone members with few somatic mutations were detected. Second, the population of memory B cells generated from a GC B cell clone is often very diverse in terms of V gene sequences. This implies that memory B cells with a wide spectrum of affi nities and antigen-binding characteristics is generated from a GC B cell clone. This might facilitate the recognition of slightly modifi ed pathogens in recall responses by the memory B cell pool. Third, although IgM and IgG cells are often found intermingled in the genealogical trees, there is a clear tendency that IgM + memory B cells were generally generated earlier than IgG + memory B cells and hence show a lower overall mutation load (4.2% for IgM + cells versus 6.2% for IgG + memory B cells). These values are the same as those determined previously for the populations of IgM + IgD + CD27 + and IgG + memory B cells . Therefore, our analysis of clonally related IgM + IgD + CD27 + and IgG + memory B cells indicates that a main determinant for the lower mutation load of the mutated IgM + B cells is their earlier generation during GC reactions, and not, for example, a generation in separate, perhaps less effi cient GC responses.
## Concluding remarks
The results of the present study indicate that there is a large population of IgM + memory B cells in human adults. Although the lower mutation load of these cells as compared with class-switched memory B cells may imply that the antibodies produced by these cells are usually of lower affi nity that the one of IgG antibodies, this may be compensated for by the higher avidity of the pentameric IgM complexes when B cells diff erentiate into plasma cells and secrete antibody. Moreover, these memory B cells may serve as a fl exible memory B cell reservoir that may reenter GC upon encounter with the same or a similar antigen to undergo further affi nity maturation and be able to switch the isotype of their BCR to the needs of the particular recall response. Besides the importance of revealing the origin of about a quarter of the human B cell pool, the present work also has important implications regarding the origin and consequence of dysregulated B cells in autoimmune diseases and immunodefi ciencies, as well as B lymphoid malignancies. For example, many mature B cell lymphomas with somatically mutated IgV genes express IgM (and often also IgD), and the present study therefore supports the view that these are GC B cellderived lymphomas [bib_ref] Cellular origin of human B-cell lymphomas, Küppers [/bib_ref].
# Materials and methods
Cell separation. Buff y coats of healthy adult donors were obtained from the blood bank of the Institut für Transfusionsmedizin of the Medical School memory B cells from TI responses, it may well be that a small fraction of these cells were indeed generated in such immune reactions. However, the reported specifi c dependence of IgM memory B cells from the spleen with its marginal zone, and hence the association with TI type II responses [bib_ref] Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated..., Kruetzmann [/bib_ref] , was not seen in several other studies [bib_ref] Human blood IgM "memory" B cells are circulating splenic marginal zone B..., Weller [/bib_ref] [bib_ref] Memory B cells and pneumococcal antibody after splenectomy, Wasserstrom [/bib_ref] [bib_ref] Role of the spleen in peripheral memory B-cell homeostasis in patients with..., Martinez-Gamboa [/bib_ref] , and there is evidence from studies in rodents that the marginal zone is also a reservoir for post-GC memory B cells [bib_ref] Memory B cells in T celldependent antibody responses colonize the splenic marginal..., Liu [/bib_ref] [bib_ref] Functional heterogeneity of marginal zone B cells revealed by their ability to..., Song [/bib_ref] , so a relationship between PB IgM + CD27 + B cells and splenic marginal zone B cells does not argue against a GC derivation of the cells. Regarding the evidence for a prediversifi cation mechanism in IgM + IgD + CD27 + B cells, the recent description of a small population (3-5% of IgD + B cells) of somatically mutated IgM + IgD + CD27 + B cells in fetal tissues indeed argues for a GC-independent development of these cells [bib_ref] T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+..., Scheeren [/bib_ref]. Importantly, however, only ف 20% of these fetal IgM + CD27 + B cells harbor somatic mutations, and the mutation load in these mutated cells was only about half of that of IgM + IgD + CD27 + B cells in adults . Another main argument for a GC-independent origin of mutated IgM + IgD + CD27 + B cells relates to their presence in children aff ected by the x-linked hyper IgM syndrome [bib_ref] CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversifi cation..., Weller [/bib_ref]. However, in these patients only a fraction of IgM + IgD + CD27 + B cells carried mutated V genes, and a recent study indicated that the mutation frequency of the mutated V genes is often considerably lower than that of the corresponding B cells from healthy individuals [bib_ref] Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specifi c defi..., Longo [/bib_ref]. Moreover, in x-linked hyper IgM patients, there could be an unphysiological expansion of the prediversifi ed IgM + IgD + CD27 + B cells in the absence of competition with a normal population of memory B cells for survival niches. Finally, it may also be that GC-like structures exist in such patients and that memory B cells generated in such structures preferentially give rise to IgM + IgD + CD27 + B cells in the absence of CD40-triggering for class-switching. Taking these considerations together, a picture is emerging in which some IgM + IgD + CD27 + B cells are initially generated during early development in a primary antigen-independent diversifi cation process, with a relatively low mutation load. Later in life, the majority of cells with this phenotype appear to derive from GC reactions and hence represent post-GC memory B cells.
## Dynamics of memory b cell generation in the gc
The detection and characterization of rearranged IgV genes from memory B cell clones not only showed that IgG + and IgM + IgD + CD27 + memory B cells can derive from the same GC B cell clone, but provided additional insights into the dynamics of memory B cell generation in the human. First, GC B cell clones give rise to memory B cells throughout the GC reaction, and begin to do so very early in the response, as evident from PB memory B cells with very few or even a lack of IgV gene mutations. This fi nding provides evidence that lowly mutated memory B cells are not derived from par-donors 1-8 and of naive B cells from donors 7 and 8, cDNA was prepared as described in the previous section and amplifi ed in 1.5 mM MgCl 2 , 250 μM dNTPs, 0.125 μM each primer ( V H 6 FR1 5 Ј -GGTGCGACAGGCCCCT-GGACAA-3 Ј or V H 3-23 -leader peptide sequence 5 Ј -TTTGGGCT-GAGCTGGCTTTTTCTTGTG-3 Ј and either a C ␥ RT-primer or nested primers for C 5 Ј -TCGTATCCGACGGGGAATTCTCACAG-3 Ј and C ␦ 5 Ј -GTTATCCTTTGGGTGTCTGCACCCTG-3 Ј ), and 1.2 U High Fidelity DNA polymerase mix (Roche) for 35 cycles. The cDNA of IgG + B cells was cloned using the pGEM-T-Easy cloning kit (Promega), and multiple plasmids per cloning were sequenced. For 8-30 sequences of each donor, CDRIII-specifi c primers were generated. PCR conditions were determined that were suitable to amplify for most of the clone-specifi c primers products from the corresponding diluted plasmid preparations (5 ng/μl) in no more than 35 cycles. PCR for the detection of clonally related sequences from cDNA of IgM + IgD + CD27 + B cells and naive B cells from donors 7 and 8 was performed with the following conditions: 30 μl reaction mixture containing 1.5 mM MgCl 2 , 250 μM dNTPs, 0.125 μM each primer (V H 6FR1 or VH3-23 leader peptide primer and CDRIII-specifi c primer), primary denaturation step of 95°C for 4 min, followed by 35 cycles of 30 s at 95°C, 30 s at 62°C, 60 s at 72°C, and 5 min at 72°C. Resulting amplifi cates were cloned and multiple plasmids were sequenced. CDRIII-specifi c primers that amplifi ed clonally related sequences in IgM + B cells were in parallel used to amplify further clone members from IgG + cDNA (see fl owchart in [fig_ref] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences... [/fig_ref]. All distinct V H gene sequences are available at Genbank/EMBL/DDBJ under accession nos. FN562934 -FN562983 , FN564030 , and FN563492 -FN563831 .
Mutation tree analysis. Sequences with identical CDRIII were aligned by Clustal W (Megalign; DNAStar). The number of expected DNA polymerase errors per sequence was determined using the following formula: ف 200 bp sequenced × 70 PCR cycles × polymerase error rate (8.3 × 10 Ϫ 6 /bp/cycle [Roche]) = 0.1. Neglecting the low number of 1 expected polymerase induced error per 10 sequences, all nucleotides that did not match germline confi guration of the respective V H gene were considered as mutations introduced by SHM. To create the IgV gene lineage trees, the sequences were manually positioned in genealogic order, rooted by the germline confi guration and assuming that a high number of shared mutations in a sequence determines a late ancestor and vice versa. Each set of sequences was analyzed in parallel with the IgTree program [bib_ref] IgTree: creating immunoglobulin variable region gene lineage trees, Barak [/bib_ref] ; provided by R. Mehr, Bar-Ilan University, Ramat Gan Israel). Each genealogic tree was constructed to obtain a minimal amount of branching points and mutation events. In case the particular sequence of single mutation events could not be determined (e.g., possible back mutations were not outweighed by shared mutations or shared mutations could as well be interpreted as independent events), the solution in favor of a higher number of shared mutations is shown. Importantly, the basic structure of the tree was not aff ected.
Calculation of clonal sizes. Assuming all members of a clone in the sorted cell aliquots were detected by the PCR-based strategy and counting repeatedly identifi ed sequences as one, we calculated the potential clone size in the PB. For example, for the largest clone [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] for which we detected 37 members in the 3 × 10 6 sorted CD27 + B cells, in the total PB with ف 1 × 10 9 CD19 + B cells (10 8 B cells in 500 ml PB, total blood volume of ف 5 liter), of which 29% were CD27 + , this clone would have in 5 liter of PB a size of ف 3,600 B cells (37 × 10 9 x 0.29: 3 × 10 6 ). The sizes of the other clones were analogously determined.
Online supplemental material. [fig_ref] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences... [/fig_ref] shows the V H sequences obtained from the naive B cells of donors 7 and 8 using V H 3-23-and CDRIII-specifi c primers for clones M, N, and O. [fig_ref] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or... [/fig_ref] shows fi ve selected sequences of each clone (A-P), aligned to the germline confi guration of the corresponding V-, D-, and J-segments. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20091087/DC1. We thank Kerstin Heise for excellent technical assistance, Kai-Michael Toellner for sharing unpublished data and Ramit Mehr for providing the IgTree© software.
of the University of Duisburg-Essen. Donors for full blood donations were recruited from the Medical School. All samples were collected with informed consent of the donors. The study protocol was approved by the Internal Review Board in Essen. PB mononuclear cells were isolated by Ficoll-Paque density centrifugation, and CD19 + B cells were enriched to >98% by magnetic cell separation using the MACS system (Miltenyi Biotec).
Cell sorting. The B cell-enriched cell suspensions were stained with CD27-APC (eBioscience), anti-human IgD-PE, and IgM-FITC or IgG-FITC antibodies (all from BD). B cell subpopulations were sorted with a FACSDiva cell sorter (BD) as naive B cells (IgM + IgD + CD27 -), class-switched memory B cells (IgG + CD27 + ), IgM + IgD + CD27 + B cells (IgM + IgD + CD27 + ), and IgM-only B cells (IgM + IgD -/low CD27 + ). Purity was >99% for each population as calculated by reanalysis on a FACSCanto fl ow cytometer (BD) in combination with FACSDiva software.
Bcl6 mutation analysis. Single cells were sorted into Expand High Fidelity PCR buff er (Roche) and incubated with 0.25 mg/ml proteinase K for 2 h. A semi-nested PCR strategy was used to amplify the Bcl6 -MMC with a length of 744 bp [bib_ref] BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation..., Pasqualucci [/bib_ref]. The fi rst round of amplifi cation was performed in 2.5 mM MgCl 2 , 125 μM dNTPs, 0.125 μM each primer (5 Ј -CGCTCTTGCCAAATGCTTTGGC-3 Ј and 5 Ј -CTCTCGTTAG-GAAGATCACGGC-3 Ј ), and 1.2 U High Fidelity DNA polymerase mix (Roche). For the second round of amplifi cation, 1.5 μl of the fi rst round reaction were used as template. PCR conditions were 1.75 mM MgCl 2 , 67 μM dNTPs, 0.125 μM each primer (5 Ј -CGCTCTTGCCAAATGCTTTG-3 Ј and 5 Ј -GACACGATACTTCATCTCATC-3 Ј ), and 1.2 U Fermentas Taq DNA polymerase. For sequence analysis, PCR products were purifi ed with EZNA Cycle pure kit (VWR International GmbH) and sequenced from both strands with second round amplifi cation primers. The procedure was accomplished using the ABI Cycle Sequencing Kit, version 3.1 on an ABI3130 Sequencer (Applied Biosystems). Sequence analysis was performed using the SeqScape software version 2.5 (Applied Biosystems) and the Gen-Bank data library. To calculate whether one or two alleles were amplifi ed from a cell, donors were prescreened for heterozygosity of a known monoallelic SNP in the Bcl6 -MMC, a 1-bp deletion (rs3832246).
## Clonal relation in pb b cells: page strategy.
IgG + CD27 + B cells and IgM + IgD + CD27 + B cells were sorted (as described in the Cell sorting section) in 30,000 cell aliquots. RNA was extracted with RNeasy micro kit (QIAGEN) and RT was performed with Superscript-III-RT (Invitrogen) according to the manufacturer's protocol. For gene-specifi c RT of RNA from class-switched B cells, a primer was used that recognizes C ␥ 1 and C ␥ 3 (5 Ј -AGGCAGCCCAGGGCCGCTGTGCC-3 Ј ), and for RNA from IgM + IgD + CD27 + B cells a mixture of a C (5 Ј -AGCCAACGGCCAC-GCTGCTCGTAT-3 Ј ) and a C ␦ primer (5 Ј -CATGCCAGGACCA-CAGGGCTGTTATC-3 Ј ) was used. The distance to the 3 Ј end of the rearranged J H gene is identical for the three primers. V H 1 family-specifi c cDNA was amplifi ed in 1.5 mM MgCl 2 , 250 μM dNTPs, 0.125 μM each primer (VH1FR2 5 Ј -GGTGCGACAGGCCCCTGGACAA-3 Ј and constant primers as in RT), and 1.2 U High Fidelity DNA polymerase mix (Roche) in 35 cycles. PCR products were precipitated, resolved in 10 μl formamide and heat-denatured at 95°C for 2 min. Samples were loaded in neighboring lanes of a 6% denaturing PAGE gel (19:1 acrylamide/bisacrylamide, 7 M urea in TBE buff er). The gel was stained with the Silver sequence DNA staining kit (Promega) according to the manufacturer's instructions. PCR products of equal size in both lanes were excised and watered O/N in 50 μl H 2 O. 5 μl were reamplifi ed in additional 35 PCR cycles and sequenced with the VH1FR2 primer. Products with identical, monoclonal CDRIII in both populations were cloned using pGEM-T-Easy cloning kit (Promega) and sequenced to identify clone members [fig_ref] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences... [/fig_ref].
Clonal relation in PB B cells: CDRIII-primer strategy. From 1 to 1.5 × 10 6 cell aliquots of IgG + CD27 + B cells and IgM + IgD + CD27 + B cells from
[fig] Figure 1: Flow charts of the experimental approaches used to identify clonally related sequences in PB IgM + and IgG + B cells. (A) PAGEbased approach. (B) CDRIII-specifi c primer−based approach. [/fig]
[fig] Figure 2: Genealogic trees of V H 1-18 -, V H 6 -, or V H 3-23 -expressing memory B cell clones, reconstructed from clonally related IgM + and IgG + sequences. Abbreviations in the circles represent single sequences from IgM + (M) or IgG + (G) B cells, empty circles represent hypothetical intermediates. [/fig]
[table] Table I: Bcl6 mutations in human PB B cells [/table]
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Recurrent Bilateral Breast Abscess Due to Nontuberculous Mycobacterial Infection
# Introduction
Mastitis is a common disease with both infectious and noninfectious causes. Most cases occur during lactation and are caused by Staphylococcus aureus and Streptococcus species; parasites and Mycobacterium tuberculosis have rarely been reported to cause breast infections [bib_ref] Classification of inflammatory breast disorders and step by step diagnosis, Kamal [/bib_ref] [bib_ref] Nontuberculous mycobacterial breast implant infections, Macadam [/bib_ref]. Noninfectious inflammations include plasma cell, granulomatous, and lymphocytic mastitis [bib_ref] Classification of inflammatory breast disorders and step by step diagnosis, Kamal [/bib_ref].
Recurrent mastitis and breast abscesses may be due to delayed, incomplete, or inappropriate therapy as well chronic Staphylococcus infections [bib_ref] Breast infections in non-lactating women, Abdelhadi [/bib_ref]. They may also be due to underlying breast lesions. However, the disease is clinically and radiologically very difficult to diagnose. We report a case of recurrent bilateral mastitis shown pathologically to be due to nontuberculous mycobacterial (NTM) infection.
## Case report
A 35-year-old Korean woman admitted to the in-patient clinic of our institution presented with swelling, pain, and ri-gidity of the left breast that began 4 days previous. She reported a fever that had lasted 1 day. Physical examination revealed a palpable tender mass, erythema, and swelling in the upper half of the left breast, extending to the nipple. There was no nipple discharge or pyrexia. A clinical impression of mastitis was made. She had no previous history of trauma, operation, or family history of breast cancer; further, she was not immunocompromised.
Prior to admission, the patient had a history of recurrent breast infections beginning in July 2007. She was treated for mastitis of her right breast with pus formation, and she made complete recovery. The second infection occurred 8 months later, in March 2008. She underwent breast biopsy for a mass on her right breast, and a fluid sample was cultured. Based on the culture results, the lesion was diagnosed as an NTM infection, and the patient was treated with combined antibiotic therapy for 12 months. The third infection appeared 9 months after the last treatment, in December 2009. She was admitted for a palpable mass in the left breast. The patient underwent biopsy for this lesion as well, which was also diagnosed as an NTM infection. After 4 months of combined antibiotic therapy, she made a complete recovery. The patient was admitted to the hospital for her fourth mastitis event.
We performed routine breast ultrasonography, which revealed a 2.8 cm, ill-defined, partially multilobulating contoured mass with heterogeneous internal echogenicity in the subareolar area, with diffuse edema of the left breast [fig_ref] Figure 1: B-mode gray scale ultrasonography of the left breast [/fig_ref]. No significant vascularity was observed. Several small lymph nodes less than 1 cm in short diameter were visible in
## Recurrent bilateral breast abscess due to nontuberculous mycobacterial infection
Since recurrent bilateral breast infection due to nontuberculous mycobacterium (NTM) is rare, its diagnosis is easily overlooked; in addition, complete recovery is often difficult to achieve. We report a case of recurrent bilateral infection in a 35-year-old woman who had completed treatment for NTM. Although various infec-tious diseases show similar clinical conditions and imaging findings, recurrences should raise suspicion of NTM infection, and this possibility should be considered in differential diagnoses.
the left axilla level I.
The patient also underwent bilateral dynamic contrast-enhanced magnetic resonance imaging of the affected breast to further evaluate the extent of the lesion and the potential for hidden malignancies [fig_ref] Figure 2: Magnetic resonance imaging [/fig_ref]. Dynamic contrast-enhanced magnetic resonance imaging revealed a 2.4 × 2.4 cm iso-to high-signal intense mass on T2 WI and iso-intense on T1 WI, which showed peripheral rim enhancement after contrast injection. Associated findings included diffuse parenchymal enhancement of the upper outer to central portion of the left
## A b
breast with thickening of the overlying skin. This lesion appeared to be an abscess associated with mastitis.
To exclude the possibility of hidden malignancies, a biopsy was performed. Considering the previous histopathologic findings in 2008, 2010, and 2012, granulomatous mastitis was suspected [fig_ref] Figure 3: Histopathologic findings [/fig_ref] , with no acid-fast bacilli visible on Ziehl-Neelsen staining. Although we could not confirm the identity by specimen culture, paraffin-embedded tissue samples were positive for NTM by polymerase chain reaction. Based on the clinical course of recurrent infections over 5 years, previous histopathologic findings, and positive polymerase chain reaction results, a clinical diagnosis of NTM infection was made. Clarithromycin (1,000 mg/day) was administered in combination with ciprofloxacin (1,000 mg/day) for 1 year. Because the lesion progressed to pus formation, surgical drainage was also performed. The patient underwent a follow-up breast ultrasonography after 1 month that showed lesion improvement. She has been followed up for 1 year with no clinical or radiological evidence of recurrence.
# Discussion
Rarely, NTM organisms can cause breast infections [bib_ref] Nontuberculous mycobacterial breast implant infections, Macadam [/bib_ref]. NTM infections may be acute, subacute, or late-onset. Interestingly, previous reports have typically described bilateral NTM breast infections. The most common organisms found in these infections included Mycobacterium fortuitum, M. avium, M. abscessus, and less commonly, M. chelonae. NTM should be considered as a source of infection when standard bacterial culture results are negative.
Initial treatment for infectious and noninfectious mastitis
## A b
includes appropriate antimicrobial therapy. Generally, patients are administered antibiotics at an early stage without an observation period. It can be difficult to differentiate noninfectious and infectious mastitis at early stages; hence, antibiotics are used in either case. When M. tuberculosis (TB) is suspected, anti-TB therapy may be required. A lack of response to anti-TB therapy or a diffusely deformed breast with draining sinuses may require surgical intervention [bib_ref] Breast tuberculosis: diagnosis, clinical features & management, Tewari [/bib_ref].
Once an NTM organism is isolated by culture, the infection should be treated with targeted combined antibiotic therapy. The most commonly reported cause of skin and soft tissue disease in NTM infections is M. fortuitum. Several published studies have reported M. fortuitum to be associated with breast infections. The majority reported an association between onset of infection and breast implants or reconstructive surgery [bib_ref] Cutaneous Mycobacterium fortuitum complicating breast reconstruction, Coney [/bib_ref] [bib_ref] Breast implant infection with Mycobacterium fortuitum group: report of case and review, Vinh [/bib_ref] [bib_ref] Recurrent mycobacterial breast abscesses complicating reconstruction, Rimmer [/bib_ref] [bib_ref] Mycobacterium fortuitum infection in prosthetic breast implants, Haiavy [/bib_ref]. They recommended removal of the infected implant in addition to appropriate antimicrobial therapy for the management of implant-associated NTM infections. For postoperative wound infections, the removal of implants or infected foreign bodies was required. Patients treated according to in vitro susceptibilities of isolated colonies have shown good results in a previous study; these targeted therapies are considered essential for infection management and antimicrobial stewardship. A primary panel of drugs for susceptibility testing may include amikacin, cefotoxin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and a sulphonamide.
However, few studies have reported NTM breast infections not associated with implants [bib_ref] Chronic breast abscess due to Mycobacterium fortuitum: a case report, Betal [/bib_ref]. To our knowledge, there has been only one published report of spontaneous breast abscess due to M. fortuitum [bib_ref] Spontaneous breast abscess due to Mycobacterium fortuitum, Cooke [/bib_ref] ; another report described infection following nipple piercing [bib_ref] Mycobacterium fortuitum breast infection following nipple-piercing, mimicking carcinoma, Lewis [/bib_ref] [bib_ref] Mycobacterium fortuitum and anaerobic breast abscess following nipple piercing: case presentation and..., Bengualid [/bib_ref]. When M. tuberculosis complex species are suspected as the source of infection, targeted antibacterial therapy is recommended [bib_ref] Chronic breast abscess due to Mycobacterium fortuitum: a case report, Betal [/bib_ref].
Our case demonstrated continuous recurrent infections despite appropriate treatments for NTM infection. Although we could not confirm the diagnosis by culture, the initial biopsy requested by the regular physician was positive for NTM. The patient was not immunocompromised and had no history of breast surgery, including implant insertion. Therefore, the exact cause of the recurrent NTM infection could not be determined. In conclusion, although it is difficult to differentiate from other causes due to nonspecific imaging findings, NTM should be considered for breast infections that recur despite standard antibiotic therapy.
[fig] Figure 1: B-mode gray scale ultrasonography of the left breast. (A) Ultrasonogram shows an approximately 2.8 cm ill-defined and partially multilobulating contoured mass with heterogeneous internal echogenicity in the subareolar area. (B) Additional findings of diffuse edematous changes in the left breast, with marked heterogeneous underlying parenchymal echogenicity and skin thickening. [/fig]
[fig] Figure 2: Magnetic resonance imaging: sagittal view of the left breast. (A) Contrast-enhanced magnetic resonance imaging shows an ill-defined and partially multilobular contoured lesion with rim-like enhancement in the subareolar area of the left breast (arrowheads) with additional diffuse parenchymal enhancement. The lesion (arrowheads) visible in the subareolar area correlates to the lesion detected by B-mode gray scale ultrasonography(Figure 1). This lesion appeared to be an abscess consistent with mastitis (1.5-T MR scanner, 3D fat-suppressed T1-weighted Gradient Echo Sequences; contrast injection of 0.2 mL/kg gadodiamide [OmniscanTM; GE Healthcare] was administered by manually followed by a 20-mL saline flush). (B) T2weighted image showing high signal intensity diffuse edematous changes with skin thickening (arrows) and an ill-defined high signal intensity lesion in the subareolar area (arrowhead), correlated with the lesion with rim-like enhancement. [/fig]
[fig] Figure 3: Histopathologic findings. Histologic sections of the lesion specimen obtained by ultrasound-guided core needle biopsy (A, lower magnified image, × 20; B, higher magnified image, × 100) show inflamed breast tissue with granulomatous reaction on hematoxylin and eosin staining (arrows). [/fig]
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Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter
CLC secondary active transporters exchange Clfor H + . Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Glu ex ) upon its protonation. Using 19 F NMR, we show that as [H + ] is increased to protonate Glu ex and enrich the outward-facing state, a residue~20 Å away from Glu ex , near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
# Introduction
CLC transporters catalyze the exchange of Clfor H + across cellular membranes [bib_ref] A structural perspective on ClC channel and transporter function, Dutzler [/bib_ref] [bib_ref] The CLC 'chloride channel' family: revelations from prokaryotes, Matulef [/bib_ref] [bib_ref] CLC chloride channels and transporters: a biophysical and physiological perspective, Zifarelli [/bib_ref] [bib_ref] CLC chloride channels and transporters: from genes to protein structure, pathology and..., Jentsch [/bib_ref] [bib_ref] CLC channels and transporters: proteins with borderline personalities, Accardi [/bib_ref] [bib_ref] In the beginning: a personal reminiscence on the origin and legacy of..., Miller [/bib_ref] [bib_ref] Structure and gating of CLC channels and exchangers, Accardi [/bib_ref] [bib_ref] Discovery of CLC transport proteins: cloning, structure, function and pathophysiology, Jentsch [/bib_ref]. In humans, they are critical to a wide variety of physiological processes and constitute therapeutic targets for treating diseases [bib_ref] CLC chloride channels and transporters: from genes to protein structure, pathology and..., Jentsch [/bib_ref] [bib_ref] CLC-7: a potential therapeutic target for the treatment of osteoporosis and neurodegeneration, Zhao [/bib_ref] [bib_ref] Cell biology and physiology of CLC chloride channels and transporters, Stauber [/bib_ref] [bib_ref] Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule, Devuyst [/bib_ref] [bib_ref] ClC-5: physiological role and biophysical mechanisms, Pusch [/bib_ref] [bib_ref] A tale of two CLCs: biophysical insights toward understanding ClC-5 and ClC-7..., Zifarelli [/bib_ref]. In bacteria and yeast, CLCs are virulence factors and therefore could serve as drug targets to protect against food poisoning and fungal infections [bib_ref] A biological role for prokaryotic ClC chloride channels, Iyer [/bib_ref] [bib_ref] A CLC-type chloride channel gene is required for laccase activity and virulence..., Zhu [/bib_ref].
From bacteria to humans, Cl -/H + exchange by CLC transporters occurs with a strict stoichiometry of 2 Clfor every H + [bib_ref] Secondary active transport mediated by a prokaryotic homologue of ClC cl-channels, Accardi [/bib_ref] [bib_ref] Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5, Picollo [/bib_ref] [bib_ref] Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins, Scheel [/bib_ref] [bib_ref] Structure of a slow CLC Cl/H+ antiporter from a cyanobacterium, Jayaram [/bib_ref] [bib_ref] ClC-7 is a slowly voltage-gated 2Cl(-)/1H(+)-exchanger and requires Ostm1 for transport activity, Leisle [/bib_ref]. To achieve this stoichiometric exchange, CLCs must follow an alternating access mechanism, in which bound substrate ions access either side of the membrane alternately, i.e., they cannot access both sides simultaneously [bib_ref] Contributions to the theory of active transport: II. the gate type non-carrier..., Patlak [/bib_ref] [bib_ref] Simple allosteric model for membrane pumps, Jardetzky [/bib_ref] [bib_ref] Active transporters as enzymes: an energetic framework applied to major facilitator superfamily..., Shilton [/bib_ref]. The alternating access mechanism can only be realized by coupling of ion binding, translocation, and unbinding events to conformational changes in the transporter protein. Specifically, movement of ions between solution and the ion-binding sites of the transporter, as well as ion movement between binding sites, needs to be coupled to conformational changes between "outward-facing" (in which the external, but not internal, solution is accessible to ions), "occluded" (in which neither solution is accessible), and "inward-facing" (in which the internal, but not external, solution is accessible) states [bib_ref] The structural basis of secondary active transport mechanisms, Forrest [/bib_ref] [bib_ref] How do transporters couple solute movements?, Rudnick [/bib_ref].
In all other active transporters that have been structurally (or biophysically) characterized, the conformational changes governing the interconversion between these major functional states involve significant protein motions, including reorientation of helices or even entire domains [bib_ref] Common folds and transport mechanisms of secondary active transporters, Shi [/bib_ref]. For the CLC transporters, in contrast, it has been proposed that the transport mechanism may be fundamentally different and involve only localized side chain motions [bib_ref] Structure of a eukaryotic CLC transporter defines an intermediate state in the..., Feng [/bib_ref] [bib_ref] DEER distance measurements on proteins, Jeschke [/bib_ref]. However, this proposed mechanism is based largely upon the observation that no large-scale CLC conformational change could be detected crystallographically. Given the strong constraining forces in a crystal environment, which often prevent the protein from populating all naturally accessible, functionally relevant conformational states [bib_ref] Thinking outside the crystal: complementary approaches for examining transporter mechanism, Elvington [/bib_ref] [bib_ref] Mutations that stabilize the open state of the erwinia chrisanthemi ligand-gated ion..., Gonzalez-Gutierrez [/bib_ref] [bib_ref] Structure of sugar-bound LacY, Kumar [/bib_ref] , alternative approaches for detecting CLC conformational change during its function are strongly motivated.
CLC transporters are homodimers in which each subunit independently catalyzes Cl -/H + antiport (exchange) [bib_ref] Design, function and structure of a monomeric ClC transporter, Robertson [/bib_ref]. There are two key Cl --binding sites within the protein lumen, known as S cen and S ext . The central anion-binding site (S cen ) is stabilized by a positive electrostatic potential created by the N-termini of Helices F and N as well as by interactions with conserved Ser and Tyr residues, which physically occlude the anion from the intracellular side [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Using cross-linking as an alternative approach to crystallography, demonstrated that the conserved Tyr contributes to an intracellular "gate" that opens to generate an inward-facing state eLife digest Cells have transporter proteins on their surface to carry molecules in and out of the cell. For example, the CLC family of transporters move two chloride ions in one direction at the same time as moving one hydrogen ion in the opposite direction.
To be able to move these ions in opposite directions, transporters have to cycle through a series of shapes in which the ions can only access alternate sides of the membrane. First, the transporter adopts an 'outward-facing' shape when the ions first bind to the transporter, then it switches into the 'occluded' shape to move the ions through the membrane. Finally, the transporter takes on the 'inward-facing' shape to release the ions on the other side of the membrane. However, structural studies of CLCs suggest that the structures of these proteins do not change much while they are moving ions, which suggests that they might work in a different way.
Khantwal, have now used techniques called "nuclear magnetic resonance" and "double electron-electron resonance" to investigate how a CLC from a bacterium moves ions. The experiments suggest that when the transporter adopts the outward-facing shape, points on the protein known as Y419 and D417 shift their positions. Chemically linking two regions of the CLC prevented this movement and inhibited the transport of chloride ions across the membrane.
Khantwal, Abraham et al. then used a computer simulation to model how the protein changes shape in more detail. This model predicts that two regions of the transporter undergo major rearrangements resulting in a gate-opening motion that widens a passage to allow the chloride ions to bind to the protein. Khantwal, Abraham et al.'s findings will prompt future studies to reveal the other shapes and how CLCs transition between them. . The bound Cl -(one in each identical subunit at site S cen ) is coordinated by conserved Ser and Tyr residues (shown as spacefilled). The N-termini of helices F and N (shown in purple and yellow respectively) point towards this site and provide a positive electrostatic environment for the anion. The H + -permeation pathways are delineated by two key residues, Glu ex and Glu in . Glu ex also acts as a "gate" that blocks the Cl --permeation pathway (green arrows) from the extracellular solution. (B) CLC structure highlighting helices discussed: F (purple), N (yellow), O (pink), P (blue), Q (brown), and R (aquamarine). (C) Close-up of the Cl --binding region in WT (left) and E148Q (right) ClC-ec1, highlighting intracellular and extracellular gate residues S107, Y445, and E148 (Glu ex ). In the E148Q mutant (pdb: 1OTU), the Gln side chain, mimicking the protonated Glu ex , swings away from the Cl --permeation pathway and is replaced at S ext with a Clion. The structure of this mutant is otherwise indistinguishable from the WT structure. (D) Cartoon of the Cl --binding region, illustrating the hypothesis that the E148Q structure represents an "outward-facing occluded" rather than an "outward-facing open" conformation. The following figure supplement is available for figure 1: [bib_ref] Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter, Basilio [/bib_ref]. While this inward-facing state has not yet been structurally characterized in detail, the elegantly designed cross-linking studies demonstrated that movement of neighboring Helix O [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] is required in conjunction with movement of the Tyr-gate residue.
At the extracellular side, a highly conserved glutamate residue, "Glu ex ", sits above the anion at S cen and blocks it from the extracellular solution [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , left panel). Localized side-chain motions of this residue represent the sole differences distinguished in crystallographic studies of CLC transporters [bib_ref] Gating the selectivity filter in ClC chloride channels, Dutzler [/bib_ref] [bib_ref] Structure of a eukaryotic CLC transporter defines an intermediate state in the..., Feng [/bib_ref]. In the structure of a mutant in which Gln is used as a proxy for the protonated Glu ex , the side chain swings upwards and the site previously occupied by the side chain is occupied by an anion [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Thus, the structure of this mutant has been thought to represent an outward-facing (OF) CLC conformational state. However, in this structure the pathway to the extracellular solution is very narrow -too narrow to accommodate Clor other permeant ions [bib_ref] Antiport mechanism for cl(-)/H(+) in ClC-ec1 from normal-mode analysis, Miloshevsky [/bib_ref] [bib_ref] Partial least-squares functional mode analysis: application to the membrane proteins AQP1, Aqy1,..., Krivobokova [/bib_ref] -suggesting that additional conformational changes are required for the formation of the OF state in order for external anions to access the external anion-binding site (S ext ). We therefore hypothesize that the state identified in the E148Q crystal structure is an "outward-facing occluded" state and that a distinct "outward-facing open" state may exist to permit access of external Clto the Glu ex -vacated S ext site [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Addressing this hypothesis is crucial to understanding the CLC transport mechanism and how it relates to those of canonical transporters.
Various experimental approaches have been used to evaluate whether CLC conformational changes beyond Glu ex are involved in the transition to an OF open state. Since the pK a of Glu ex is~6 [bib_ref] Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic h+/ClÀ..., Picollo [/bib_ref] , a change in pH from 7.5 to 5.0 will cause Glu ex to transition from mostly deprotonated to mostly protonated, and therefore from its position occupying S ext outward towards the extracellular solution. Such pH manipulations can therefore be used to enrich the OF state and probe for changes in protein conformation. Although crystallization at pH 4.6 failed to reveal any conformational change (Figure 1-figure supplement 1) [bib_ref] X-ray structure of a ClC chloride channel at 3. 0 Å reveals..., Dutzler [/bib_ref] , spectroscopic approaches have shown that H + -dependent changes do occur outside the restraints of crystallization. Using environmentally sensitive fluorescent labels, Mindell and coworkers showed that Helix R, which lines the intracellular vestibule to the Cl --permeation pathway [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , undergoes H +dependent conformational change during the transport cycle [bib_ref] Site-directed fluorescence studies of a prokaryotic ClC antiporter, Bell [/bib_ref]. Using site-specific NMR labeling schemes, our lab has identified H + -dependent structural change at Helix R and also at the linker connecting Helices P and Q (P/Q linker) [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] -a region~20 Å distant from the Cl -permeation pathway [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref] [bib_ref] 13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1, Abraham [/bib_ref]. Clearly, CLCs undergo H +dependent conformational changes beyond those revealed by crystallography. The question remains whether and how these conformational changes are involved in regulating ion binding and translocation during Cl -/H + transport.
Here, we study the conformational change in Helix P and the P/Q linker region [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] in ClC-ec1, a well-studied prokaryotic CLC, using a combination of 19 F NMR, double electron-electron resonance spectroscopy, chemical cross-linking, crystallography, molecular dynamics (MD) simulations, and analysis of cross-linking in mutant transporters. Our results show that rearrangement of Helices N and P occurs to widen the extracellular vestibule and generate a previously uncharacterized "outward-facing open" CLC conformational state, thus establishing the involvement of structural changes beyond the rotation of Glu ex .
# Results
Sensitivity of 19 F spectra to the paramagnetic probe TEMPOL The 19 F NMR nucleus is an advantageous reporter of conformational change because of its sensitivity to chemical environment, its small (non-perturbing) size, and the lack of endogenous 19 F in proteins [bib_ref] Fluorine NMR of proteins, Gerig [/bib_ref] [bib_ref] Use of 19F NMR to probe protein structure and conformational changes, Danielson [/bib_ref] [bib_ref] Current applications of 19F NMR to studies of protein structure and dynamics, Kitevski-Leblanc [/bib_ref]. Using ClC-ec1, a prokaryotic CLC homolog that has served as a paradigm for the family [bib_ref] Structure and function of clc channels, Chen [/bib_ref] [bib_ref] A structural perspective on ClC channel and transporter function, Dutzler [/bib_ref] [bib_ref] The CLC 'chloride channel' family: revelations from prokaryotes, Matulef [/bib_ref] [bib_ref] Structure and gating of CLC channels and exchangers, Accardi [/bib_ref] , we previously showed that we could replace native Tyr residues with 19 F-Tyr and observe conformational changes reported by changes in 19 F chemical shift [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. Our strategy to enrich the OF conformational state of ClC-ec1 involved lowering the pH of the solution from 7.5 to 4.5-5.0, as described above. Of the five buried Tyr residues in ClC-ec1, two reported [H + ]-dependent changes in chemical environment. The first, as expected, was at Y445, which is within 6 Å of Glu ex ; the second, strikingly, was in a region~20 Å away, at Y419 near the dimer interface .
To better understand this conformational change, we performed an accessibility experiment, reasoning that global protein conformational changes often result in solvent exposure of previously buried regions. For this experiment, we exploited the sensitivity of 19 F relaxation rates (and hence spectral line widths) to the water-soluble paramagnetic probe TEMPOL [bib_ref] NMR studies of lysozyme surface accessibility by using different paramagnetic relaxation probes, Bernini [/bib_ref] [bib_ref] Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule..., Venditti [/bib_ref]. In this experimental setup, movement of a 19 F-labeled residue from a buried to a solvent accessible location would be detected by line-broadening and peak attenuation. We first examined whether any of the five buried tyrosine residues in ClC-ec1 exhibits sensitivity to TEM-POL. In "BuriedOnly" ClC-ec1, a mutant in which all five buried Tyr residues have been labeled with 19 F effects of TEMPOL were observed at both low and high [H + ], with apparently greater sensitivity at high [H + ] ; however, because the 19 F resonances are overlapping, we were unable to unambiguously assign the observed changes specifically to effects on chemical shift or line-broadening of a particular resonance. Therefore, to clearly identify the residue(s) sensitive to TEMPOL, we generated ClC-ec1 constructs containing only one 19 F-Tyr label per subunit (either Y445 or Y419, , replacing all other Tyr residues with Phe. Although the "Y445only" mutant was unstable and could not be further examined, the "Y419only" mutant was stable and showed robust, fully coupled Cl -/H + exchange activity [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. The functionality of the Y419only mutant may seem startling, given that it involves mutating the highly conserved Cl --coordinating Tyr445 [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] to Phe, but it is consistent with previous structural and functional studies demonstrating wild-type behavior of the Y445F mutant [bib_ref] Synergism between halide binding and proton transport in a CLC-type exchanger, Accardi [/bib_ref] [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref].
## H + -dependent accessibility of y419
Prior to investigating the effect of [H + ] on solvent accessibility of Y419 using TEMPOL, we acquired 19 F spectra for Y419 only as a function of pH. The 19 F NMR spectrum of Y419 only at pH 7.5 shows a single 19 F peak centered at 60 ppm . This peak shifts upfield and splits into two peaks when [H + ] is increased, indicating that the 19 F nucleus has experienced a change in chemical environment. This result is consistent with our previous findings [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref] further supporting the notion that conformational changes occur in the vicinity of Y419 as increasing [H + ] promotes occupancy of the OF state in which Glu ex is protonated. The appearance of two peaks, at -61 and -63 ppm, indicates that the 19 F label on Y419 is experiencing two different environments. This could arise from two conformational states of ClC-ec1 or from a tyrosine ring flip that occurs slowly on the NMR timescale (<<1000 s -1 ) [bib_ref] Ring flips revisited: 13 c relaxation dispersion measurements of aromatic side chain..., Weininger [/bib_ref]. While this information is useful in identifying Y419 as being in a region involved in H + -dependent conformational change, the lack of comprehensive theory for interpreting 19 F chemical shifts in terms of structure motivates additional studies to provide more details on the nature of the conformational change. To evaluate whether there might be a change in solvent accessibility of Y419, we examined the effect of TEMPOL on the 19 F spectra of Y419only. Because of the steep distance dependence of nuclear relaxation enhancements mediated by paramagnets like TEMPOL, significant line-broadening requires the paramagnetic center to approach the target nucleus within less than~10 Å [bib_ref] Spin relaxation measurements of electrostatic bias in intermolecular exploration, Teng [/bib_ref]. At pH 7.5, there is little sensitivity of the 19 F-Y419 signal to 100 mM TEMPOL which is consistent with the largely buried position of Y419 in the crystallographically captured state of ClC-ec1, i.e., 12-13 Å from the protein surface . In contrast, at pH 4.5, significant line-broadening is observed , indicating exposure of Y419 to the bulk solution allowing a close approach, or direct contact, of the TEMPOL probe with the fluorine atom [bib_ref] Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra, Esposito [/bib_ref] [bib_ref] NMR studies of protein surface accessibility, Niccolai [/bib_ref]. This H + -dependent change in accessibility is reversible, as demonstrated by the reappearance of the Y419 signal when pH is returned to 7.5 from 4.5 in the presence of 100 mM TEMPOL . The reproducibility of these experiments is shown in H + -independent accessibility of Y419 in channel-like ClC-ec1
The outer-and inner-gate residues of ClC-ec1 (Glu ex and Y445 respectively, [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] can be replaced by smaller residues Ala, Ser, or Gly, to yield "channel-like" ClC-ec1 variants [bib_ref] Ion permeation through a cl-selective channel designed from a CLC cl-/H+ exchanger, Jayaram [/bib_ref]. This excavation of the gates yields a narrow water-filled conduit through the transmembrane domain, which allows rapid Clthroughput and abolishes H + coupling. Thus, it . H + -dependent solvent accessibility of Tyr residues in ClC-ec1, detected by 19 F NMR. (A) "BuriedOnly" ClC-ec1, a mutant in which the five buried Tyr residues (spacefilled in yellow) have been labeled with 19 F. The seven solvent-exposed Tyr residues have been mutated to Phe. Residues Y445 (on Helix R, shown in aquamarine) and Y419 (linker between Helices P and Q, blue and brown respectively) were previously identified as undergoing H + -dependent changes in chemical shift [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. (B) 19 F NMR spectra of BuriedOnly ClC-ec1. Top data panel: low pH was used to enrich the outward-facing conformational state. Changes in chemical shift reflect changes in chemical environment experienced by the 19 F nuclei. Middle data panel: spectral changes in response to addition of TEMPOL (inset) at pH 7.5. Bottom data panel: spectral changes in response to addition of TEMPOL at pH 5.0. DOI: 10.7554/eLife.11189.005 . 19 F NMR detects H + -dependent solvent accessibility at Y419. (A) Y419only ClC-ec1. In this variant, all native Tyr residues except for Y419 have been mutated to Phe, so that only Y419 will carry a 19 F label. Y419 is highlighted in the ClC-ec1 structure shown from the point of view of the membrane (left) and from the extracellular side (right). The lower panels illustrate that Y419 lies in a buried position (left: thin slice through the protein at Y419; right, surface representation viewed from the extracellular side. (B) 19 F NMR spectra of Y419only. The prominent peak centered at -60 ppm shifts upfield (-61 and -63 ppm) when the pH is shifted from 7.5 to 4.5 to enrich the OF state. (C) Y419 becomes substantially more exposed to solvent at increased [H + ], as indicated by susceptibility to line-broadening by the water-soluble TEMPOL at pH 4.5 (bottom spectra, green vs black trace) compared to pH 7.5 (top spectra, orange vs cyan trace). (D) The change in the Y419 exposure to solvent is reversible, as revealed by return of the signal (to the expected chemical shift) when the pH is raised to 7.5 (bottom trace, orange). (E) Y419 in the channel-like ClC-ec1 background is accessible to TEMPOL at both pH 7.5 and 4.5. DOI: 10.7554/eLife.11189.006
The following figure supplements are available for figure 3: continued on next page appears that the mechanism of Clflux through these variants involves channel-like diffusion that is independent of substrate-dependent conformational change. Consistent with this picture, our previous 19 F NMR data showed that E148A/Y445S ClC-ec1 (exhibiting the highest Clpermeability among the channel-like variants) does not undergo the substrate-dependent spectral changes observed in the coupled ClC-ec1 transporters [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. In this channel-like background, Y419only exhibits a single NMR peak at~-61 ppm, and this signal is sensitive to line-broadening by TEMPOL at both pH 4.5 and 7.5 . The accessibility at pH 7.5 is surprising given that the crystal structure of channel-like ClC-ec1 variant E148A/Y445A superposes closely with WT (RMSD 0.52 Å ) and indicates a buried position for Y419 [bib_ref] Ion permeation through a cl-selective channel designed from a CLC cl-/H+ exchanger, Jayaram [/bib_ref]. While in these studies we used variant E148A/Y445S, which has not been crystallized, the two variants are functionally similar [bib_ref] Ion permeation through a cl-selective channel designed from a CLC cl-/H+ exchanger, Jayaram [/bib_ref]. In channel-like E148A/Y445S, the accessibility of Y419 to TEMPOL indicates that the channel-like ClC-ec1 variant adopts a conformation in solution different from that observed in the crystal structure and similar to the conformation adopted by WT at low pH.
## Cross-linking at helix p slows transport
We investigated the functional relevance of the conformational change detected at Y419 by introducing cysteines into this region and examining the effects of inter-subunit cross-linking. We reasoned that this cross-linking would restrict the conformational changes responsible for the increased solvent accessibility of Y419 at low pH, and, if these conformational changes are functionally important, cross-linking should also reduce the efficiency of Cl -/H + transport. In the X-ray crystal structure, the Ca-Ca distance between the two Y419 residues (one in each subunit) is 8.8 Å , within striking range for potential disulfide bond formation. We found that Y419C forms spontaneous inter-subunit cross-links and, to our surprise, that these cross-links have no detectable effect on function (Figure 4-figure supplement 1). Since Y419 lies in the middle of a loop (the P/Q linker), we reasoned that loop flexibility may thwart the intended restriction of motion by the disulfide cross-link. To test this possibility, we examined cross-linking at D417, the residue immediately following Helix P, which also has an inter-subunit Ca-Ca distance of 8.8 Å . Like Y419C, D417C forms spontaneous inter-subunit disulfide cross-links, with~50% of the protein migrating as a dimer on nonreducing SDS-PAGE , top panel). To determine the effect of the cross-link on function, we purified D417C transporters under reducing conditions, thereby obtaining a sample in which the majority (>90%) of the protein was not cross-linked, and then induced varying amounts of cross-link by titrating with copper-phenanthroline (CuP) , bottom panel). We assessed the functional effect of cross-linking using a Clefflux assay [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref]. These assays show that crosslinking at 417C correlates directly with a decrease in transport activity [fig_ref] Table 1: D417C activity extrapolated to 0 and 100% cross-link [/fig_ref].
Since our NMR results indicated that the Helix P-Q region of channel-like ClC-ec1 adopts a conformation similar to that of WT at low pH , we hypothesized that the conformational changes underlying the increased solvent accessibility of Y419 may also move the two D417 side chains out of the range for inter-subunit cross-linking. To test this hypothesis, we generated the D417C mutant in the channel-like background and evaluated its sensitivity to cross-linking. Consistent with our hypothesis, D417C in channel-like background does not form spontaneous cross-links and is only minimally cross-linked even when treated with up to 100 mM CuP , top panel). Because this limited cross-linking of D417C/channel-like ClC-ec1 could be due to non-availability of the cysteines (due to oxidation) rather than lack of structural proximity of the two cysteine residues, we used a spectrophotometric assay to quantify the free thiols. Immediately after purification and before CuP treatment, essentially all of the 417C residues are available as free thiols , bottom panel). Therefore, the deficiency in cross-linking of 417C in the channel-like background compared to 417C in the WT background is not due to unavailability of the free thiols but rather because of a change in proximity of the two cysteines. After treatment with 100 mM CuP, 40% of the cysteines are available as free thiols . Since only~25% had been cross-linked, this result indicates that~35% became oxidized to other (non-disulfide) species. Thus, oxidation to non-disulfide species competes with disulfide bond formation and therefore thwarts any attempt to increase the extent of cross-linking beyond~25% with longer CuP treatments. To rule out the possibility that the crosslinking might be due to inter-dimer (rather than inter-subunit) disulfide bond formation, we examined D417C proteins on a Superdex 200 gel filtration column both before and after treatment with CuP and found that they ran as dimers (and not tetramers, as would occur in the case of interdimer cross-linking) . To the degree it can be cross-linked, D417C in channel-like ClC-ec1 is inhibited similarly to D417C in WT . Thus, movement of Helix P away from the position observed in the crystal structures is necessary for maximal activity in both transporter and channel-like ClC-ec1.
To evaluate whether this movement of D417C/Helix P is H + -dependent (as is the movement detected by 19 F NMR, , we labeled D417C ClC-ec1 with the nitroxide spin label MTSSL (1-Oxyl-2,2,5,5,-tetramethylpyrroline-3-methyl methanethio-sulfonate) and used double electron-electron resonance (DEER) spectroscopy [bib_ref] DEER distance measurements on proteins, Jeschke [/bib_ref] to deduce distance changes as a function of pH. At pH 7.5, the distribution is dominated by a single peak at a distance shorter than 20 Å . Lowering the pH to 4.5 induces a shift to a peak at 20 Å . The channel-like D417C-MTSSL exhibits an altered distance distribution profile compared to the WT background , suggesting that the protein adopts a different conformation. Notably, the D417C-D417C distance in channel-like is increased relative to that observed in the WT background at pH 7.5 , consistent with the resistance of the channel-like protein to cross-linking . A decrease in pH does not shift the distance distribution as observed in the WT background, consistent with the loss of pH dependence in 19 F NMR experiments on channel-like ClC-ec1 , [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. Together, these results link the conformational changes observed via NMR to those prevented by the cross-link.
Crystallization of cross-linked D417C (WT background) confirms that the cross-link has trapped the conformation seen in the crystal structures and not some other (non-native) conformation. Our crystal structure, determined at 3.15 Å resolution, reveals a backbone that superimposes on WT ClC-ec1 with a Ca RMSD of 0.57 Å , [fig_ref] Table 2: Data collection and refinement statistics a [/fig_ref]. Extra density connecting the 417C residues confirms the formation of an inter-subunit disulfide bridge . The regions around both the Cland the H + permeation pathways are intact and not notably distinguishable from WT . To confirm the integrity of the Cl --permeation pathway in cross-linked D417C ClC-ec1, we directly measured Cl --binding affinity using isothermal titration calorimetry (ITC) (Picollo et al., 2009). Both in the absence and presence of cross-link, D417C binds Clrobustly, with an affinity somewhat stronger than observed with WT (K d~0 .1-0.2 mM vs 0.6 mM) .
## Helix p cross-link specifically affects the cl --permeation pathway
The inhibition of channel-like ClC-ec1 activity by the D417C cross-link suggests that inhibition occurs via an effect on the Cl --permeation pathway, given that channel-like ClC-ec1 transports only Cland not H + . But since this conclusion is based on experiments with the atypical channel-like ClC-ec1with high transport rate and a continuous water passageway connecting the two sides of the membrane -we sought to strengthen the conclusion by examining uncoupled transporters that display typical transport rates and lack a continuous passageway: (1) E148A, which lacks the critical Glu ex residue that acts both as an extracellular gate for Cland as a transfer-point for H + permeation [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , and (2) Y445S, which is mutated at the intracellular gate [bib_ref] Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter, Basilio [/bib_ref] [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. The E148A (Glu ex ) mutant is similar to channel-like ClC-ec1 in that it transports only Cl -; however, it has a much lower turnover rate, comparable to WT [bib_ref] Secondary active transport mediated by a prokaryotic homologue of ClC cl-channels, Accardi [/bib_ref] [bib_ref] Ion permeation through a cl-selective channel designed from a CLC cl-/H+ exchanger, Jayaram [/bib_ref]. This slow turnover suggests that despite being uncoupled E148A still depends on conformational changes to catalyze transport, a view supported by both 19 F NMR and fluorescence-based experiments, which detect H + -dependent conformational change in this mutant [bib_ref] Site-directed fluorescence studies of a prokaryotic ClC antiporter, Bell [/bib_ref] [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. In contrast to D417C/channel-like ClC-ec1, we found that D417C in the E148A mutant background is readily cross-linked by CuP [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref]. Therefore, uncoupling through E148A alone does not alter the protein conformations sampled in solution as substantially as observed with channel-like (E148A/Y445S). The turnover rate of un-crosslinked D417C/E148A is quite low -as low, in fact, as the extrapolated value for the turnover of fully cross-linked D417C [fig_ref] Table 1: D417C activity extrapolated to 0 and 100% cross-link [/fig_ref]. Nevertheless, cross-linking of D417C/E148A is associated with significant inhibition of activity [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref] , [fig_ref] Table 1: D417C activity extrapolated to 0 and 100% cross-link [/fig_ref]. This result is consistent with the conclusion that inhibition occurs via an effect on the Cl --permeation pathway. The second uncoupled transporter examined, the inner-gate mutant Y445S, differs from the Glu ex mutant in that it is only partially uncoupled, with a Cl -/H + stoichiometry of~39:1 instead of the 2:1 stoichiometry observed with WT transporters [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref]. The double mutant D417C/ Y445S transports Cl -at~850 s -1 [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref] and H + at~20 s -1 (Figure 6-figure supplement 1) yielding a Cl -/H + stoichiometry of~43, similar to that of the Y445S single mutant [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref]. Cross-linking of D417C/Y445S proceeds to~70% and inhibits Cltransport by~60% [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref] , with extrapolation to 100% cross-linking yielding a turnover of~0 (± 100 s -1 ) [fig_ref] Table 1: D417C activity extrapolated to 0 and 100% cross-link [/fig_ref]. For H + turnover, it is difficult to judge whether there is a significant effect of the cross-link (Figure 6-figure supplement 1). Given the uncertainty in measuring such low H + fluxes (~20 s -1 ), it may be that H + is inhibited to the same extent as Cl -, to a lesser degree, or not at all. In the latter cases, the cross-link would in effect "rescue" ClC-ec1 coupling; such rescue could arise from an increase in Cloccupancy at S cen , which is known to facilitate H + coupling [bib_ref] Synergism between halide binding and proton transport in a CLC-type exchanger, Accardi [/bib_ref] [bib_ref] Uncoupling of a CLC cl-/H+ exchange transporter by polyatomic anions, Nguitragool [/bib_ref] [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. However, we cannot distinguish these possibilities within the uncertainty of our measurements.
The results with channel-like and uncoupled transporters [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref] support the conclusion that the D417C cross-link inhibits the Clbranch of the Cl -/H + transport mechanism but do not rule out an effect on H + transport. As an approach to examine the effect of the cross-link on H + transport, we used MD simulations to examine water entry into the hydrophobic region between Glu in and Glu ex , which is essential to connect these two major H + -binding sites and thus support H + transport [bib_ref] Proton pathways and h+/Cl-stoichiometry in bacterial chloride transporters, Kuang [/bib_ref] [bib_ref] Proton transport pathway in the ClC cl-/H+ antiporter, Wang [/bib_ref] [bib_ref] Molecular dynamics investigation of cl-and water transport through a eukaryotic CLC transporter, Cheng [/bib_ref] [bib_ref] Intracellular proton access in a cl(-)/H(+) antiporter, Lim [/bib_ref] [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. Water entry occurs via a narrow portal on the cytoplasmic side of the protein, lined by Glu in together with E202 and A404 [bib_ref] Intracellular proton access in a cl(-)/H(+) antiporter, Lim [/bib_ref] [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. Previously, we showed that constricting this portal by introducing large side chains at position 404 inhibits water entry detected computationally and H + transport detected experimentally [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. Since A404 is on the intracellular end of Helix P , restricting movement of this helix via the D417C cross-link might restrict water entry. To determine whether the D417C cross-link affects water entry, we compared the number of water molecules entering the central hydrophobic region during the simulation of cross-linked D417C compared to WT. In contrast to the A404L mutation, which greatly reduces water permeation through the portal [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref] , the D417C cross-link has no effect on water entry . This result suggests that the cross-link reduces ClC-ec1 transport predominantly via an effect on the Cl --permeation pathway rather than on the H + -permeation pathway.
## Potential gate-opening motions in wt and cross-linked clc-ec1
Our experimental results suggest that there could be functionally important motions of ClC-ec1 that open the Cl --transport pathway and are impeded by the D417C cross-link. To investigate the molecular basis of such motions, extensive MD simulations were conducted either in the absence or in the presence of the D417C cross-link. Note that even the hundreds of nanoseconds of simulations performed here can probe mainly conformational fluctuations of ClC-ec1 near its reference conformation (in this case, the crystal structure), which did not permit direct observation of the opening of the gates. Nevertheless, the sampled dynamics and fluctuations can provide information that can be used to derive collective motions, which are often functionally relevant [bib_ref] Normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins, Bahar [/bib_ref]. Collective motions are defined as those involving concerted movements of a large number of atoms distributed throughout the protein, and are therefore distinguished from localized conformational changes. A series of collective motions of a protein can be obtained in general by decomposing the fluctuations of a protein sampled through MD simulations, e.g., through principal component analysis, or by analyzing normal modes of the protein that underlie protein motions [bib_ref] Normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins, Bahar [/bib_ref]. Structural integrity of cross-linked D417C. (A) The cross-linked D417C backbone (PDB 5HD8, green) superposes with WT (PDB 1OTS, blue) (RMSD 0.57 Å for 862 Ca atoms). (B) Extra density between residues 417 on the two subunits was modeled as a disulfide bridge, shown in stereo. (C) Close up stereo view of key residues around the Cl -(upper panel) and H + (lower panel) permeation pathways. In the upper panel, the residues shown (E148, S107, and Y445) are the same as those depicted in [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. In the lower panel, also shown are E203, the internal H + -transfer site [bib_ref] Separate ion pathways in a cl-/H+ exchanger, Accardi [/bib_ref] and A404, a residue lining the portal for H + entry from the intracellular solution [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. Clmodeled in the central binding site is depicted as green and blue spheres. [bib_ref] Global motions exhibited by proteins in micro-to milliseconds simulations concur with anisotropic..., Gur [/bib_ref]. Collective motions can further be used to probe how larger-magnitude conformational change along the identified displacement vectors (modes) might involve crucial, functionallyrelevant protein motions, such as opening-closing movements of enzymatic active sites, ligand-binding sites on receptors and channel pores [bib_ref] Analysis of a 10-ns molecular dynamics simulation of mouse acetylcholinesterase, Tai [/bib_ref] [bib_ref] Molecular dynamics of apo-adenylate kinase: a principal component analysis, Lou [/bib_ref] [bib_ref] Common mechanism of pore opening shared by five different potassium channels, Shrivastava [/bib_ref] [bib_ref] Mechanics of channel gating of the nicotinic acetylcholine receptor, Liu [/bib_ref] [bib_ref] Large collective motions regulate the functional properties of glutamate transporter trimers, Jiang [/bib_ref] [bib_ref] Identification of motions in membrane proteins by elastic network models and their..., Isin [/bib_ref] [bib_ref] Ubiquitin dynamics in complexes reveal molecular recognition mechanisms beyond induced fit and..., Peters [/bib_ref] [bib_ref] Connecting protein conformational dynamics with catalytic function as illustrated in dihydrofolate reductase, Fan [/bib_ref] [bib_ref] Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals..., Yao [/bib_ref]. For example, collective motions obtained from normal mode analysis (NMA) were used to project opening movements of potassium-channel pores [bib_ref] Common mechanism of pore opening shared by five different potassium channels, Shrivastava [/bib_ref] , and these predicted movements are consistent with those seen in single-molecule and X-ray crystallographic experiments [bib_ref] Global twisting motion of single molecular KcsA potassium channel upon gating, Shimizu [/bib_ref] [bib_ref] Structural analysis of ion selectivity in the NaK channel, Alam [/bib_ref]. In this study, we identified collective motions in ClC-ec1 using principal component analysis (PCA) of the equilibrium MD simulations (see Methods), which in general identifies similar collective motions to those derived from NMA [bib_ref] An analysis of core deformations in protein superfamilies, Leo-Macias [/bib_ref] [bib_ref] Close correspondence between the motions from principal component analysis of multiple HIV-1..., Yang [/bib_ref] [bib_ref] Principal component and normal mode analysis of proteins; a quantitative comparison using..., Skjaerven [/bib_ref]. We then introduced deformations in the reference protein structure along each of the top 20 collective motions identified in our analysis (~75% of the motions observed in the equilibrium MD simulation). We then examined whether increasing the amplitude of these collective motions (which overcomes timescale limitations of the simulation) confer conformational change to the Cl --transport pathway. We specifically examined regions around the extracellular and intracellular gates to the Cl -transport pathway, where motions may lead to opening of either gate (which are both closed in the reference protein structure).
The extracellular gate is formed by the juxtaposition of Helix F (which contains Glu ex ) and Helix N [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. To scrutinize opening of this gate, we examined Ca distance changes (Dr) between several residue pairs on these helices: I356-G149, F357-E148, and A358-R147 [fig_ref] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1... [/fig_ref]. A search over the 20 dominant collective motions obtained through the PCA of the entire WT MD simulation revealed that deformations along some of the collective motions increase the distances between these pairs by >1.5 Å and thus are coupled to gate opening. To conduct a statistical analysis of these motions, we divided the entire simulation trajectory into six blocks and determined the number of times such collective motions occur in each block [fig_ref] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1... [/fig_ref] , blue bars). An identical analysis performed on the MD simulation trajectory obtained from the cross-linked D417C mutant revealed that the motions that open the extracellular gate are dampened due to the cross-linking (P=0.002 -0.008) [fig_ref] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1... [/fig_ref] , orange bars). The intracellular gate is formed by two key residues S107 and Y445 [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref] [bib_ref] CLC channels and transporters: proteins with borderline personalities, Accardi [/bib_ref] [bib_ref] Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter, Basilio [/bib_ref]. To scrutinize opening of this intracellular gate, we examined distance changes between these two residues as a result of collective motions. As with the extracellular gate, we observed some collective motions that lead to distance changes (Dr) of > 1.5 Å . Unlike the extracellular gate, however, the cross-link at residue 417 does not significantly dampen the distance changes around the intracellular gate (P=0.338) [fig_ref] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1... [/fig_ref].
## Collective motions in channel-like clc-ec1
The comparison of dominant gate-opening motions between WT and cross-linked forms described above suggests that the cross-link at residue 417 likely cripples the opening of the extracellular gate, thereby slowing Cltransport. However, along this line of reasoning, one must reconcile why the E148A mutants, in which the extracellular gate has ostensibly been removed, are inhibited when the cross-link is introduced. To address this question, we first investigated the bottleneck for Cltransport in ClC-ec1 based on the crystal structures. The radius profile of the ClC-ec1 Cltransport tunnel, calculated using the program HOLE [bib_ref] HOLE: a program for the analysis of the pore dimensions of ion..., Smart [/bib_ref] , shows an extracellular bottleneck with a minimum radius of~0.2 Å . Interestingly, the calculated radius profile for both the E148A mutant (lacking Glu ex ) and the channel-like variant E148A/Y445A also reveal extracellular bottlenecks. (E148A/Y445A was evaluated rather than the E148A/Y445S construct used here because this is the only channel-like variant for which there is a crystal structure.) With minimum radii of 0.9 Å these bottlenecks are still too narrow to allow Clpermeation (r(Cl -) » 1.81 Å ) [bib_ref] Revised effective ionic radii and systematic studies of interatomic distances in halides..., Shannon [/bib_ref]. Thus, additional opening motions in the gate region are needed for Cltransport.
To test the idea that additional gate-opening motions occur in the absence of Glu ex , the computational analysis discussed above was applied to characterize and analyze the collective motions of channel-like ClC-ec1. The analysis revealed that there are fewer collective motions that can open the extracellular gate after the cross-link is introduced to the channel-like mutant (P=0.001-0.070) [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , whereas the intracellular gate was not significantly affected (P=0.354) [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. This result is consistent with that obtained in the WT background. Taken together, our MD results suggest that the cross-link at residue 417 hinders the opening of the extracellular gate -beyond the Glu ex motions -in both the WT and channel-like ClC-ec1.
## Helix n connects helix p to the extracellular gate
How are motions at Helix P transmitted to the extracellular gate? Visual inspection reveals an obvious potential transduction pathway: Helix N, which forms part of the extracellular gate [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , [fig_ref] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1... [/fig_ref] , makes direct contacts to Helix P through side-chain packing of conserved residues in each Helix [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. We hypothesized that disrupting these contacts would disrupt transduction of Helix-P motions to the extracellular gate, thereby abolishing the inhibitory effect of the Helix-P cross-link. To test this hypothesis, we generated Helix-N mutants F357A and L361A, in which the inter-helical coupling of motion is expected to be weakened by removing bulky side chains contributing to the contact area. The mutant transporters are slow compared to WT but retain the ability to couple Cl -/H + exchange [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Strikingly, the D417C cross-link only weakly inhibits L361A activity and completely fails to inhibit F357A [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. The sluggish turnover of the F357A mutant suggests that it might be insensitive to the D417C cross-link because it is already maximally inhibited. To evaluate this possibility, we examined another slow mutant, A404L. A404 lines an intracellular "portal" for water (and hence H + ) entry into the transporter [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. This residue is located at the N-terminal end of Helix P [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , which does not contact Helix N. We found that the activity of the A404L mutant, despite being similarly sluggish to F357A, is reduced further yet by the D417C cross-link [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Thus, the lack of sensitivity of F357A to the D417C cross-link appears due to the weakened interaction with Helix P and not to its already-low turnover. These results provide strong support for the hypothesis that Helix-P motions are transmitted to the extracellular gate via side-chain contacts to Helix N. . Computational analysis of water entry through the portal lined by A404 (Helix P). (A) ClC-ec1 structure highlighting the location of the A404 "portal" residue at Helix P. (B) The D417C cross-link does not affect water entry into the pathway connecting Glu in and Glu ex . The aggregate number of water molecules entering the region between the two residues was determined as described previously [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref] and compared for wild-type (WT) and cross-linked mutant (D417C) over the same timescales. DOI: 10.7554/eLife.11189.021
# Discussion
Our results describe a previously unidentified protein conformational state and suggest a new framework for understanding the CLC transport mechanism, introducing two key concepts. First, the structure of the E148Q mutant, with the side chain rotated away from S ext [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] represents an "outward-facing occluded" (OF occluded ) state [bib_ref] Stö lting G, Fischer M, Fahlke C. 2014. CLC channel function and..., Stein [/bib_ref] , in which bound Cldoes not have full access to the extracellular solution. Second, H + binding promotes an "outward-facing open" (OF open ) state, involving conformational rearrangement of Helices N and P [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , that widens the extracellular ion-permeation pathway in comparison to the known crystal structures.
The first clue to conformational change at Helix P came from our NMR studies of Y419, on the short P/Q linker, where unambiguous changes in both chemical shift and solvent accessibility of 19 Flabeled Y419 are observed when the pH is lowered from 7.5 to 4.5 . At pH 7.5, the lack of accessibility is consistent with the crystal structure of the occluded conformational state, which depicts Y419 in a buried position. At pH 4.5, the increased accessibility of Y419 indicates a conformational state different from that captured in crystal structures. This state (with Y419 exposed to solution) is observed in channel-like ClC-ec1 at both pH 7.5 and 4.5 . This shift in equilibrium distribution of conformational states for channel-like ClC-ec1 is useful because it enables comparison of the disulfide cross-linking of the two states, which must be done at a pH that is caused by a collective motion. Following a collective motion, a native structure (red helices) undergoes structural transition (peach helices). As a result, the distance between the helices increases by Dr = r' -r. (C) Opening motions of the extracellular gate. The number (N) of collective motions that lead to distance changes (Dr > 1.5 Å ) at each of the extracellulargate residue pairs was determined from analysis of MD simulations for WT and cross-linked ("D417C x-link") ClC-ec1, as described in the text. The data are shown in a box-and-whisker plot where the whiskers denote minimum and maximum of the data and the box denotes the range of 25th percentile to 75th percentile of the data when sorted. The horizontal line in the box denotes the median of the data. (D) The number (N) of collective motions that lead to distance changes (Dr > 1.5 Å ) at the intracellular gate pair 107-445 is not significantly different between WT and cross-linked ClC-ec1. amenable to disulfide bond formation (7.5 rather than 4.5). In the WT background, cross-links near Y419, at D417C, form readily , as expected based on the crystal structure of the occluded conformational state . In contrast, in the channel-like E148A/Y445S background, D417C is resistant to cross-linking . These results suggest that the pH-dependent conformational change detected by NMR involves a change in inter-subunit proximity of D417 residues in addition to the change in solvent accessibility of Y419. DEER experiments confirm such pHdependent change at D417 .
Inter-subunit cross-linking of D417C restricts the conformational transition to the OF open state and inhibits activity. The inhibition occurs not only in the WT background but also in uncoupled E148A, Y445S, and E148A/Y445S (channel-like) backgrounds [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref]. Therefore, the conformational change being restricted is something other than the localized movements of side-chain gates, as these gates (E148 and Y445) are missing altogether in the uncoupled transporters. To gain insight into how conformational change near the subunit interface affects activity, we performed MD simulations on WT and channel-like ClC-ec1, with and without the D417C crosslink. We found that the major motions of both WT and channel-like involve opening of the extracellular vestibule and that these opening motions are dampened by the cross-link at D417 [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Further, our mutagenesis experiments show that removing side-chain interactions between Helices N and P eliminates the effect of the cross-link on Cltransport [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Therefore, we conclude that rearrangement of these helices facilitates a widening of the extracellular ion-permeation pathway.
The residual activity remaining with maximal cross-linking at D417 (ranging from 0-300 s -1 , Table 1) suggests that the OF occluded state may allow some minimal level of Clflux. However, an alternative interpretation is that the OF occluded is completely impermeant to Cland that the residual transport observed with the cross-link is either (1) not distinguishable from zero (due to compounding uncertainties in the various steps involved in the experimental measurement, including quantification of the fraction cross-linked) or (2) occurs because the cross-link does not completely prevent movement of Helix N and opening of the extracellular vestibule to the OF state. We favor the alternative interpretation as it is in keeping with the general principles of transporter function, in which protein conformational change plays a key role in sustaining coupling stoichiometry. In support of this idea, we note that Helix N motions have been strongly implicated not only in ClC-ec1 (the results presented here) but also in the mammalian antiporter CLC-4 [bib_ref] Insights into the ClC-4 transport mechanism from studies of Zn2+ inhibition, Osteen [/bib_ref]. Experiments on this homolog identified an inhibitory Zn 2+ -binding site at the top of Helix N that appears to transmit conformational change to the Cl --permeation pathway at the other end of Helix N [bib_ref] Insights into the ClC-4 transport mechanism from studies of Zn2+ inhibition, Osteen [/bib_ref].
While it is clear that rearrangement of Helices N and P is required for opening the extracellular vestibule, the precise molecular details of this rearrangement remain to be determined. Nevertheless, several pieces of information suggest that the overall motions, though long-range in effect, may involve rearrangements/reorientations of only a few Angstroms in magnitude. First, the crosslinking of Y419C, just 5 Å away from D417C, does not inhibit function [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. Second, any large movement of Helix P would likely have a major effect on water entry via the narrow portal that is the rate-limiting barrier for formation of water wires and H + transport [bib_ref] Intracellular proton access in a cl(-)/H(+) antiporter, Lim [/bib_ref] [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. Since our computational analysis indicates that cross-linking does not significantly affect water entry , Helix-P motion may involve only a small tilt or rotation that exerts a "lever-arm" effect on Helix N and the Cl --entryway. Third, previous inter-subunit cross-linking studies targeting Helices I and Q, and the H-I and I-J loops showed that simultaneously cross-linking these regions had no significant effect on function [bib_ref] CLC cl /H+ transporters constrained by covalent cross-linking, Nguitragool [/bib_ref] and therefore argue against a major restructuring of the inter-subunit interface. Together, these results suggest that the rearrangements at Helices N and P are likely small in magnitude and do not involve the entire inter-subunit interface. This conclusion is in line with computational studies using normal-mode and functional-mode analysis, which showed the subunit interface remaining largely intact even as other regions of ClC-ec1 underwent global conformational changes to alternately expose Cl --and H + -binding sites during the exchange process [bib_ref] Antiport mechanism for cl(-)/H(+) in ClC-ec1 from normal-mode analysis, Miloshevsky [/bib_ref] [bib_ref] Partial least-squares functional mode analysis: application to the membrane proteins AQP1, Aqy1,..., Krivobokova [/bib_ref]. One of the mobile helices identified in these computational studies was Helix R, which has also been pinpointed in experimental studies of H + -dependent conformational change [bib_ref] Site-directed fluorescence studies of a prokaryotic ClC antiporter, Bell [/bib_ref] [bib_ref] 13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1, Abraham [/bib_ref]. Since Helix R extends from the center of the protein (where Y445 coordinates Cl -, [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] out to the cytoplasmic solution [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , the H +dependent conformational change characterized here, while not large in magnitude, may extend well beyond the immediate region around Helices P and N.
To integrate the OF open state into a model of the CLC transport cycle, we build on the model of Basilio et al. [bib_ref] Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter, Basilio [/bib_ref]. Starting with the OF occluded state (State 1 in [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , reflecting the state captured in the E148Q crystal structure, [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] , a conformational change generates the OF open state (State 2). This conformational change is pH dependent but need not be promoted solely by the protonation of Glu ex , as suggested by previous observations of H + - . Purple, yellow and blue each represent data obtained from a separate protein preparation. (E) Detection of inter-subunit disulfide cross-links on D417C/A404L (F) Effect of crosslinking on activity of D417C/A404L. Purple, yellow and blue represent data obtained from separate protein preparations. DOI: 10.7554/eLife.11189.026 dependent conformational changes in Glu ex mutants [bib_ref] Site-directed fluorescence studies of a prokaryotic ClC antiporter, Bell [/bib_ref] [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. The conformational change allows 2 Clions to exit to the extracellular side (State 3). Entry of the protonated Glu ex into the vacated permeation pathway (State 4) facilitates transfer of one H + to the intracellular side, via water wires and the internal H + -transfer site Glu in [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] [bib_ref] Separate ion pathways in a cl-/H+ exchanger, Accardi [/bib_ref] [bib_ref] Intracellular proton-transfer mutants in a CLC cl-/H+ exchanger, Lim [/bib_ref] [bib_ref] Intracellular proton access in a cl(-)/H(+) antiporter, Lim [/bib_ref] [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. Upon unbinding of H + , the protein adopts the apo occluded conformation (State 5) which can then undergo conformational change to the inward-facing state (State 6, [bib_ref] Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter, Basilio [/bib_ref]. Binding of 2 Clfrom the intracellular side knocks Glu ex out of the S ext -binding site (State 7), which then allows H + binding from the extracellular side (back to State 1). This revised model is completely consistent with previous experimental observations, and the addition of new conformational states adds potentially key control points to the mechanism. First, the extracellular occlusion in State 7 assures no extra Clslips through during the step in which Clbinds from the intracellular side. Second, we hypothesize that the OF open state lowers Claffinity and promotes Clrelease, as suggested by the increase in Cl --binding affinity observed when formation of the OF open state is inhibited by the D417C cross-link .
Our revised model also sheds light on the mechanism of channel-like ClC-ec1. Previously, it was recognized that the narrow pathway depicted by the crystal structures of channel-like ClC-ec1 is not sufficiently wide to allow rapid ion conduction and that protein dynamics (either breathing or conformational change) must play an important part in the mechanism of ion conduction [bib_ref] Ion permeation through a cl-selective channel designed from a CLC cl-/H+ exchanger, Jayaram [/bib_ref]. Our results clarify the issue by showing that channel-like ClC-ec1 populates a conformation different from that seen in the crystal structure and exhibiting similarities to the new OF open state characterized in these studies. In this state, the region of the narrowest constriction -just above S ext -is significantly widened [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref]. The population of this state explains why channel-like ClC-ec1 can conduct Clrapidly at pH 7.5.
The long-range conformational change described here improves our understanding of CLC mechanisms by providing a first glimpse of an "outward-facing open" CLC conformational state and its mechanistic implications. In future studies, it will be important to investigate the transition between the OF open , OF occluded and inward-facing conformational state(s). Using a cross-linking strategy, Basilio et al. showed that transition to the inward-facing state involves motion of the intracellular half of Helix O. This motion is thought to be limited in scope, as it is relayed directly to the intracellular gate via a steric interaction between intracellular-gate residue Y445 and Helix O residue I402 [bib_ref] Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter, Basilio [/bib_ref]. Nevertheless, since Helix O also makes direct contacts to Helices N and P (studied here), it seems likely that intracellular and extracellular gate-opening motions will be linked through the interaction of these three helices. Understanding these interactions will be critical to providing a molecularly detailed view of the CLC transport mechanism.
# Materials and methods
Expression, purification, reconstitution and flux assays Expression and purification of unlabeled ClC-ec1 WT and mutant proteins was performed as documented in detail [bib_ref] Secondary active transport mediated by a prokaryotic homologue of ClC cl-channels, Accardi [/bib_ref] except that the final purification step was by size exclusion chromatography on a Superdex gel filtration column [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref] rather than ionexchange chromatography. Point mutations introduced by conventional PCR methods were confirmed by sequencing. D417C constructs were made in a previously characterized cysteine-less background C85A/C302A/C347S [bib_ref] CLC cl /H+ transporters constrained by covalent cross-linking, Nguitragool [/bib_ref] , which here is referred to as the "WT background". For preparing ClC-ec1 under reducing conditions, 20 mM b-mercaptoethanol (b-ME) and 1 mM dithiothreitol (DTT) (Fisher Scientific, Pittsburgh, PA) were added to cell pellets during resuspension, and 1 mM DTT was included in subsequent purification steps. DTT was removed in the final purification step over a Superdex 200 size exclusion column.
To measure turnover rates in flux assays, ClC-ec1 variants were reconstituted into liposomes by dialysis [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref] into buffer R (300 mM KCl, 40 mM Na-citrate, pH 4.5) using 0.2 -5 mg protein per mg of E. coli polar lipids (Avanti Polar Lipids, Alabaster, AL). For the high-turnover channel-like variant, the lower end of this range (0.2 mg protein per mg lipids) was used. For experiments to determine stoichiometry, protein to lipid ratio was 0.4-10 mg protein per mg lipid (with higher ratios used for low-turnover mutants). Reconstituted liposomes were subjected to 4 freeze-thaw cycles and were extruded through 400-nm filters 15 times using an Avanti Mini-Extruder. Liposomes were buffer-exchanged through Sephadex G-50 spin columns [bib_ref] A proteoliposome-based efflux assay to determine single-molecule properties of clchannels and transporters, Basilio [/bib_ref] into fluxassay buffer (300 mM K-isethionate, 50 mM KCl, buffered with 2 or 40 mM Na-citrate pH 4.5). (The 2 mM Na-citrate buffer was used in experiments in which Cland H + transport were measured in parallel; the 40 mM Na-citrate buffer was used in experiments in which only Cltransport was measured.) Transport was initiated by addition of 2 mg/mL valinomycin (for dual Cl -/H + -transport measurements) or 3 mg/mL CCCP + 7 mg/mL valinomycin (for Cl --transport measurements) [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. At the end of each flux-assay experiment, total liposomal Clwas determined by disrupting the liposomes with Triton X-100 (0.01%; from a 10% stock solution); flux-assay traces shown in [fig_ref] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds [/fig_ref] and 11 show normalization to this value. Transport turnover rates were calculated by measuring the initial velocity of the Cland/or H + transport [bib_ref] Uncoupling and turnover in a cl-/H+ exchange transporter, Walden [/bib_ref]. Stoichiometry was determined from the ratio of the Clto the H + turnover rate. Flux assays were performed in sets of 20-40 (2). This step is pH-dependent but may be promoted by protonation of residues other than Glu ex (see Discussion). Two Clions leave (3) and then entry of the protonated Glu ex into the permeation pathway (4) facilitates H + -transfer to the inside (via Glu in , [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] (5). Conformational change to the inward-facing state (6) allows 2 Clions to enter from the intracellular side, knocking Glu ex out of the pathway (7). The cycle is reversible, with protonation favoring conformational change to the OF open state. (B) Channel-like CLC states. The crystal structure of channel-like ClC-ec1 reveals a narrow constriction at the extracellular-gate region, depicted at left. However, results here demonstrate that the major conformation adopted in solution more closely resembles the OF open state (equilibrium shifted to right). This finding is consistent with the high Clthroughput observed in channel-like ClC-ec1. samples; within each set, an assay was discarded if the total liposomal [Cl -] (a measure of the yield of reconstituted liposomes, which affects the accuracy of the unitary-turnover calculation) was >30% outside of the mean. Flux-assay measurements were performed on at least 4 samples for each condition. This sample size and selection method is based on previous experience with flux-assay measurements [bib_ref] A designed inhibitor of a CLC antiporter blocks function through a unique..., Howery [/bib_ref] [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref].
## F nmr
19 F-Tyr labeling was performed as described [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. Labeled ClC-ec1 was purified into Buffer A (150 mM NaCl, 10 mM HEPES (Fisher Scientific, Pittsburgh, PA), pH 7.5 and 5 mM ndecyl b-D maltopyranoside (DM) (Anatrace, Maumee, OH), then concentrated to approximately 50 mM. E. coli polar lipids were added in a 1:80 lipid:detergent molar ratio to the BuriedOnly construct to enhance stability [bib_ref] Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR, Elvington [/bib_ref]. The Y419Only construct was more stable without the addition of lipids. 10% D 2 O was added prior to NMR experiments. Samples (~300 mL starting volumes) were placed in the outer tube of Shigemi symmetrical microtubes in order to reduce the volume of sample required for data acquisition. The Shigemi tube insert was not used so as to avoid generating froth from adjusting the plunger in the detergent containing sample. Data were collected using a 5 mm H/F probe on a Bruker Avance 500 MHz spectrometer running Topspin version 1.3 with variable temperature control. Data represent acquisition of 30 -50k transients at 470 MHz; 12 kHz spectral width; 45˚pulse; 0.17s acquisition time; 1.8 -2.8s relay cycle; 20˚C; 15 Hz linebroadening; referenced to TFA. The pH of the samples was lowered to 4.5 using a 1 M citric acid solution (EMD Millipore, Billerica, MA) and raised to 7.5 using a 1M Tris-acetate pH 9.0 solution. TEMPOL (4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, Fluka Analytical, Ronkonkoma, NY) was added to the sample by carefully weighing out and adding the solid reagent required to attain a final concentration of 100 mM in the NMR sample.
## Cysteine cross-linking
All procedures were carried out at room temperature (21-23ºC). Stock solutions of CuP at 10x were made from 1:3 mixtures of CuSO 4 (aqueous) (MCB Reagents, Cincinnati, OH) and 1,10-phenanthroline (in ethanol) (Sigma-Aldrich, St. Louis, MO). ClC-ec1 eluted from the Superdex 200 column in Buffer A was diluted to 0.2 mg/mL (1.9 mM homodimer; 3.8 mM Cys-containing subunits) before addition of CuP. After an hour of incubation, 1 mM Na-EDTA (Fisher Scientific, Pittsburgh, PA) was added to terminate the cross-linking reaction. Cross-linking was visualized using SDS/PAGE (4-15% gradient gels) and staining with Coomassie brilliant blue (TCI America, Portland, OR). Cross-linking was documented using an Odyssey Infrared Imaging System (LI-COR Biosciences) using the 700 nm channel. ClC-ec1 band intensities were quantified using NIH ImageJ software. Un-cross-linked ClC-ec1 runs as a monomer (apparent molecular weight~36 kD) and cross-linked mutant as a dimer (apparent molecular weight~64 kD). The fraction cross-linked was calculated based on the relative intensities of the dimer and monomer bands. For channel-like ClC-ec1, which exhibited a low efficiency of cross-linking, free thiols were quantified colorimetrically (Life Technologies Thiol and Sulfide quantification kit, T6060). During reconstitution into liposomes, most CuP-treated samples were dialyzed in buffer containing 1 mM DTT in order to avoid additional cross-linking during the dialysis step; this level of DTT was sufficiently low that it did not reduce D417C disulfide bonds that had already been formed. Mutant proteins D417C/F357A and D417C/L361A were reconstituted in the absence of DTT when crosslinked with 100 mM CuP, as an extra precaution to avoid disulfide-bond reduction in these samples.
## Deer
For preparing D417C ClC-ec1 (WT and channel-like backgrounds) for DEER experiments, 20 mM b-ME was added to cell pellets during resuspension. b-ME was removed during washing and elution from the cobalt column. Proteins eluted from the cobalt column were incubated with 50x molar excess of the paramagnetic spin label MTSSL (Enzo Life Sciences, Farmingdale, NY) that was dissolved in small volume of dimenthylformamide (DMF) (Fisher Scientific, Pittsburgh, PA) such that the final DMF concentration was < 0.1%. The protein solution was sealed under argon and mixed by slow rotation (~20 rpm) for 2 hr at room temperature. The remaining steps of the purification were identical to our usual ClC-ec1 purifications. Thus, the 6-His tag was then removed by a one-hour incubation with endoprotease Lys-C (Roche Diagnostics, Indianapolis, IN). The ClC-ec1 samples were then purified from the cleaved 6-His tag and excess MTSSL by size exclusion chromatography on a Superdex 200 column. Glycerol (23% v/v) was added to the protein solution as cryoprotectant. This was achieved by adding an 80% (v/v) glycerol stock solution (prepared in buffer A) to the purified protein. The samples were then concentrated to a final concentration of 50-100 mM, and E. coli polar lipids were added at 1:80 lipid:detergent molar ratio. A stock solution of 25 mM citrate was used to adjust the sample at pH 7.5 to pH 4.5. Functional assays were performed on EPR samples that had been exposed to pH 4.5 for one hour before reconstitution. CW-spectra were collected on a Bruker EMX at 10 mW power with a modulation amplitude of 1.6G. Spectra were normalized to the double integral. DEER experiments were carried out using a standard four-pulse protocol [bib_ref] DEER distance measurements on proteins, Jeschke [/bib_ref]. Samples were maintained at 83K. DEER distributions were obtained from fitting the DEER decays to a sum of Gaussian distributions [bib_ref] The global analysis of DEER data, Brandon [/bib_ref] [bib_ref] A straightforward approach to the analysis of double electron-electron resonance data, Stein [/bib_ref].
## Structure determination
For crystallization, the D417C mutant was put into a deletion construct (DNC) lacking N-terminal residues 2-16 and C-terminal residues 461-464 [bib_ref] Intracellular proton access in a cl(-)/H(+) antiporter, Lim [/bib_ref]. Purified DNC-D417C was cross-linked with 100 mM CuP for 1 h, incubated with excess Fab fragment [bib_ref] Gating the selectivity filter in ClC chloride channels, Dutzler [/bib_ref] for 30 min, then purified by size exclusion chromatography (Superdex 200) into buffer containing 100 mM NaCl, 5 mM DM, 10 mM Tris (Fisher Scientific, Pittsburgh, PA), pH 7.5. The complex was concentrated to 10-12 mg/mL and mixed with 30% PEG 400 (Hampton Research, Aliso Viejo, CA), 0.075 M K/Na-tartrate (Fluka Analytical, Ronkonkoma, NY), 0.1 M Tris HCl (MP Biomedicals, Santa Ana, CA) (pH 9.0). Crystals were grown by the sitting drop method for 2-4 weeks at 20 o C and were directly harvested from the reservoir, flash frozen and stored in liquid N 2 . Diffraction data were collected to 0.9795 Å at the BL12-2 beamline (SLAC) and processed using XDS. Phases were obtained by molecular replacement with the WT protein in complex with Fab (PDB 1OTS) using the MOLREP program [bib_ref] Molecular replacement with MOLREP, Vagin [/bib_ref]. Refinement was done using the refmac program [bib_ref] Refinement of macromolecular structures by the maximumlikelihood method, Murshudov [/bib_ref]. Atomic coordinate and structure factors are deposited in the Protein Data Bank under accession code 5HD8.
## Isothermal titration calorimetry
ITC was carried out using a MicroCal VP-ITC instrument. Chloride binding to WT and mutants were carried out as described previously [bib_ref] Basis of substrate binding and conservation of selectivity in the CLC family..., Picollo [/bib_ref] [bib_ref] A designed inhibitor of a CLC antiporter blocks function through a unique..., Howery [/bib_ref]. Briefly, ClC-ec1 (WT or D417C or D417 cross-linked using 100 mM CuP) was purified over a Superdex 200 size exclusion column pre-equilibrated with Buffer B (100 mM K + -Na + -tartrate, 20 mM HEPES, 5 mM DM, pH 7.5) and then concentrated to 25-50 mM. Percent cross-link following treatment with 100 mM CuP was 92.0 ± 0.6% (n=2). The injection syringe was filled with Buffer B containing 20 mM KCl. Each experiment consisted of 30 10-mL injections of the Cl --containing solution at 5 min intervals, to achieve a final molar ratio of 50-160. The chamber was kept at 25 o C with constant stirring at 350 rpm. All solutions were filtered and degassed before use. ITC data were fit to a single-site isotherm as described with Origin 7 MicroCal program.
## Molecular dynamics (md) simulations
The ClC-ec1 crystal structure at 2.51 Å (PDB ID: 1OTS) [bib_ref] Gating the selectivity filter in ClC chloride channels, Dutzler [/bib_ref] was used to prepare for the MD simulations of all the systems studied in the present work -WT, D417C, channel-like (E148A/Y445S), and D417C/channel-like. The system setup for the WT ClC-ec1 is detailed in our previous work [bib_ref] Water access points and hydration pathways in CLC h+/Cltransporters, Han [/bib_ref]. In short, to have the protein hydrated properly, all the crystallographic water molecules were maintained and 49 additional water molecules were added using DOWSER [bib_ref] Hydrophilicity of cavities in proteins, Zhang [/bib_ref]. One additional water molecule was placed between Glu ex (E148) and the Clion bound to the central ion-binding site of ClC-ec1 [fig_ref] Figure 1: Structure of CLC transporters [/fig_ref] in order to stabilize the two closely (within~4 Å ) positioned negative charges, as suggested in previous simulation studies [bib_ref] Exterior site occupancy infers chloride-induced proton gating in a prokaryotic homolog of..., Bostick [/bib_ref] [bib_ref] Mechanism of anionic conduction across ClC, Cohen [/bib_ref] [bib_ref] Proton transport pathway in the ClC cl-/H+ antiporter, Wang [/bib_ref]. Glu ex (E148) and Glu in (E203) were both deprotonated, while E113 was modeled in its protonated form according to previous Poison-Boltzmann electrostatic calculations . The protein was embedded into a POPE lipid bilayer, fully equilibrated TIP3P water [bib_ref] Comparison of simple potential functions for simulating liquid water, Jorgensen [/bib_ref] and buffered in 150 mM NaCl, resulting in a 105 Â 105 Â 110 Å 3 box with~110,000 atoms.
The mutant systems were constructed on the basis of that of the WT. For each mutant, residue substitutions were done using the Mutator plugin of VMD [bib_ref] VMD: visual molecular dynamics, Humphrey [/bib_ref]. Disulfide bonds were constructed by introducing geometric restraints on two cysteine residues, including a distance restraint between the sulfur atoms and angular restraints involving C b atom of either cysteine and the two sulfur atoms. To avoid structural disruption of the protein due to sudden introduction of restraints, the disulfide restraints were turned on gradually over 20-ns simulations. Note that the systems prepared as such are not significantly different from the cross-linked D417C crystal structure. In fact, during the equilibrium simulation of the mutant containing the disulfide bond (see below), the RMSD of the protein to the D417C crystal structure is on average~1.6 Å , even smaller than its RMSD (~1.9 Å ) to the WT crystal structure that the simulation started from.
All MD simulations were carried out with NAMD 2.9 [bib_ref] Scalable molecular dynamics with NAMD, Phillips [/bib_ref] using the CHARMM-CMAP [bib_ref] Extending the treatment of backbone energetics in protein force fields: limitations of..., Mackerell [/bib_ref] and CHARMM36 force fields [bib_ref] Update of the CHARMM all-atom additive force field for lipids: validation on..., Klauda [/bib_ref] to model the proteins and lipids, respectively. The particle mesh Ewald (PME) [bib_ref] Particle mesh ewald: an nÁlog(N) method for ewald sums in large systems, Darden [/bib_ref] method was used to calculate long-range electrostatic forces without truncation. All simulation systems were subjected to Langevin dynamics and the Nosé -Hoover Langevin piston barostat [bib_ref] Canonical dynamics: equilibrium phase-space distributions, Hoover [/bib_ref] for constant pressure (P = 1 atm) and temperature (T = 310 K) (NPT). Each system was energy-minimized for 5,000 steps, followed by a 1-ns MD run with positions of all protein atoms and oxygen atoms of the crystallographic water molecules restrained. Each system was simulated without any restraints for~300 ns.
## Analysis of collective motions of protein
The collective motions of the protein were analyzed through principal component analysis (PCA) of the equilibrium MD trajectories [bib_ref] Essential dynamics of proteins, Amadei [/bib_ref]. Specifically, we first constructed the covariance matrix C of C a atoms of select parts of the proteins for each subunit based on equilibrium MD trajectories. The covariance matrix C was calculated as c ij = <(x in -<x in >)(x jn -<x jn >)>, where X n ={x in } are the coordinates of C a atoms of select parts of protein in the n th sampled structure and the brackets <> denote the averages over all the sampled structures. The first 50 ns of each MD trajectory were discarded to remove any initial bias. Only the transmembrane helical regions were selected for this analysis as they define the overall architecture of the protein and most relevant to the functionally relevant global motions. We then derived orthonormal eigenvectors R={R k } of the covariance matrix C. Each eigenvector R k ={r ik } defines relative movement (r ik ) of each select atom in a collective motion of the protein represented by the eigenvector. The 20 eigenvectors with the largest eigenvalues were chosen for further analysis. These eigenvectors correspond to the collective motions that account for >75% of protein motion observed in the simulations.
Following the approach by Bahar and co-workers [bib_ref] Mechanism of signal propagation upon retinal isomerization: insights from molecular dynamics simulations..., Isin [/bib_ref] , conformational deformation driven by a given collective motion can be calculated according to the associated eigenvector R k as follows:
[formula] X 0 ¼ X 0 AE AR k(1) [/formula]
where X 0 and X' denote the coordinates of the reference structure and the structure of the protein deformed by the collective motion, and A is an arbitrary scaling factor determining the extent of structural deformation to be examined. The value of A is related to the RMSD between the reference and the deformed structures through the relationship RMSD = A/M 1/2 , where M is the number of atoms selected to calculate RMSD (here M=538, the number of C a atoms located in the transmembrane helical region of the protein). To make a meaningful comparison of all collective motions investigated, the value of A was chosen such that the structure of the protein is altered by each motion to the same extent, targeting always a total RMSD of 3.5 Å with respect to the original structure. Thus, the distance change (Dr) between two sites (x i and x j ) of interest [fig_ref] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1... [/fig_ref] can be calculated according to Dr ¼ jx 0
i À x 0 j j À jx i À x j j. Finally, we quantified the protein's ability of opening its gates via collective motions by counting the dominant collective motions that involved an increase in the distance between residues lining the gates by Dr > 1.5 Å . To achieve a statistical estimate of such counts, each~300-ns simulation trajectory of the homodimer was divided evenly into three time blocks, each being analyzed through the procedure described above, providing a dataset of six segments (three time blocks for each subunit x 2 subunits). Statistical comparisons between datasets were made using the Wilcoxon-Mann-Whitney test [bib_ref] On a test of whether one of two random variables is stochastically..., Mann [/bib_ref].
[fig] Figure 1: Structure of CLC transporters. (A) Structure of [/fig]
[fig] Figure supplement 1: Comparison of CLC structures determined at high and low pH. DOI: 10.7554/eLife.11189.004 [/fig]
[fig] Figure 6: Cross-linking D417C in uncoupled transporter backgrounds. (A) D417C/E148A -detection of intersubunit disulfide cross-links by non-reducing SDS-PAGE. (B) Effect of cross-linking on activity of D417C/E148A. Left: Representative data traces showing Cl --transport activity. Right: Summary data showing Cl --transport activity as a function of disulfide cross-linking. Each data point represents one flux-assay measurement, with error bars indicating the uncertainty in curve-fitting to the primary data. Purple, yellow, blue, and dark red each represent data from a separate protein preparation. (C) D417C/Y445S -detection of inter-subunit disulfide cross-links. (D) Effect of cross-linking on activity of D417C/Y445S, as in panel B. Data are from three separate protein preparations (indicated in purple, yellow, and blue). DOI: 10.7554/eLife.11189.019 The following figure supplement is available for figure 6: Figure supplement 1. H + turnover of D417C/Y445S. DOI: 10.7554/eLife.11189.020 [/fig]
[fig] Figure 8: Coupling of extracellular and intracellular gating motions to collective motions in ClC-ec1 detected computationally. (A) Key inter-Ca distances were employed to detect functional motions. The left panel shows the location of the Clgates (dashed box) and transport pathways (dashed green line) in ClC-ec1. Right panel shows a close-up of the Clgates where key inter-Ca distances for both the extracellular and intracellular gates are denoted by dashed double arrows. (B) Scheme for determining distance change (Dr) [/fig]
[table] Table 1: D417C activity extrapolated to 0 and 100% cross-link.Values for turnover at 0 and 100% D417C cross-link were estimated from extrapolation of fits to data inFigures 4, 7 and 11. The uncertainties report the 95%confidence interval in the extrapolated values. DOI: 10.7554/eLife.11189.016 [/table]
[table] Table 2: Data collection and refinement statistics a . [/table]
|
A case of amyotrophic lateral sclerosis which was diagnosed with progressive dysphagia and muscle atrophy
This 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.AbstractA 79-year-old man presented to our outpatient clinic with symptoms of dysphagia, dysarthria, and muscle atrophy of the trunk and upper extremities. These symptoms were gradually progressive, and he had lost substantial weight-20 kg in 2 years. One month later, he was admitted due to dehydration and received tube feeding. The presence of "split hand" suggested amyotrophic lateral sclerosis (ALS). Finally, the patient was diagnosed with ALS by two neurologists. When elderly patients present with progressive dysphagia and muscle atrophy, especially with "split hands," ALS should be included as a differential diagnosis.K E Y W O R D Samyotrophic lateral sclerosis, dysarthria, dysphagia, muscle atrophy, split hand, weight loss
## | introduc ti on
Amyotrophic lateral sclerosis (ALS) is a common and well-recognized motor neuron disease that involves degeneration of both upper and lower motor neurons, leading to progressive muscular paralysis. Death typically occurs within 1-5 years after the onset of the disease. [bib_ref] Evaluation and rehabilitation of patients with adult motor neuron disease, Francis [/bib_ref] Here, we describe an elderly patient who exhibited "split hand" 2,3 with progressive dysphagia and muscle atrophy, which suggested a diagnosis of ALS.
## | c a s e rep ort
A 79-year-old man developed dysarthria, dysphagia, and weakness of the hands 1 year before admission, which gradually progressed. Therefore, 6 months later, he was referred to a neurosurgical hospital. A definitive diagnosis was not confirmed even after brain computed tomography (CT). Five months later, he presented to our outpatient clinic. One month postpresentation, he was admitted to our hospital owing to appetite loss and dehydration. He had lost substantial weight (20 kg; starting weight: 75 kg; final weight: 55 kg; body height: 158 cm) in 2 years. He had no family history of neurodegenerative disease.
On admission, he was alert (Glasgow Coma Scale score: 15).
We observed atrophy and fasciculation of the tongue; dysphagia; and dysarthria. Other cranial nerves were normal. Muscle atrophy was observed . The first dorsal interosseous muscles and thenar eminence muscles showed "split hand" 2,3 signs bilaterally Neither sensory impairments nor cerebellar ataxias were observed.
Deep tendon reflexes were as follows: upper extremities (2+/2+) and lower extremities (3+/3+). Abnormal reflexes/fasciculation details are shown in . No bladder or rectal disturbances, nor extrapyramidal disorders were observed. He could walk using a U-shaped walker. All other physical examination results were unremarkable.
The differential diagnoses were ALS, brain-stem tumor, cervical spondylosis, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, progressive muscular atrophy, myasthenia gravis, and myopathy. Precontrast brain CT and cervical X-ray film excluded other intracranial or cervical abnormalities. Electrophysiological examinations of the bilateral median and ulnar nerves revealed mild reduction in amplitude in motor nerve velocity tests; no abnormality was recorded in sensory nerve velocity tests. Brain MRI was not performed because he had a DDD pacemaker. Lumbar puncture was not executed because he had lumbar spondylosis. Hence, the aforementioned differential diagnoses could not be completely excluded.
The final diagnosis was made by two neurologists based on the Awaji algorithm, combined with the El Escorial criteria, which are considerably useful for early ALS diagnosis. [bib_ref] Awaji diagnostic algorithm increases sensitivity of El Escorial criteria for ALS diagnosis, Carvalho [/bib_ref] The diagnostic grade was "probable." 4 Regarding rehabilitation, because there is no effective training for symptoms of ALS, we modified his daily environment. 1,5 For 1 month after admission, his muscle weakness progressed noticeably. He could not walk, despite staff assistance. He experienced breathing difficulty and received frequent intratracheal aspiration.
Two months after admission, he was transferred to a center for intractable neural disease. Three months after admission, he died of acute hypercapnic respiratory failure.
## | d iscuss i on
To definitively diagnose ALS, initial diagnosis should be confirmed by a neurologist. However, it is important for the attending physician to collect relevant medical information before consultation with the neurologist. [bib_ref] Evaluation and rehabilitation of patients with adult motor neuron disease, Francis [/bib_ref] [bib_ref] Comprehensive rehabilitative care across the spectrum of amyotrophic lateral sclerosis, Paganoni [/bib_ref] In this case, positive signs were apparent and progressive: Negative signs were consistent with ALS. Therefore, when ALS is suspected, diagnosis may not be difficult. An important consideration is the need for suspicion of this disease at an earlier stage of its progressive clinical course. Chio suggested that the diagnostic process for ALS may be excessively prolonged: The median time from onset to diagnosis was 12-17 months. [bib_ref] Update on ISI survey: Europe, North America and South America, Chiò [/bib_ref] The reasons included the presence of other diseases, misinterpretation of examination findings, and lack of familiarity with ALS. [bib_ref] Update on ISI survey: Europe, North America and South America, Chiò [/bib_ref] In our case, "lack of familiarity" was thought to have delayed the diagnosis. However, the suspicion of ALS was greatly enhanced owing to the observed "split hand" sign, which is a highly specific ALS symptom. [bib_ref] The split hand syndrome, Wilbourn [/bib_ref] [bib_ref] Dissociated small hand muscle involvement in amyotrophic lateral sclerosis detected by motor..., Kuwabara [/bib_ref] Murphy et al 8 reported that ALS incidence rates steadily increased by 3% per year over the 22 years (from 1985 to 2006). They confirmed older age, male sex, and bulbar onset as adverse prognostic factors. [bib_ref] Increasing incidence of, Murphy [/bib_ref] Furthermore, Tanaka et al 9 suggested that patients with late-onset ALS showed more rapid disease progression than those with early-onset ALS using the progression rate. Our case is "late-onset ALS with bulbar onset," which showed rapid disease progression.
In summary, when physicians encounter elderly patients with progressive dysphagia and muscle atrophy, they should consider that those patients may have neurodegenerative diseases: Thus, they should examine whole-body condition and function, including muscle mass and strength.
## Ack n owled g em ents
I especially thank all the nursing and rehabilitation staff for their devoted and capable nursing care and rehabilitation. I greatly appreciate Dr. Keiko Tanaka and Dr. Toru Imamura for the diagnosis
[fig] F: I G U R E 1 Picture of dorsal side of both hands showing atrophy especially of the first dorsal interosseous muscles and thenar eminence muscles [/fig]
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Lip synechiae: A rare complication of azithromycin-associated Stevens–Johnson syndrome
Stevens-Johnson syndrome (SJS) is a severe form of erythema multiforme, is a self-limiting acute inflammatory disease of multifactorial origin, but can also present as a chronic recurrent lesion. It causes a whole plethora of lesions, mostly mucocutaneous. It is a dermatologic emergency that occurs with a spectrum of severity and can result in severe morbidity and mortality. Lip adhesion is an unusual complication of healing in the lesions of SJS, for which only a few cases have been reported till date which not only causes esthetic morbidity but also impairs the proper functioning of the patient. The importance of this lesion also lies in its multifactorial and varied origin, this being the first case to report azithromycin as a causative drug, leading to SJS associated with lip adhesion. In this paper, we present a case report of SJS with lip adhesion, azithromycin being the causative drug, which was treated surgically with chalinoplasty. Along with it, the clinical features, its pathogenesis, the preventive measures, and the treatment modalities for the same including conservative as well as surgical have also been extensively discussed with a review of the existing English literature to date.
# Introduction
Stevens-Johnson syndrome (SJS) is a severe form of erythema multiforme. It is a disease of multifactorial origin. This self-limiting acute inflammatory disease affects the skin and oral mucous membranes, especially the lips due to which speech, eating, and swallowing becomes very difficult and painful. [bib_ref] Lip adhesion: Unusual complication of Stevens-Johnson syndrome, Royan [/bib_ref] Its causative agents including various triggering agents such as infections, food, drugs (azithromycin being one of the rarest), immunizations, malignancies, and certain systemic diseases are also implicated in its pathology. [bib_ref] Lip adhesion: An unusual complication of erythema multiforme. Oral Surg Oral Med..., Marinho [/bib_ref] An unusual complication of healing lesions in azithromycin-induced SJS is lip adhesion, and only a few cases have been reported in the World English Literature so far.
## Case report
An 18-year-old female reported to us with restricted mouth opening due to adhesion of lips. On recording the history, it was found out that the patient was treated with azithromycin for common cold 2 months back, following which she developed skin target lesions on her trunk, limbs, and face, mild conjunctivitis, and severe desquamative gingivostomatitis. The patient was diagnosed with SJS that developed due to azithromycin administration [ [fig_ref] Figure 1: Stevens-Johnson syndrome developed in an 18-year-old female due to azithromycin administration [/fig_ref] ] and was successfully managed at a tertiary care center.
## Lip synechiae: a rare complication of azithromycin-associated stevens-johnson syndrome
## 10.4103/njms.njms_24_19
This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. On the day of the presentation to our department, the patient had no systemic disability and the skin lesions were resolving. On examination, the patient was well and had mucosa-lined adhesions close to the corners of her mouth, one on each side, between the upper and lower lips that restricted her mouth opening [ ]. These bands were pink, 0.2 cm in diameter, and about 1 cm in length on either side (at maximal opening). Although her mouth opening was restricted, the interincisal opening was almost 2 cm.
Bilateral chalinoplasty was performed that involved the release of adhesions between the lips at the corners of mouth using a knife under local anesthesia and lips were applied with petroleum jelly so as to prevent re-attachment. The postoperative period was uneventful, with no recurrence of lip adhesions [ ].
# Discussion
In 1922, Stevens and Johnson described SJS as a combination of symptoms reported in two patients who had continuous fever, inflamed buccal mucosa, and severe conjunctivitis. [bib_ref] A new eruptive fever associated with stomatitis and ophthalmia, Stevens [/bib_ref] The various features associated with the syndrome include target lesions of the skin, erosions of the oral mucosa and lips, and formation of pseudomembranous tunica conjunctiva palpebrarum. SJS, also known as erythema multiforme major, [bib_ref] Burket's Oral Medicine. 10, Greenberg [/bib_ref] is an immune complex-mediated disease which can cause significant morbidity given that it includes the skin and mucous membrane and may even lead to death (10%-34%). [bib_ref] Causes and treatment outcomes of Stevens-Johnson syndrome and toxic epidermal necrolysis in..., Kim [/bib_ref] [bib_ref] Comprehensive survival analysis of a cohort of patients with Stevens-Johnson syndrome and..., Sekula [/bib_ref] The disease usually begins with a nonspecific upper respiratory tract prodrome, which lasts for up to 14 days. Mucocutaneous lesions then develop abruptly, and these may continue to erupt for around 2-4 weeks. Some of the complications that can occur are corneal ulceration, anterior uveitis, panophthalmitis, penile scarring, vaginal stenosis, esophageal webs and strictures, renal tubular necrosis, renal failure, tracheobronchial shedding, respiratory failure, and finally scarring, deformity, and infections that mostly occur in slow-healing ulcers. [bib_ref] Erythema multiforme: A critical review of characteristics, diagnostic criteria, and causes, Huff [/bib_ref] The oral mucosal lesions are painful, and the progression of the disease is such that the vesiculobullous lesions rupture soon after their appearance causing the raw exudate to further limit the function.Wherever broad erosions of the labial epithelium are seen, lip adhesions, although uncommon, may occur in severe cases. This is followed by crusting, which occurs in approximately 2-3 days. [bib_ref] Burket's Oral Medicine. 10, Greenberg [/bib_ref] The lips often seal at the angle region due to crusting, most probably by the scarred healing of the coalescence of pseudomembranes and oral mucosal ulcers, and so was the scenario in our case.
To the best of our knowledge, five other cases of lip adhesion have been reported previously, out of which three occurred as a complication of erythema multiforme, all of which affected children or adolescents, one of them reported as a complication of a herpetic infection in an adult, and only one case was related to complication of a drug, phenytoin (anticonvulsant). [bib_ref] Lip adhesion: Unusual complication of Stevens-Johnson syndrome, Royan [/bib_ref] [bib_ref] Lip adhesion: An unusual complication of erythema multiforme. Oral Surg Oral Med..., Marinho [/bib_ref] [bib_ref] Angular webbing associated with Stevens-Johnson syndrome, Sakamoto [/bib_ref] [bib_ref] An unusual complication of erythema multiforme and its treatment, Karincaoglu [/bib_ref] [bib_ref] A complication of primary herpetic gingivostomatitis, Thomas [/bib_ref] With a review of the current literature, we can appreciate the rarity of this condition which can also be attributed to the paucity of the reported cases due to lack of awareness because of the multifactorial origin of the disease.
When we talk about the etiology of the cases that have been mentioned in the English literature, we conclude that the causes of angular webbing [bib_ref] Angular webbing associated with Stevens-Johnson syndrome, Sakamoto [/bib_ref] that have been reported so far are concurrent with the etiological factors mentioned in the literature, including erythema multiforme, infections, and anticonvulsant drugs such as phenytoin; [bib_ref] Lip adhesion: Unusual complication of Stevens-Johnson syndrome, Royan [/bib_ref] however, ours is the first case that has azithromycin as an etiological factor, which is in fact a very commonly prescribed drug for common cold and hence raises a point of concern for the physician before prescribing it.
Although rare, exposure to azithromycin may result in immune-mediated dermatologic emergency known as SJS. The exact pathogenesis of SJS is not well established so far. Aihara et al. measured serum cytokines and eosinophilic cationic protein (ECP) levels at different time intervals in a patient of SJS due to azithromycin. Serum cytokines, ECP, and interleukin-6 levels were increased initially on day 8 and then decreased dramatically on day 28. Based on their findings, they suggested that activated eosinophil might play some role in SJS. [bib_ref] Stevens-Johnson syndrome associated with azithromycin followed by transient reactivation of herpes simplex..., Aihara [/bib_ref] More or less, its management is supportive and symptomatic, and the use of corticosteroids is still a topic of debate. Controversy exists as to whether newer treatments such as intravenous immunoglobulin, plasmapheresis and hemodialysis, cyclophosphamide, cyclosporine, acetylcysteine, and thalidomide actually decrease mortality. [bib_ref] Drug-induced Stevens-Johnson syndrome/toxic epidermal necrolysis, Fritsch [/bib_ref] Although lip adhesion is not a serious complication and can be treated appropriately with chalinoplasty which was the treatment of choice, in this case, prevention is always better; hence, precautionary measures can be taken such as application of a lubricant-like petroleum jelly over the lips.
Another method to encourage rapid healing is the use of hydrocortisone ointment over the ulcerations.
## Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given her consent for her images and other clinical information to be reported in the journal. The patient understands that her name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed.
## Financial support and sponsorship
Nil.
[fig] Figure 1: Stevens-Johnson syndrome developed in an 18-year-old female due to azithromycin administration [/fig]
[fig] Figure 2, Figure 3: Bilateral lip adhesions after healing of lip lesions Lips 6 weeks after bilateral chalinoplasty [/fig]
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The Impact of Load Carriage on Measures of Power and Agility in Tactical Occupations: A Critical Review
The current literature suggests that load carriage can impact on a tactical officer's mobility, and that survival in the field may rely on the officer's mobility. The ability for humans to generate power and agility is critical for performance of the high-intensity movements required in the field of duty. The aims of this review were to critically examine the literature investigating the impacts of load carriage on measures of power and agility and to synthesize the findings. The authors completed a search of the literature using key search terms in four databases. After relevant studies were located using strict inclusion and exclusion criteria, the studies were critically appraised using the Downs and Black Checklist and relevant data were extracted and tabled. Fourteen studies were deemed relevant for this review, ranging in percentage quality scores from 42.85% to 71.43%. Outcome measures used in these studies to indicate levels of power and agility included short-distance sprints, vertical jumps, and agility runs, among others. Performance of both power and agility was shown to decrease when tactical load was added to the participants. This suggests that the increase in weight carried by tactical officers may put this population at risk of injury or fatality in the line of duty.
# Introduction
Tactical personnel are defined as professionals whose sworn duty is to protect their community or country; that duty that can place them at risk of injury [bib_ref] The impact of physical training programs on the fitness of tactical populations:..., Cocke [/bib_ref]. These men and women include, but are not limited to, military, fire and rescue, and law enforcement personnel [bib_ref] The impact of occupational load carriage on carrier mobility: A critical review..., Carlton [/bib_ref]. Due to the nature of their occupations, these personnel may be required to perform tasks that require them to react and move very quickly, often at a moment's notice and in life-threatening situations, such as when seeking cover when they come under enemy fire [bib_ref] Effects of Military Load Carriage on Susceptibility to Enemy Fire During Tactical..., Billing [/bib_ref] [bib_ref] Relationship between selected measures of power and strength and linear running speed..., Dawes [/bib_ref]. These personnel are also subjected to tasks that require a level of mobility; for example, the ability to negotiate obstacles like walls or fencesor perform 'fire and maneuver tasks' and 'break contact' drills [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref]. 'Fire and maneuver' tasks and 'break contact' drills, for example, require personnel to perform short explosive sprints and often start and end in a lying prone position. On this basis, a degree of power and agility would be needed for tactical personnel to get to their feet from a lying prone position, sprint forward a short distance and return to a lying prone position as quickly as possible. As such, success at accomplishing tactical tasks and survival in the field is dependent, in part, on the ability of men and women who serve in tactical populations to perform tasks requiring power and agility to a high standard, or risk injury, fatality, or mission failure. Tactical populations, by the nature of their occupations, are also required to wear and carry additional loads on a daily basis [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref]. Law enforcement officers are often required to wear stab-resistant body armour, as well as other accessories on their duty belts [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref]. This additional equipment can add as much as 8 kg [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] to 10 kg [bib_ref] Physiological responses of police officers during job simulations wearing chemical, biological, radiological..., Blacker [/bib_ref] of load to their person, with officers in specialist police units carrying as much as an additional 22 kg of load on their body [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref]. Firefighters are required to carry similar loads of around [bib_ref] Repeated-sprint ability-Part I: Factors contributing to fatigue, Girard [/bib_ref] [bib_ref] Repeated-sprint performance and vastus lateralis oxygenation: Effect of limited O(2) availability. Scand, Billaut [/bib_ref] [bib_ref] The effects of compression garments on recovery of muscle performance following high-intensity..., Duffield [/bib_ref] [bib_ref] Quantifying the effects of load carriage and fatigue under load on sacral..., Mcginnis [/bib_ref] [bib_ref] The feasibility of creating a checklist for the assessment of the methodological..., Downs [/bib_ref] kg when on duty [bib_ref] Wildland firefighter load carriage: Effects on transit time and physiological responses during..., Leadbetter [/bib_ref] , while military personnel typically carry much heavier loads that may be in excess of 45 kg as part of their occupation [bib_ref] The history of the soldier's load, Orr [/bib_ref]. The requirement to perform tasks while wearing this load may negatively impact the ability of the tactical personnel to perform their tasks effectively and safely [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref]. Holewijn and Lotens [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] found that, on average, physical performance decreased by 1% per 1 kg of additional load, while Dempsey et al. [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] established that police officers decrease in performance by 13-42% while wearing~10 kg of body armour.
It has been well established that repeated or sustained high intensity bouts of physical activity negatively affect the ability to maintain power, speed, and agility performance among athletic populations [bib_ref] Repeated-sprint ability-Part I: Factors contributing to fatigue, Girard [/bib_ref] [bib_ref] Repeated-sprint performance and vastus lateralis oxygenation: Effect of limited O(2) availability. Scand, Billaut [/bib_ref] [bib_ref] The effects of compression garments on recovery of muscle performance following high-intensity..., Duffield [/bib_ref]. This may not only reduce an athlete's opportunity for success in their respective sports, but also contributes to an increased risk of injury, as neuromuscular control tends to diminish with increasing levels of fatigue. Similarly, as the 'occupational athlete' is required to sustain prolonged activity, their ability to express force rapidly may also diminish. Furthermore, this loss of force generation ability may be exacerbated by the increased physiological burden associated with their need to carry the aforementioned loads [bib_ref] Quantifying the effects of load carriage and fatigue under load on sacral..., Mcginnis [/bib_ref]. For tactical personnel, any factors that reduce these physical capacities of power and agility may place personal safety and mission success at risk.
The literature suggests that load carriage can impact on tactical task performance, most notably, in this case, mobility [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref]. The literature also suggests that this mobility may be relied upon by tactical personnel for survival in the field [bib_ref] Effects of Military Load Carriage on Susceptibility to Enemy Fire During Tactical..., Billing [/bib_ref] , especially the ability to perform the high-intensity movements described above. Furthermore, repeated high-intensity efforts can create significant muscular fatigue, which may be accentuated as load carriage demands increase [bib_ref] Repeated-sprint performance and vastus lateralis oxygenation: Effect of limited O(2) availability. Scand, Billaut [/bib_ref]. Considering this, how load carriage might impact the discrete measures of power and agility, and as such mobility, would allow for the informing of means to mitigate the potential negative impacts associated with increased load and aid in the implementation of targeted risk mitigation strategies. On this basis, the aims of this review were to critically examine the literature investigating the impacts of load carriage on measures of power and agility in tactical occupations and to synthesize the findings.
# Methods
## Developing search strategy
A three-stage approach was used to identify and obtain studies that were potentially relevant to this critical review. The first stage consisted of a rapid literature review (conducted on 14 August 2017), which helped formulate the search strategy. Key search terms were identified and selected by extracting commonly used terms in the known research. Final research terms were then established by the researchers through joint collaboration. In the second stage, the aforementioned search terms were entered into the following databases: PUBMED, EMBASE, CINAHL and SPORTDiscus. These terms were modified as required to meet the individual search strategies within each database (see [fig_ref] Table 1: Databases and search terms used during literature search [/fig_ref]. Where available, the 'humans-only' filter was applied to rule out studies that did not include human participants. Where this option was not available as a filter, it was manually applied. (Load* OR Equipment OR "body armor" OR "body armour") AND (Power OR Sprint OR "vertical jump" OR jump OR Agility OR "Obstacle course" OR mobility) AND (Tactical OR Military OR "military personnel" OR police OR officer* OR firefighter* OR "law enforcement" OR soldier* OR army OR navy) * is part of the search terms and symbols. There is no actual meaning it just tells the search engine to look for any versions of that word.
## Inclusion and exclusion criteria
Once duplicates were removed, the articles were subjected to vigorous screening using carefully selected inclusion criteria. All articles were screened by title and abstract to meet the relevance of the aims of the review. Criteria for inclusion were as follows: (a) Study available in English or able to be translated into English; (b) study available in full text; (c) study used adult human participants only; (d) study involved participants carrying added load; and (e) study used a power and/or agility outcome measure.
For the purposes of this review, power was defined as the product of force on a subject and the subject's velocity in the direction in which the force was exerted. This differs from strength, as there is a speed component involved in power. Agility, on the other hand, can be defined as the skills and abilities needed to explosively change movement velocities or modes. In the case for both power and agility, speed is a major component. However, accurately measuring power and agility can be difficult, and there are often disputes on the best ways to measure each. Where there was uncertainty of whether an outcome measure used in a particular study did meet the definition for power or agility, the study was reviewed, and its potential inclusion was agreed upon by consensus. After the studies were subjected to the above inclusion criteria, the remaining studies were screened using criteria for exclusion listed [fig_ref] Table 2: Exclusion criteria and examples of excluded studies [/fig_ref]. In an effort to limit bias and accurately screen the studies derived from the literature search, two authors (A.J., A.W.) again reviewed and screened the studies separately using the criteria above. Disagreements regarding the inclusion or exclusion of any article were discussed and mediated by a third author (R.O.) before continuing the process. Through this approach, search bias, inclusion and exclusion bias, and duplication bias were limited. Finally, as part of the third stage of the search strategy, additional relevant studies, as well as grey literature, were sourced from references found in the studies retrieved from the database search and from known researchers in this field identified through the references or known to the reviewers through previous collaborations.
## Critical appraisal and data extraction
After subjecting the studies to all inclusion and exclusion criteria, the remaining studies were critically appraised using the Downs and Black checklist [bib_ref] The feasibility of creating a checklist for the assessment of the methodological..., Downs [/bib_ref]. The checklist has 27 items designed to assess the quality for randomized control trials and non-randomized studies and outline the strengths and weaknesses of these studies and has been used in previous reviews within tactical populations [bib_ref] The impact of physical training programs on the fitness of tactical populations:..., Cocke [/bib_ref]. The majority of the items are scored on a 'yes' or 'no' scale, awarding one point for a 'yes' answer and zero points for a 'no' answer. Item 5 on the checklist, however, is scored on a two-point scale, awarding two points for 'yes', one point for 'partially', or zero points for 'no' result. The final question in this checklist, which assessed statistical power of the study, is normally scored on a scale of 0-5 based on the study's sample size. This question was modified to give one point for a 'yes' answer when the authors of the study reported a power analysis or zero points for a 'no' answer when the authors did not provide a power analysis. This modified approach to the checklist has been previously used in the literature to limit subjectivity to the question [bib_ref] A profile of injuries sustained by law enforcement officers: A critical review, Lyons [/bib_ref]. Through this approach, the maximum possible raw score became a 28, as opposed to the original maximum score of 32.
The appraisal process described above was completed by two authors (Aaron Joseph, Amy Wiley) individually, so as to limit bias. Using the calculation of a Cohen's kappa coefficient (k), the level of interrater agreement of the raw scores was then determined by a third author (Robin Orr). This method followed previously published guidelines that are currently used in the literature [bib_ref] Understanding interobserver agreement: The kappa statistic, Viera [/bib_ref]. The Critical Appraisal Score (CAS) was then determined by the third author (Robin Orr) by settling any discrepancies in scores between the two raters. Following this, the scores given for each study were converted to percentages and subjected to the grading system proposed by Kennelly. Kennelly's system awards a rating based on the Downs and Black raw score given by the raters; however, the authors of this review modified the system to be presented as percentages to make it relevant to the modified Downs and Black checklist as follows: >61% as 'good' quality, 45-61% as 'fair' quality, and <45% as 'poor' quality.
Once the critical appraisal of the studies was completed, pertinent data were extracted from the included studies and tabled. Information extracted included all authors, title of study, year of publication, aim of the study, participant details, and main findings that were relevant to the aims of this review.
# Results
## Study selection and demographics
The PRISMA flow diagram details the refinement of research articles through the critical review process. It also provides a list of the databases and search results prior to screening and removal of duplicates. In total, 1042 studies were identified across four databases, with a further four articles acquired outside the database search through other sources. Studies that used the same data set as another study were treated as duplicates and were removed. There were 254 articles removed as duplicates, resulting in 792 studies to be reviewed against the inclusion criteria. Through implementation of the inclusion criteria, 728 articles were removed, leaving 64 studies to be reviewed against the exclusion criteria. Of those studies, 50 were removed following implementation of exclusion criteria. In total, 14 studies were deemed eligible for review and were subject to critical review. Of these studies, seven were conducted in the USA [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref] [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref] [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] , four were conducted in Australia [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , two in New Zealand [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] , and one in the Netherlands [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref]. Nine of the studies used only male participants [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , one study used only female participants, three studies used both male and female participants [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] , and one study did not specify gender of participants [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref]. Ten of the studies [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref] [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] subjected military personnel to the tests and four studies [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref] [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] tested police officers. Two of the studies [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] measured the impact of load on power, five studies [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref] [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] measured the impact of load on agility, and seven studies measured both power and agility [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] ].
## Critical appraisal of studies
The final CAS percentage scores, indicating the methodological quality of each study, are presented in [fig_ref] Table 3: Participants, loading conditions, outcome measures, and main findings [/fig_ref] , as well as information regarding the outcome measures that were used in the study and the study's findings. The Cohen's kappa analysis (k = 0.728) revealed an interrater agreement of 'substantial agreement' as per Viera and Garrett's interpretation [bib_ref] Understanding interobserver agreement: The kappa statistic, Viera [/bib_ref]. Four studies were graded as 'good' quality studies [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] , nine were graded as 'fair' quality [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] [bib_ref] Development and evaluation of a soccer focused complexity test, Schwesig [/bib_ref] , and one was graded as 'poor' quality. The mean CAS percentage for methodological quality of the included studies was 58.16%, ('fair' quality) with a high score of 71.43% ('good' quality) [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] and a low score of 42.85% ('poor' quality). Simulation was significantly slower in tiers-two, -three, -four than control, with tier-four also being sig. slower than tier-one (p < 0.05). There was no significant difference in time to complete the Obstacle Avoidance Test between the control and tier-one (p > 0.05), times for tiers-two, -three, and -four were sig slower than the control state (p < 0.05).
Time for the Combat-Rush Simulation was sig. slower for the tier-four state compared to the control and tier-one states (p > 0.05). There were no sig differences in Vertical Jump among tiers-one through to four (p > 0.05), tiers-two, -three, -four were sig. less than the control state (p < 0.05). Common weaknesses were observed in the included studies in certain areas of the Downs and Black checklist [bib_ref] The feasibility of creating a checklist for the assessment of the methodological..., Downs [/bib_ref]. Questions dealing with external validity were often given a score of '0' due to an overwhelming number of studies using only male participants, which is not representative of the whole population from which they were recruited. The facilities in which the measures were taken (for example, in fitness centers or training areas) were also not representative of the environment in which they would be performing these measures in their occupations (for example on the street, battlefield of fire ground). Questions dealing with internal validity were also often given a score of '0', since most of the included studies did not make an attempt to blind the participants or assessors. This was mainly due to the nature of the studies, as it would be difficult to blind in the study given that participants would be aware of when they were or were not wearing additional load on their bodies. [fig_ref] Table 3: Participants, loading conditions, outcome measures, and main findings [/fig_ref] outlines the data extracted from the included studies, with information on the participants, specific outcome measures used, and main findings of the study. The outcome measurements for power and agility varied across the included studies. When assessing power, some studies used a sprint as the outcome measure; either a 10 m [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] , 25 m [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] , or 30 m [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] ] sprint with load. Other studies measured power through a loaded vertical jump test [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref]. Agility was also measured through various techniques. Some studies used an agility run as the primary outcome measure, while others used an obstacle course or maneuverability tasks that incorporated various agility measures [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref]. Agility was also measured in the form of a sprint but from a prone starting position [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref]. All of the included studies used one of the measures listed above while carrying added load.
## 50.00%
## Study characteristics and findings
## Short distance sprints
Six of the included studies measured power in the form of short distance sprints. Of the studies that measured 30-m sprints [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , all of them showed a significant decrease in performance when additional load was added. It should be noted that in the study conducted by Pandorf et al., the 30 m sprint was one leg of an obstacle course the participants had to traverse, so a slower time to complete this sprint may be due in part to fatigue from the other sections of the course or participants were conserving energy to optimize overall time to completion. In the study completed by Martin et al. [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] , the 25 m sprints were conducted under five loaded conditions (1: 0.77 kg, 2: 9.41 kg, 3: 17.59 kg, 4: 29.93 kg, 5: 36.73 kg) for each participant. Each condition showed a significant decrease in performance when compared to the unloaded condition, and all loaded conditions showed a significant difference in performance from each other except for conditions 4 and 5 (29.93 kg and 36.73 kg, respectively). There was not a significant difference in the loaded (approximately 22 kg) and unloaded conditions in the 10 m sprint conducted by Carlton et al. [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] , but increases in time required to complete the sprint in the loaded condition were observed.
## Vertical jump
Three studies included in this review measured power via a vertical jump test [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref]. The studies conducted by Dempsey et al. [bib_ref] Body armour: The effect of load, exercise and distraction on landing forces, Dempsey [/bib_ref] and Holewijn and Lotens [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] both showed a significant decrease in the height of vertical jump when loads of between 7.65 kg and 16 kg was added to the participant; Dempsey et al. found a decrease of 13% when loaded with 7.65 kg while Holewijn and Lotens showed a 27% loss in their loaded condition with loads of 16 kg. Taylor et al. [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] found that there were no significant decreases between each of their four tiers of loaded conditions (1: 21.6 kg, 2: 25.0 kg, 3: 26.0 kg, 4: 29.2 kg) but they did find a significant decrease in each tier when compared to the control (19.1 kg) state. Maximal effort vertical jump was only collected for descriptive purposes in the study conducted by Lewinski et al. [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref] , however, a 17% decrease in performance was observed while participants were wearing the 9-kg weight belt.
## Maneuverability tasks
Numerous studies used certain maneuverability tasks to measure the performance loss in agility with added load. These tasks included traversing obstacle courses that incorporated various agility measures [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , fire and movement simulations [bib_ref] Tactical combat movements: Inter-individual variation in performance due to the effects of..., Hunt [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , and agility drills [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] [bib_ref] Changes in combat task performance under increasing loads in active duty marines, Jaworski [/bib_ref]. Obstacle course times across the studies showed significant decreases in completion times when load was added. However, in the study conducted by Taylor et al. [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , obstacle course times were only significantly slower in weight tiers two-four (2: 25.0 kg, 3: 26.0 kg, 4: 29.2 kg) when compared to the control state (19.1 kg). Holewijn and Lotens [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] divided their obstacle course into three segments , and while only obstacle course A showed a significant decrease on its own, overall the combination of the three courses showed a significant decrease in completion time when participants were wearing loads of 16 kg. Fire and movement simulations all showed significant decreases in time as well. Agility drills, such as the modified MANUF test and acceleration tasks simulating exiting a vehicle, often showed loss of agility performance; however one studydid not observe this result. DeMaio et al.used a box drill that incorporated sprinting forward, side shuffling, and running backwards four times around a 10 × 10 m box, but this box agility test was not significantly affected by personal protective equipment (PPE) (9.8 ± 0.9 kg).
## Prone-start sprint
Sprints from a prone starting position require a considerable amount of agility. This outcome measure was used to observe the performance of agility under load as opposed to power. Three studies used this measure [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] , and these studies unanimously observed significant effects of load on the agility of the participant. The distance of the sprints varied from 5 m [bib_ref] Balancing ballistic protection against physiological strain: Evidence from laboratory and field trials, Taylor [/bib_ref] to 30 m [bib_ref] Use of body armor protection with fighting load impacts soldier performance and..., Loverro [/bib_ref] [bib_ref] Effect of load carriage on performance of an explosive, anaerobic military task, Treloar [/bib_ref] , showing that agility is affected over a variety of distances, with loads ranging from 12.1 to 30.4 kg.
## Agility run
Two of the included studies measured agility through the use of an agility or shuttle run [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref]. Both studies observed that the time to complete increased when load was added to a significant standard. Martin [bib_ref] The effect of carried loads on the combative movement performance of men..., Martin [/bib_ref] states that with respect to load, significant differences were found between the performance for all loads for the men and for all loads (1: 0.77 kg, 2: 9.41 kg, 3: 17.59 kg, 4: 29.93 kg, 5: 36.73 kg) except load conditions 4 and 5 (29.29 and 36.09 kg respectively) for the women.
# Discussion
This critical review aimed to identify and critically appraise the methodological quality of studies investigating impacts of load carriage on measures of power and agility and to synthesize their findings. Four main areas of discussion were formed based off the results gathered: (1) the quality of the included studies; (2) the impact of added load on outcome measures of power; (3) the impact of added load on outcome measures of agility; and (4) implications of these findings to tactical population based on the volume of evidence reviewed and potential recommendations to mitigate these implications.
## Quality of research
The methodological quality of the included studies as a whole was deemed 'moderate' based on the grading system by Kennelly, with the mean CAS percentage at 58.16%. While this score is not considerably high, it should be noted that the mean score was largely influenced by the majority of the included studies being marked lower in certain areas of the Downs and Black checklist [bib_ref] The feasibility of creating a checklist for the assessment of the methodological..., Downs [/bib_ref] dealing with blinding the participants and assessors [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref] [bib_ref] A profile of injuries sustained by law enforcement officers: A critical review, Lyons [/bib_ref]. Due to the nature of these studies, it is very difficult to blind the participants, as the participants were either carrying additional load or not. As such, these questions typically scored a '0', causing a considerable reduction in the overall score. Similarly, Question 13 on the checklist, which relates to the environment in which the participants were tested being representative of the environment in which they normally work in, had the majority of studies score a '0' due to the difficulty of replicating these measures in an operational tactical environment. This disparity is highlighted when heightened senses and emotions of the officer under enemy fire are taken into account; a feat difficult to truly replicate when taking the safety of the officer into consideration.
Bearing these considerations in mind, it should be noted that the quality of these studies was acceptable overall. Furthermore, based on the volume of research available, the findings of added load carriage on measures of power and agility can be considered with confidence.
## The impact of load carriage on power
Power was shown to decrease across the included studies when additional tactical load was added to the participant. Both short-distance sprints and vertical jump tests were shown to have significantly lower results in the loaded conditions in the majority of the studies. Carlton et al. [bib_ref] The impact of occupational load carriage of the mobility of the tactical..., Carlton [/bib_ref] did not find a significant difference when measuring added load carriage over a 10 m sprint, but this may be largely due to the small sample size of their study (n = 6). This study did show an increase in time to complete the 10-m sprint when the 22.8 kg (±1.8 kg) load was added to the members of the specialist police unit, and it can be inferred that if more officers participated in this study, the results may have shown statistically significant decreases in performance. Overall, additional load was shown to have the most effect on short distance sprints in respect to completion time. This indicates that when under heavy load, the tactical officer will typically require a significantly greater amount of time to reach his or her destination safely.
The ability to generate power is necessary for explosive movements that the tactical officer performs in the field, such as sprinting to seek cover or jumping to negotiate a high fence. Increases in tactical load may come at the expense of the officer's ability to successfully perform these movements quickly, and could mean risking the officer's safety or the success of the mission. While this time period (i.e., seconds) may be considered very small it must be considered in context. For example, the cyclic rate of an AK 47 automatic assault rifle is around 600 rounds per minute and, on this basis, covering a distance one second slower could leave tactical personnel exposed to an additional 10 rounds while seeking cover from an armed offender or enemy combatant utilizing one of these weapons. This information should be taken into consideration when sending an officer into a situation in which explosive maneuvers may be required to survive.
## The impact of load carriage on agility
As was the case with power, agility was shown to suffer when the tactical officer was subjected to additional load. As a whole, the performance of all of the outcome measures used to observe agility decreased as load carriage increased. This was observed as many studies used tiers of weight in their experiments, and the heavier tiers typically showed increasingly significant differences from the lighter tiers. DeMaio et al.observed that their box agility drill was not significantly affected under load, however, this may be due in part to the relatively low weight of the PPE worn during the experiment (9.8 ± 0.9 kg). Incidentally, time to complete the drill did increase.
It should be taken into account, however, that while increases in times to complete obstacle courses were observed across the included studies, this may be due in part to the amount of space that the increased load occupied. For example, the increase in completion times for crawling underneath wires in the study by Holewijn and Lotens [bib_ref] The influence of backpack design on physical performance, Holewijn [/bib_ref] could have been due to the fact that there was reduced space for the officer to crawl through due to the large backpack they carried.
Apart from the agility to traverse or circumvent obstacles rapidly, which may give an opponent an advantage, it should be noted that reduction in agility may increase the tactical officer's risk of a slip, trip, or fall. Research by Park et al. [bib_ref] Effect of load carriage on gait due to firefighting air bottle configuration, Park [/bib_ref] identified that firefighter foot clearance when stepping over a 30 cm hurdle decreased and contacts with the hurdle increases with they were loaded (9.1 kg). This finding is of note given that slips, trips, and falls are a leading mechanism of injury in tactical personnel.
## Implications and recommendations
These results are especially pertinent for the tactical population. Load carriage represented by the weight of additional equipment carried by tactical personnel significantly decreased their power and agility, and therefore their mobility. Although the equipment and armour that tactical populations are required to carry may offer additional protection or necessary supplies to the person, the load may reach a point to where mobility is suffering [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref]. This decrease in mobility is also directly correlated with an increase in exposure to enemy fire in the field [bib_ref] Effects of Military Load Carriage on Susceptibility to Enemy Fire During Tactical..., Billing [/bib_ref]. This information suggests that these personnel are placed at a much higher risk for injury or fatality if the loads are such that they reach a point where the detrimental effects of load carriage on mobility are greater than the potential protection they provide.
Considering this, it is critical that training procedures and policies for tactical personnel include physical conditioning to specifically increase and optimize the carrier's ability to generate power and move with agility [bib_ref] Effects of Military Load Carriage on Susceptibility to Enemy Fire During Tactical..., Billing [/bib_ref] [bib_ref] The influence of officer equipment and protection on short sprinting performance, Lewinski [/bib_ref]. It is also recommended that, where possible, loads carried be reduced as much as possible prior to any tasks that require power and/or agility [bib_ref] Impact of police body armour and equipment on mobility, Dempsey [/bib_ref].
# Limitations
Key limitations of this review identified included a potential language bias and restriction to the majority of research to male participants. Given that only English databases were searched, in conjunction with English search terms, the potential for a language bias is present. Furthermore, while the quality of the studies was of good standard, many studies only used young male participants in their research. Considering that female personnel serve in tactical populations and perform the same operational tasks as male personnel, there was very little research into the effects of load carriage on female performance of power and agility. Given the relationship between fat mass and the ability to generate power [bib_ref] Relationship between selected measures of power and strength and linear running speed..., Dawes [/bib_ref] and that, in general, females have a higher fat mass than males, female personnel may be more adversely impacted by loads than male personnel. As such, more research is required to understand the impact load may have on the mobility of female personnel to identify whether any differences exist due to the sex of the load carrier. Finally, it should be noted that the majority of this research was conducted in military populations, with a limited number of studies in law enforcement and no studies in firefighter populations. Considering this, with all these tactical populations required to carry loads, it is anticipated that the impacts of load carriage on measures of power and agility identified in this study will transcend to all tactical personnel.
# Conclusions
In conclusion, this review observed that added load may have significant impacts on the ability of tactical personnel to perform activities that require power and agility. This may in turn reduce their mobility and increase their risk of injury and potentially mortality and operational success. On this basis, measures that optimize the ability of tactical personnel to generate power and agility while carrying load, such as physical conditioning and load reduction, is of importance. More research is required to take measures to reduce the weight carried by tactical personnel without compromising the safety or utility the load may offer.
Author Contributions: Aaron Joseph and Amy Wiley performed the systematic search of the literature, critical appraisal analysis, and data extraction, analyzed the data and synthesized the findings, and drafted the manuscript. Robin Orr conceived the critical review topic; conducted the Kappa analysis; moderated final scores and reviewed the manuscript. Benjamin Schram and J. Jay Dawes aided in the development of the search terms and strategies, aided in elements of drafting the manuscript, and reviewed and edited the final manuscript.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] 1, Figure 1: PRISMA diagram detailing the screening process of the literature review. [/fig]
[table] Table 1: Databases and search terms used during literature search. Mesh]) OR tactical OR military OR police OR firefight* OR "law enforcement") AND (load* OR "body armor" OR "body armour" OR equipment) AND (power OR agility OR mobility OR sprint OR jump OR obstacle) Tactical OR Military OR "military personnel" OR police OR officer* OR firefighter* OR "law enforcement" OR soldier* OR army OR navy) Tactical OR Military OR "military personnel" OR police OR officer* OR firefighter* OR "law enforcement" OR soldier* OR army OR navy) [/table]
[table] Table 2: Exclusion criteria and examples of excluded studies. [/table]
[table] Table 3: Participants, loading conditions, outcome measures, and main findings. times between UL and TL conditions, mean increases in time were noted There was a significant increase in mean time to complete the 10-m dummy drag during the TL condition compared to the UL condition (p = 0.009). Mean age: 24.2 ± 3.7-yrs Mean height: 180.1 ± 5.4-cm Mean weight: 82.9 ± 11.0 kg Females: Mean age: 23.0 ± 0-yr Mean height: 161.3 ± 14.4-cm Mean weight: 56.1 ± 6.7-kg Box Drill 4 × 10-yd (9.1-m): sprint forward, side shuffle, run backwards, carioca Upper Extremity Power: rope pull and dummy drag Shuttle Run was significantly affected by added PPE (p < 0.001) Box Agility Test was not significantly affected by added PPE (p = 0.28) Rope Pull and Dummy Drag was not significantly affected by added PPE (p = 0.42) Mean Age: 37 ± 9.16-yrs Mean Height: 180.68 ± 6.12-cm Mean Weight: 90.21 ± 11.59-kg Mean BMI: 27.61 ± 3.09 +0.2-s, p < 0.001). Maneuverability Task was significantly effected in loaded condition (+2.1-s, p < 0.001). Mean Age: 37 ± 9.16-yrs Mean Height: 180.68 ± 6.12-cm Mean Weight: 90.21 ± 11.59-kg Mean BMI: 27.61 ± 3.09 [/table]
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Origin of Pressure-induced Superconducting Phase in KxFe2−ySe2 studied by Synchrotron X-ray Diffraction and Spectroscopy
Pressure dependence of the electronic and crystal structures of K x Fe 2−y Se 2 , which has pressure-induced two superconducting domes of SC I and SC II, was investigated by x-ray emission spectroscopy and diffraction. X-ray diffraction data show that compressibility along the c-axis changes around 12 GPa, where a new superconducting phase of SC II appears. This suggests a possible tetragonal to collapsed tetragonal phase transition. X-ray emission spectroscopy data also shows the change in the electronic structure around 12 GPa. These results can be explained by the scenario that the two SC domes under pressure originate from the change of Fermi surface topology. Our results here show the pronounced increase of the density of states near the Fermi surface under pressure with a structural phase transition, which can help address our fundamental understanding for the appearance of the SC II phase.
Recently, single phase non-superconducting K 2 Fe 4 Se 5 was synthesized, and the pressure-temperature phase diagram was revealed [bib_ref] Role of the 245 phase in alkaline iron selenide superconductors revealed by..., Gao [/bib_ref]. By comparing the K 2 Fe 4 Se 5 and K x Fe 2−y Se 2 phase diagrams, the phase separation in the SC II region was suggested, and the superconducting phase attributed to the 122 phase. Therefore, this means that superconducting phase with KFe 2 Se 2 and non-superconducting phase with K 2 Fe 4 Se 5 co-exist in the SC II phase.
A theoretical study of the SC I and SC II phases in the 122* system suggested that superconducting symmetry is d-wave without Γ -point hole pocket at SC I and s ± -pairing at SC II. In these systems, however, since no experimental study of not only the electronic structure, but also the crystal structure under pressure has been reported so far, the issue of the appearance of SC II dome under pressure remains unclear.
In this paper we report a systematic study of K x Fe 2−y Se 2 with x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) under pressure. The purpose of this work is to reveal both the crystal and electronic structures of K x Fe 2−y As 2 under pressure to clarify the origin of the two superconducting domes. XES technique has made it possible to probe local magnetic moment under pressure by detecting Fe Kβ emission spectra for iron-based superconductor [bib_ref] The x-ray non-diagram lines Kβ′ of some compounds of the iron group, Tsutsumi [/bib_ref] [bib_ref] Probing the 3d spin momentum with x-ray emission spectroscopy: the case of..., Vankó [/bib_ref] [bib_ref] Pressure dependence of the electronic structure and spin state in Fe 1.01..., Chen [/bib_ref] [bib_ref] Revealing the dual nature of magnetism in iron pnictides and iron chalcogenides..., Gretarsson [/bib_ref] [bib_ref] Spin-state transition in the Fe pnictides, Gretarsson [/bib_ref]. We also performed the bulk sensitive x-ray absorption (XAS) measurements with partial fluorescence (PFY) mode at the Fe K absorption edge [bib_ref] Elimination of the inner-shell lifetime broadening in x-ray-absorption spectroscopy, Hämäläinen [/bib_ref]. We have used the PFY-XAS method where a decay process with shorter life time is selected, resulting spectra are narrower, and making fine electronic structure near the absorption edge better visible [bib_ref] Elimination of the inner-shell lifetime broadening in x-ray-absorption spectroscopy, Hämäläinen [/bib_ref] [bib_ref] New spectroscopy solves an old puzzle: the kondo scale in heavy fermions, Dallera [/bib_ref] [bib_ref] Role of valence fluctuations in the superconductivity of Ce122 compounds, Yamaoka [/bib_ref]. Our results show the change in the c-axis compressibility around boundary pressure of the SC I and SC II phases, suggesting a crystal structure change at this pressure, probably a tetragonal (T) to collapsed tetragonal (cT) transition. The Fe Kβ XES also shows a pressure-induced change in the electronic structure at the transition pressure.
# Results
## P-t phase diagram.
We prepared two kinds of K x Fe 2−y Se 2 single crystals: a sample quenched at 550 °C (quenched sample) and one cooled slowly (slow-cooled sample). A P-T phase diagram of the quenched and slow-cooled samples is shown in [fig_ref] Figure 1: A P-T phase diagram of K x Fe 2−y Se 230 [/fig_ref]. T c was determined from the onset temperature of the electrical resistivity measurements. Both samples show that T c decreases with pressure monotonically in the SC I phase. This behavior agrees well with the reports published [bib_ref] Re-emerging superconductivity at 48 kelvin in iron chalcogenides, Sun [/bib_ref] [bib_ref] Role of the 245 phase in alkaline iron selenide superconductors revealed by..., Gao [/bib_ref]. However, the maximum T c of SC II phase depends on the samples. T c of the quenched and slow-cooled samples are ~5 K and ~20 K at the SC II phase, respectively 30 , while T c of SC II was ~50 K in the reports published [bib_ref] Re-emerging superconductivity at 48 kelvin in iron chalcogenides, Sun [/bib_ref] [bib_ref] Role of the 245 phase in alkaline iron selenide superconductors revealed by..., Gao [/bib_ref]. These results suggest that the T c of SC II depends strongly on the sample preparation. Actually, island-and mesh-shape morphology were observed in the back-scattered electron (BSE) image in the slow-cooled and the quenched samples, respectively [bib_ref] Origin of the Higher-T c Phase in the K x Fe 2−y..., Tanaka [/bib_ref]. These morphologies were caused by the difference of iron concentration 31 .
## X-ray diffraction.
We measured x-ray diffraction patterns under pressure up to 19.1 GPa for the quenched sample and 18.0 GPa for the slow-cooled sample at room temperature as shown in [fig_ref] Figure 2: XRD pattern of [/fig_ref]. Both samples consist of a I4/m symmetry of the 245 phase and a I4/mmm symmetry of the 122 phase at ambient pressure. Fe vacancy order-disorder transition was reported in the non-superconducting 245 phase at SC II, and crystal symmetry after the transition becomes I4/mmm which is the same as the superconducting phase [bib_ref] Pressure-driven quantum criticality in iron-selenide superconductors, Guo [/bib_ref] [bib_ref] Interplay of electronic and lattice degrees of freedom in A 1−x Fe..., Bendele [/bib_ref]. [fig_ref] Figure 2: XRD pattern of [/fig_ref] (a,c) show the XRD patterns of the quenched and slow-cooled samples, and the enlarge views are shown in [fig_ref] Figure 2: XRD pattern of [/fig_ref]. Intensity of the superstructure peak (110) attributed to the Fe vacancy order disappears around 12 GPa, indicating a clear structural phase transition from I4/m to I4/mmm symmetry at 245 phase. The same feature has been observed previously [bib_ref] Pressure-driven quantum criticality in iron-selenide superconductors, Guo [/bib_ref] [bib_ref] Interplay of electronic and lattice degrees of freedom in A 1−x Fe..., Bendele [/bib_ref]. Seemingly, the above structural transition pressure of 12 GPa coincides with the appearance of the SC II phase as seen in [fig_ref] Figure 1: A P-T phase diagram of K x Fe 2−y Se 230 [/fig_ref].
Although a Rietveld refinement was not performed because of the restriction of the observed Q range, we performed peak fits by using the several peaks with the Voigt functions in order to derive the lattice constants. [fig_ref] Figure 2: XRD pattern of [/fig_ref] indicates (002) and (110) peak position vs pressure. Trend of the pressure evolution of (002) peak position changes around 12 GPa. This system consists of the 122 and 245 phases and thus only the average lattice . Red circles and blue squares indicate quenched sample and slow-cooled sample, respectively. Colouring region is based on the data taken from the ref. [bib_ref] Re-emerging superconductivity at 48 kelvin in iron chalcogenides, Sun [/bib_ref].
constant of two phases could be analyzed. Since the 245 phase under pressure shows no superconductivity up to 22 GPa even though the cross over from the insulating phase to the metalic phase is found around 220 K at 0.4 GPa 20 , it is expected that only the 122 phase shows superconductivity in the whole SC region observed at ambient and high pressure. Therefore, it is likely that the volume fractions of the 122 phase should remain at 10-13% and 30-35% in the slow-cooled and quenched samples in the SC II region, respectively, (See Method section) Here, we assumed I4/m symmetry at all pressures because I4/mmm symmetry can express I4/m symmetry. [fig_ref] Figure 2: XRD pattern of [/fig_ref] (g,i) show pressure evolution of the lattice constants. Pressure evolution of the a-axis shows a monotonic decrease, while that of the c-axis changes the slope around 12 GPa. Thus the compressibility along the c-axis becomes lower above 12 GPa. This means that the bond along the c-axis at the SC II phase is stronger than that at the SC I phase. This suggests a crystal structure change at 12 GPa, probably T → cT structural phase transition analogous to EuFe 2 As 2. Pressure induced change in the Kβ emission spectra. [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref] show pressure evolution of Kβ emission spectra of the quenched and slow-cooled samples, respectively. A Kβ spectrum consists of a main peak of Kβ 1,3 and a satellite peak of Kβ′ , which correspond to low-spin and high-spin states, respectively [bib_ref] The x-ray non-diagram lines Kβ′ of some compounds of the iron group, Tsutsumi [/bib_ref]. In [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref] , pressure evolution of Kβ spectrum shows a shift from the high-spin to the low-spin state with pressure. [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref] shows a comparison among the Kβ spectra of the quenched sample, the slow-cooled sample, FeCrAs (0 μ B ), and FeSe (2 μ B ). As seen in [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref] , comparison of Kβ spectra between K x Fe 2−y Se 2 and FeCrAs concludes that K x Fe 2−y Se 2 is in the higher-spin state because of larger Kβ′ intensity. The local moment of Fe can be extracted by the the integrated absolute difference (IAD) analysis of the Fe Kβ emission spectra to a reference spectrum [bib_ref] Probing the 3d spin momentum with x-ray emission spectroscopy: the case of..., Vankó [/bib_ref] [bib_ref] Revealing the dual nature of magnetism in iron pnictides and iron chalcogenides..., Gretarsson [/bib_ref]. It is known that the IAD values are proportional to the local magnetic moments [bib_ref] Revealing the dual nature of magnetism in iron pnictides and iron chalcogenides..., Gretarsson [/bib_ref]. [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref] shows the local magnetic moment estimated by the IAD analysis of the Kβ spectra in [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref]. The local magnetic moment decreases from ~3 μ B at ambient pressure to ~1 μ B at the SC II phase with pressure. Two samples show roughly the same trend under pressure. Especially the pressure evolution of the local magnetic moment of slow-cooled sample changes the slope at 12 GPa. This coincides with the change in the compressibility along the c-axis shown in [fig_ref] Figure 2: XRD pattern of [/fig_ref]. [fig_ref] Figure 4: Pressure evolution of the PFY-XAS spectra of [/fig_ref] show a pressure evolution of the PFY-XAS spectra setting the emitted photon energy to Kβ 1,3 peak of the quenched and slow-cooled samples, respectively. The intensity is normalized to that at 7160 eV. The PFY-XAS spectra show large pre-edge peaks. The pre-edge and the main edge peaks correspond to 1s → 3d quadrupole and 1s → 4p dipole transitions, respectively. The strong pre-edge peak intensity includes the information of the hybridization between the Fe 3d and Se 4p orbitals [bib_ref] The 1s x-ray absorption pre-edge structures in transition metal oxides, De Groot [/bib_ref]. The edge position of the PFY-XAS spectra shifts toward lower energy with pressure in both samples, indicating the decrease of the Fe valence. The system includes Fe 3+ and Fe 2+ 35 and thus the above result indicates a change in the Fe valence from Fe 3+ → Fe 2+ . The decrease of the Fe valence with pressure may be due to the electron supply from K to FeSe layer caused by the shrink along the c-axis. [fig_ref] Figure 4: Pressure evolution of the PFY-XAS spectra of [/fig_ref] shows that the pre-edge peak intensity of the PFY-XAS spectra increases with pressure. Another point we would like to emphasize is that the intensity around 7125 eV changes at 12 GPa in the slow-cooled sample, although it is not clear in the quenched one [fig_ref] Figure 4: Pressure evolution of the PFY-XAS spectra of [/fig_ref]. This pressure also coincides with the pressure where the compressibility of the c-axis changes.
## Pressure induced change in the pfy-xas spectra.
# Discussion
The XRD and XES studies under pressure have been performed for the 122* system, which have pressure-induced two superconducting domes. The XRD results show that the compressibility along the c-axis changes at 12 GPa and the superlattice diffraction disappears at the same pressure. Pressure dependence of the lattice constant along the c-axis and the volume becomes gentle at the SC II phase. The same c-axis evolution has been observed in AFe 2 As 2 where A = Ca, Sr, Ba and Eu. This was interpreted as the T → cT structural phase transition. As shown in [fig_ref] Figure 2: XRD pattern of [/fig_ref] , the FWHM of the (002) peak in the XRD pattern starts to increase rather abruptly at around 12 GPa. This can be explained if we make the assumption that the (002) peak consists of two components. One peak [fig_ref] Figure 2: XRD pattern of [/fig_ref] appears to originate from the 245 phase, which remains structurally stable up to 22 GPa, by considering previous high-pressure resistivity data 20 , and the other from the 122 phase, which undergoes a cT phase transition. We note that the quenched sample shows a more sudden change than that of the slow-cooled sample. This may be related to the SC volume fraction difference between them. Since the 122 phase in K x Fe 2−y Se 2 has the same crystal structure as that of AFe 2 As 2 (A = Ca, Sr, Ba, Eu), it is reasonable to expect that the 122 phase in K x Fe 2−y Se 2 shows the same phase transition as AFe 2 As 2 under pressure. This coexistence of two phases is likely to lead to a smearing out of the T to cT phase transition, as compared, for example, to the clear discontinuity observed in EuFe 2 As 2. The change in the crystal structure affects the magnetic property. Actually, the Kβ XES results indicate that the trend of the pressure evolution of the magnetic moment and the electronic state shown in [fig_ref] Figure 3: Pressure dependence of Kβ emission spectra of the [/fig_ref] changes also at 12 GPa, which seems to correlate to the T → cT transition. The average local magnetic moment changes from ~3 μ B at ambient pressure to ~1 μ B at the SC II phase with pressure. The change in the magnetic moment at 12 GPa is not large in K x Fe 2−y Se 2 system, probably because the collapse along the c-axis at 12 GPa is small.
The PFY-XAS spectra show that the Fe valence decreases with pressure, which may correspond to the increase of the carrier density at the SC II phase due to the supply of the electrons from K to the FeSe layer caused by the shrink along the c-axis. The pre-edge peak intensity in the PFY-XAS spectra increases with pressure, indicating the increase of the hybridization between Fe 3d and Se 4p and also the density of states (DOS) near the Fermi surface. The pressure-induced change in the pre-edge peak intensity also correlates to the shift from high-spin to low-spin states (See also .
In the 122 system the superconductivity emerged suddenly at the cT phase when the T → cT structural phase transition occurred. The phase diagram of the 122 system is similar to that of the 122* system 37 . The DFT calculations showed the change in the electronic structure between the T phase and cT phase [bib_ref] Origin of the superconducting state in the collapsed tetragonal phase of KFe..., Guterding [/bib_ref]. In KFe 2 As 2 , the T → cT transition changed the superconductivity symmetry from d-wave to s-wave. This is a Lifshitz transition which is known to change the Fermi surface drastically from the electronic state with only hole pocket to that with electron and hole pockets. Other calculations of K x Fe 2−y Se 2 also showed the d-wave in the SC I and s-wave in the SC II phase 21 . Therefore, together with these theoretical calculations we conclude that K x Fe 2−y Se 2 shows the T → cT transition and the increase of the density of states near the Fermi surface under pressure, which are the key evidence for understanding the appearance of the SC II phase.
# Methods
Sample preparation and characterizations. We prepared two kinds of K x Fe 2−y Se 2 single crystals [bib_ref] Origin of the Higher-T c Phase in the K x Fe 2−y..., Tanaka [/bib_ref] [bib_ref] Evolution of superconductivity in isovalent Te-substituted KFe 2−y Se 2 crystals, Ozaki [/bib_ref].
Single crystals were grown by a simple one-step synthesis. Fe (99.9%), K 2 Se (99%) powders and Se (99.999%) grains were put into an alumina crucible and sealed into an evacuated quartz tube. The mixture was slowly heated up to 900 °C and held for 3 hours. The melting mixture was, then, cooled down to room temperature slowly (slow-cooled sample) and quenched at 550 °C (quenched sample). Back-scattered electrons (BSE) images were obtained to observe micro-structure. Island-and mesh-shape structure were shown in the slow-cooled and quenched samples, and the chemical composition determined by using energy dispersive x-ray spectrometry (EDX) were K 0.40 Fe 1.95 Se 2 and K 0.63 Fe 1.71 Se 2 , respectively [bib_ref] Origin of the Higher-T c Phase in the K x Fe 2−y..., Tanaka [/bib_ref]. The area ratios between the superconducting region and non-superconducting region is ~10-13% in the slow-cooled sample and ~30-35% in the quenched sample. T c of the present samples under pressure were measured at Osaka University 30 .
## Xrd, xes, and pfy-xas measurements under pressure. we performed xrd, xes, and pfy-xas
experiments for the slow-cooled and quenched samples. For XRD, XES, and PFY-XAS measurement, these samples with NaCl as the pressure medium were loaded into a sample chamber of the gasket in the glove box of pure Ar atmosphere because these samples are chemically unstable in the air. Pressure was monitored by ruby fluorescence method [bib_ref] High-pressure physics: The 1-megabar mark on the ruby R 1 static pressure..., Mao [/bib_ref].
Pressure dependence of the XRD patterns were measured at SPring-8 BL12B2 using a 3-pin plate diamond anvil cell (DAC, Almax Industries) with a CCD detection system at room temperature. We took an arrangement of both incoming and outgoing x-ray beams passed through the diamonds with incident photon energy of 20 keV. NaCl was loaded as the pressure medium and well-mixed with the sample because of reduction of preferred orientation of the sample. [fig_ref] Figure 1: A P-T phase diagram of K x Fe 2−y Se 230 [/fig_ref] 2D image of CCD was integrated by using FIT2D program [bib_ref] Two-dimensional detector software: From real detector to idealised image or two-theta scan, Hammersley [/bib_ref].
The PFY-XAS and XES measurements were performed at the Taiwan beam line BL12XU at SPring-8. The undulator beam was monochromatized by a cryogenically-cooled double crystal Si(111) monochromator. A Johann-type spectrometer equipped with a spherically bent Si(531) analyzer crystal (radius of ~1 m) and a Si solid state detector (Amptech) were used to analyze the Fe emission of the 3p → 1s de-excitation at the Fe K absorption edge. At the emitted photon energy of 7.6 keV the overall energy resolution was estimated to be 0.9 eV. The intensities of the measured spectra were normalized using the incident beam that was monitored just before the sample.
For the high-pressure XES experiments the x-ray beam was focused to 20-30 (horizontal) × 30-40 (vertical) μm 2 at the sample position using a toroidal and a Kirkpatrick-Baez mirror. High-pressure conditions were achieved at room temperature using a diamond anvil cell coupled with a gas-membrane. A Be-gasket with 3 mm in diameter and approximately 100 μm thick was pre-indented to approximately 35-40 μm thickness around the center. The diameter of the sample chamber in the gasket was approximately 100 μm and the diamond anvil culet size was 300 μm. We used the Be gasket in-plane geometry with a scattering angle of 90°, where both incoming and outgoing x-ray beams passed through the Be gasket. Be was used due to its higher transmittance to x-rays in comparison to other high-Z materials.
## Iad analyses.
The IAD analysis is performed in the following way: (i) match the center of mass between the sample and reference spectra, (ii) take the difference between them, and (iii) integrate the absolute value of the difference. The intensity is normalized by the area of the Kβ spectrum.
[fig] Figure 1: A P-T phase diagram of K x Fe 2−y Se 230 [/fig]
[fig] Figure 2: XRD pattern of (a) the quenched sample and (c) the slow-cooled sample. (b,d) Enlarged views of (a,c), respectively. Asterisk mark means reflection of NaCl used as the pressure medium of the diamond anvil cell. In the both quenched and slow-cooled samples, the (110) superstructure reflection disappear around 12 GPa. (e-i) Pressure evolution of the peak properties and the structure parameters of the quenched (red circle) and slow-cooled (blue square) samples. (e) Peak position of (002) and (110). (f) Full width at half maximum of the (002) peak. (g) Lattice constant along the a-axis. (h) Lattice constant along the c-axis. (i) Volume. Linear dashed-lines are guides for the eye. Scientific RepoRts | 6:30946 | DOI: 10.1038/srep30946 [/fig]
[fig] Figure 3: Pressure dependence of Kβ emission spectra of the (a) quenched and (b) slow-cooled samples. (c) Kβ spectra of FeCrAs, FeSe, the quenched sample, and the slow-cooled sample. (d) Pressure dependence of amplitude of magnetic moment per Fe estimated with the IAD values of the Kβ spectra. A linear dashed-line is a guide for the eye. Scientific RepoRts | 6:30946 | DOI: 10.1038/srep30946( [/fig]
[fig] Figure 4: Pressure evolution of the PFY-XAS spectra of (a) the quenched sample and (b) the slow-cooled sample. In both the quenched and slow-cooled samples, the pre-edge peak intensity increase with pressure and edge position move towered to low energy. Pressure evolution of (c) the pre-edge peak intensity and (d) the edge position. Red circle and blue square indicate the quenched and slow-cooled samples, respectively. [/fig]
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Inflammatory bowel disease: Looking beyond the tract
Inflammatory bowel disease is a chronic inflammatory condition that encompasses Crohn's disease and ulcerative colitis. Inflammatory bowel disease is not exclusive to the gastrointestinal system, as it has been identified to be associated with extraintestinal manifestations that encompass every other organ system in the human body. This review article will comprehensively review the current knowledge on extraintestinal manifestations of inflammatory bowel disease. In addition, it will discuss the recommendations for screening and surveillance for extraintestinal manifestations in these patients since early appropriate diagnosis is imperative in preventing morbidity and cancer development.
# Introduction
Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that includes two clinical entities, Crohn's disease (CD) and ulcerative colitis (UC).Recent research by the Centers for Disease Control and Prevention (CDC) using the 2015 National Health Interview Survey (NHIS) found that IBD is actually more prevalent than previously reported. Their analysis concluded that IBD affects 3.1 million or 1.3% of US adults, which is approximately three times more than previous estimates. Prevalence was found to be higher among adults aged 45 years or older, Hispanics, non-Hispanic Whites, and adults with less than a high school education, currently unemployed, living in poverty, and born in the United States. Contrary to earlier studies, IBD prevalence was not found to be associated with types of health insurance coverage, geographic region, or sex.Multiple factors have been found to contribute to the pathogenesis of the disease including environmental, genetic, microbial, and immune.IBD is frequently associated with the development of extraintestinal manifestations (EIMs). EIMs of IBD are so common that the disease should be regarded as a systemic disorder that is not limited to the gastrointestinal tract.EIMs may involve almost any organ system making them major contributors to the morbidity of patients with IBD. 3
## Eims
EIMs are estimated to affect approximately 6% to 47% of adult patients with IBD and around 25% to 29% of pediatric patients with IBD.They have been reported to occur more frequently in patients with CD compared with those with UC.A patient may be affected by several EIMs at the same time, given that the presence of one EIM increases the likelihood of developing another.A Swiss IBD cohort study showed that 25% of patients with IBD affected by EIMs actually suffered from multiple, in some cases up to five different EIMs. EIMs may occur prior to the diagnosis of IBD, which happens in 25.8% of cases.Patients with IBD with smoking history, perianal CD, and colonic involvement are at an increased risk of developing EIMs.The pathogenesis of EIMs of IBD is unclear; however, it is thought to include immune and genetic factors. It is believed that immune responses may be triggered at extraintestinal sites by diseased gastrointestinal mucosa due to shared epitopes at the different sites.Bacteria translocated across a leaky intestinal barrier triggers an adaptive immune response that eventually will not be able to discriminate between bacterial epitopes and epitopes of the joints and skin.In terms of genetic susceptibility, links have been demonstrated between EIMs and certain major histocompatibility complex loci.For example, EIMs in CD are found more frequently in patients with HLA-A2, HLA-DR1, and HLA-DQw5. EIMs in UC are associated with HLA-DR103. A higher risk of primary sclerosing cholangitis (PSC) in UC specifically has been associated with HLA-B8/DR3.Also, HLA-DRB10103, HLA-B27, and HLA-B58 have been found to be associated with EIMs in the joints, skin, and eyes, respectively. 7,10 Studies have found that multiple organ systems are affected by EIMs including the joints, skin, eyes, and liver.In addition to EIMs, patients with IBD may also experience extraintestinal complications. This can be further divided into those caused by the disease itself, for example, consequences of loss of function of diseased or resected bowel including malabsorption, and those caused by treatments.
## Musculoskeletal manifestations
The most common EIMs of IBD are musculoskeletal, which affects up to 40% of the patients.The use of corticosteroids, immunosuppressants, and anti-tumor necrosis factor (TNF) therapy in patients with IBD have been found to cause therapy-induced arthralgias in a number of patients requiring withdrawal of the offending agent when possible.Musculoskeletal manifestations are divided into peripheral arthropathies and axial arthropathies.
## Peripheral arthropathies
Peripheral arthropathy in patients with IBD is typically a seronegative arthropathy, which affects 5%-10% of patients with UC and 10%-20% of patients with CD. Patients who are at increased risk for peripheral arthropathies include those with colonic involvement, perianal disease, erythema nodosum, stomatitis, uveitis, and pyoderma gangrenosum (PG).Peripheral arthropathies in patients with IBD are categorized into two types with the diagnosis being made clinically since imaging of the joints is usually normal.Type I arthropathy is pauciarticular or oligoarticular, usually involving less than five large joints, acute, asymmetrical, and migratory. It is usually related to the pattern of intestinal activity of the IBD. It is self-limiting, typically lasting no longer than 10 weeks with no persistent joint damage. It has been found to be associated with HLA-B27, HLA-B35, and HLA-DR103.On the contrary, type II arthropathy is polyarticular, usually involving five or more small joints that are symmetrical. It most commonly involves the metacarpophalangeal joint. It is independent of the disease course and can persist for years. It is associated with an increased risk of uveitis and has been found to be associated with HLA-B44. 7
## Axial arthropathies
Axial arthropathies in patients with IBD include ankylosing spondylitis (AS) and isolated sacroiliitis, which affect 5%-22% of patients with CD and 2%-6% of patients with UC.Similar to type II peripheral arthropathies, axial arthropathies do not follow the intestinal course of the underlying IBD. Back pain and morning stiffness are the most common presenting axial joint symptoms and often present prior to intestinal symptoms of IBD. A strong association has been suggested between AS and IBD. AS is found to occur in up to 10% of patients with IBD, and 6.5% of patients with AS go on to develop IBD. It has been suggested that part of this association is due to a shared genetic relationship.First-degree relatives of patients with AS are three times more likely to develop IBD. HLA-B27 has been identified as the main culprit since individuals who are HLA-B27 positive have an increased risk of developing AS. Also, 25%-78% of patients who have both IBD and AS are HLA-B27 positive.Because of this strong association, physicians should remain alert to evidence of AS. Presenting symptoms include but are not limited to severe onset of back pain at a young age and morning stiffness or pain associated with long periods of rest. Physical exam findings include limitations in spine flexion and reduced chest expansion.Radiographic findings range from normal to minimal sclerosis. In advanced AS, squaring of vertebral bodies, marginal syndesmophytes, bone proliferation, and ankylosis may be evident.
Contrary to AS, sacroiliitis does not share this association with HLA-B27 with most patients being HLA-B27 negative. It is discovered radiographically in up to 25% of patients.Most patients do not progress to AS but are more likely to if there is radiographic evidence of bilateral sacroiliitis.The diagnosis of axial arthropathies is made clinically and supported by imagining.Magnetic resonance imaging (MRI) is the current gold standard since it can detect acute and chronic changes before radiographs show any abnormalities, therefore, resulting in earlier disease detection.
## Treatment
In type I arthropathy, the treatment of choice is therapy of the underlying bowel disease since it is related to the intestinal activity. 7 Nonsteroidal anti-inflammatory drugs (NSAIDs) can be used but with caution due to reports of IBD exacerbation following their use.Selective cyclo-oxygenase-2 (COX-2) inhibitors may be a safer alternative in patients with IBD with peripheral arthropathies.Sulfasalazine is usually used if NSAIDs or COX-2 inhibitors fail to provide adequate symptom relief. Sulfasalazine has been found to have superior results in peripheral arthropathies compared with axial. If these drugs are not successful at relieving symptoms, patients with CD can undergo a trial of tumor necrosis factor alpha (TNF-α) inhibitor at the same doses used for rheumatoid arthritis, whereas patients with UC may be treated with a TNF-α inhibitor or a short course of intra-articular or oral steroids.Patients with axial arthropathies should always be referred for physiotherapy. Like peripheral arthropathies, NSAIDs and COX-2 inhibitors can be used for symptom relief. If the patient does not improve or is intolerant to these therapies, early use of anti-TNF therapy is recommended.Infliximab and adalimumab have been found to be successful in improving both intestinal symptoms and axial arthropathies in several studies in patients with IBD.In severe cases of axial arthropathy, specifically in AS, the disease-modifying anti-rheumatic drug (DMARD) methotrexate proves to be effective in maintaining remission in IBD. However, it is not recommended as first line therapy due to the lack of evidence regarding its effect on AS. 7
## Screening recommendations
Patients with IBD more commonly develop reduced bone mineral density (BMD) and bony fractures.Studies have estimated the incidence of osteoporosis in IBD to range from 10% to 15%.The cause of low BMD in IBD is thought to be multifactorial.In addition to risk factors affecting the general population, risk factors specific to IBD include corticosteroid use, low physical activity, vitamin D deficiency, poor calcium intake likely related to lactose intolerance, calcium and magnesium dietary malabsorption, and inflammatorymediated bone resorption.The American Gastroenterology Association (AGA) guidelines identified age and glucocorticoid use as the strongest risk factors highlighting the fact that all efforts should be made to limit prolonged exposure to corticosteroids.This notion is supported by two European studies that found that the risk of IBD leading to major osteoporotic fractures decreased when there was adequate control of glucocorticoid use.Because the absolute risk of bone fractures is low, a more conservative, cost-effective approach to screening IBD patients for osteoporosis follows guidelines established for the general population. This means screening all patients with a preexisting fragility fractures, women aged 65 and older, men aged 70 and older, and those with risk factors that increase the likelihood of low bone mass using dual-energy X-ray absorptiometry (DEXA) scanning for BMD measurement.Regarding corticosteroid use, all patients who have used oral corticosteroids for more than 3 consecutive months at a dose ⩾7.5 mg/day should have a DEXA scan.For patients who have a T-score above −1 on DEXA, recommendations are preventive including smoking cessation, calcium and vitamin D supplementation, exercise, limiting alcohol intake, and minimizing the use of corticosteroids. Osteopenia is reflected by a T-score of −1 to −2.5 on DEXA and it is recommended that these patients implement the same preventive measures and consider a repeat DEXA in 2 years. Bisphosphonates are recommended for osteoporotic patients (T-score less than −2.5) and patients with a history of atraumatic fracture or failure to withdraw from corticosteroids therapy after 3 months.Further workup should be completed for patients who are found to be osteoporotic or who sustain a low-trauma fracture to screen for secondary causes of low BMD including celiac disease, hypogonadism, and vitamin D deficiency. 21
## Mucocutaneous manifestations
Cutaneous disorders occur in up to 15% of patients with IBD, whereas mucosal disorders are less frequent and occur in up to 10% of the cases.In addition, skin lesions may occur as complications of IBD secondary to malabsorption or to specific treatments. Immunosuppressants and anti-TNF therapies commonly used in IBD are associated with the development of cutaneous infections and cutaneous lesions, including injection-site reactions. The use of these therapies may also lead to immune-mediated complications including psoriasis, eczematous eruptions, lupus-like syndrome, and rarely skin cancers.The most common cutaneous manifestations observed in IBD are erythema nodosum and PG. Less common mucocutaneous manifestations include psoriasis, Sweet syndrome, and oral aphthous stomatitis.Erythema nodosum, PG, and psoriasis were found to be associated with TRAF3IP2 gene in IBD patients based on a case-control cohort study carried out by Ciccacci et al.
## Erythema nodosum
Erythema nodosum (EN)is more common in patients with CD, affecting 15% compared with 10% of patients with UC.It has been suggested that EN is more common in females.EN involves inflammation of the subcutaneous fat usually presenting as raised, red or violet, tender nodules with a diameter of 1-5 cm. It is most commonly found on the anterior surface of the lower extremities and less frequently can involve the face, neck, trunk, and arms. EN is usually diagnosed clinically, rarely requiring a skin biopsy.PG PGis generally more severe and less common than EN occurring in 0.1%-1.2% of patients with CD and 1%-5% of patients with UC.Up to 50% of patients with PG have underlying IBD. It is more common in females.A characteristic feature of PG is a phenomenon known as pathergy, defined as an exaggerated physiologic response to a minor trauma such as a venous puncture or biopsy. 7 PG usually begins as an erythematous pustule or nodule that spreads and develops a burrowing ulcer with irregular violaceous edges. These ulcers are usually purulent and found to be sterile when cultured.These lesions are variable and can be unilateral or bilateral, solitary or multiple, and cover several centimeters or an entire limb.The legs are most commonly affected but PG can occur anywhere on the body, including the abdominal wall adjacent to a postsurgical stoma. PG ulcers can leave behind scars after they heal.Like EN, PG is usually a clinical diagnosis but requires a biopsy in rare cases to rule out other possible causes including infection, malignancy, or vascular disease.Also, up to 50% of patients with PG have IBD.
## Psoriasis
Studies suggest that psoriasis is more prevalent among patients with CD compared with the general population.An immunologic relation is suggested due to the high concentrations of TNFα found in both psoriatic lesions and similar lesions found in patients with CD.
## Sweet syndrome
Sweet syndrome is an acute febrile neutrophilic dermatosis. It is a rare disease that is associated with IBD and other systemic diseases including malignancy.Sweet syndrome presents as papulosquamous exanthema or nodules occurring on the arms, legs, hands, trunk, or face. Associated findings on presentation include arthritis, fever, ocular symptoms including conjunctivitis, and leukocytosis. It usually follows the course of intestinal disease activity but may precede the diagnosis of IBD. The use of azathioprine may be associated with the development of Sweet syndrome. 4
## Oral lesions
Oral lesions affect up to 10% of patients with IBD and they include periodontitis, aphthous stomatitis, and pyostomatitis vegetans.Oral lesions usually follow the course of the underlying intestinal disease. Aphthous stomatitis lesions are usually painful and occur on the buccal and labial mucosa, the tongue, and the oropharynx.Diagnosis is usually clinical, and biopsy is rarely required but if done, histology usually reveals noncaseating granulomas in CD similar to those found in the colon.Pyostomatitis vegetans presents as multiple ulcerations and hemorrhagic erosions anywhere on the oral mucosa usually with a cobblestone pattern. 29
## Treatment
Erythema nodosum follows the course of intestinal disease activity and is self-limiting with treatment usually aimed at the underlying bowel disease.Supportive therapy includes leg elevation, compression stockings, and analgesics.Occasionally, EN may precede bowel exacerbations and require treatment with oral corticosteroids.In cases that are severe or resistant to therapy, alternative causes of EN should be considered including infections with streptococcus, tuberculosis (TB), sarcoidosis, coccidioidomycosis, histoplasmosis, blastomycosis, Yersinia pseudotuberculosis, Yersinia enterocolitica, syphilis and Behçet disease, or use of certain medications including sulfonamides, iodides, bromides, and estrogens.After excluding alternative causes, severe cases may require systemic corticosteroids, immunosuppressive therapy, or TNF antibodies.Prevalence of recurrent disease is thought to be nearly 20%.PG, unlike EN, is not associated with underlying intestinal disease activity; however, it may resolve with treatment of the IBD. Mild, localized disease usually responds to local and topical therapy including moist treatment with hydroactive dressings, topical or intralesional corticosteroid injections, and topical sodium cromoglycate.Widespread PG requires systemic therapy including oral sulfasalazine, dapsone, corticosteroids, and immunomodulators such as azathioprine, cyclopho sphamide, cyclosporine, methotrexate, tacrolimus, and mycophenolate mofetil.Adalimumab and infliximab are effective treatments for severe or therapy-resistant cases of PG.Surgical intervention typically worsens PG and should be avoided.When discussing psoriasis treatment options in IBD patients, one has to be cautious as some psoriasis treatment options may worsen IBD. A recent systematic review carried out by Whitlock et al.analyzed Food and Drug Administration (FDA)approved biologic medications and FDA-approved nonbiological systemic medications for psoriasis and evaluated the evidence of their use in CD and UC. Among the FDA-approved biologic medications for psoriasis, infliximab and adalimumab are also FDA-approved for CD and UC; ustekinumab is also FDA-approved for CD but not yet studied for UC; etanercept, secukinumab, and ixekizumab have the potential risk of exacerbation of both CD and UC; brodalumab is contraindicated for CD and not yet studied for UC; and guselkumab is not yet studied for CD and UC.Among the FDAapproved nonbiologic systemic medications for psoriasis, cyclosporine has moderate efficacy in UC and modest efficacy in CD; methotrexate has strong efficacy in CD and modest efficacy in UC; and acitretin and apremilast are not yet studied for CD and UC.Most cases of Sweet syndrome respond rapidly to topical or systemic corticosteroid therapy and subsequently heal without scarring.Therapy for oral lesions includes antiseptic mouthwashes, topical steroids, and treatment of the underlying disease. 4
## Screening recommendations
Studies have found that individuals with IBD, both CD and UC, have a 37% higher risk of developing melanoma compared with the general population.On the contrary, there is no suggestion that IBD itself increases the risk of non-melanoma squamous cell cancer (NMSC).In general, patients with IBD likely have the same risk factors as the general population, but additionally have risks specific to their treatment. A study by Long et al.found that the use of anti-TNF therapy increases the risk of developing melanoma. Also, individuals with prior or current use of antimetabolite therapy, including azathioprine or 6-mercaptopurine, have an increased risk of developing NMSC, including squamous cell carcinomas (SCC) and, to a lesser degree, basal cell carcinomas (BCC).The United States Preventive Services Task Force (USPSTF) reported insufficient evidence to recommend universal skin cancer screening by whole-body skin examination by a primary care clinician or patient self-examination.There are no clear guidelines available for patients with IBD regarding annual skin examinations for the secondary prevention of skin cancer; however, many argue that this is warranted in patients with IBD on chronic immunosuppressive therapy due to the increased risk.The American College of Gastroenterology (ACG) recommends patients with IBD to undergo screening for melanoma independent of the use of biologic therapy and for those on immunomodulators to undergo screening for NMSC, especially when above the age of 50, with periodic skin examination.Even though exact recommendations are unclear, there is general agreement that all patients with IBD should definitely be educated about the risk of skin cancer and have discussions with their primary care physicians or gastroenterologists about the importance of prevention and early treatment.It is highly recommended that all individuals should take proper precautions including the use of sunscreen and sun protective clothing, especially those initiating immunosuppressive therapy. 36
## Ocular manifestations
Approximately 2%-5% of IBD patients experience ocular manifestations. They occur more frequently in patients with CD (3.5%-6%) compared with patients with UC (1.6%-4.6%). 4 Ocular manifestations often occur concurrently with other EIMs, most commonly musculoskeletal manifestations.It is important to note that conjunctivitis is the most common cause of red and itchy eyes in the general population, which holds true for patients with IBD. Therefore, physicians should keep this in mind, especially since conjunctivitis can easily mimic more serious ocular conditions.In addition to ocular manifestations, ocular complications secondary to IBD treatments can be seen.Steroids can cause cataracts and open-angle glaucoma, whereas cyclosporine can lead to optic neuropathy, ophthalmoplegia, and nystagmus.The most common ocular manifestation found in patients with IBD is episcleritis. Less common manifestations include scleritis and uveitis.Episcleritis Episcleritis is defined as inflammation of the blood-rich episclera.It is more common in patients with CD compared with UC.It usually presents with acute hyperemia, burning, and irritation in one or both eyes. Patients often have tenderness to palpation but no changes in vision. 7
## Scleritis
If deeper layers of the eye are affected, a diagnosis of scleritis should be considered.Scleritis is more severe than episcleritis and has the potential to cause visual impairment if not diagnosed early. Patients usually present with severe pain and tenderness to palpation. Recurrent scleritis can lead to retinal detachment, swelling of the optic nerve, or scleromalacia.Uveitis Uveitis is defined as inflammation of the eye's middle chamber. It is less common than episcleritis, occurring in 0.5%-3% of IBD patients, and is four times more common in women.Patients usually present with pain, photophobia, headaches, and blurring of vision.Diagnosis is confirmed by slit-lamp examination, which reveals perilimbic edema in addition to an inflammatory flare in the anterior chamber.Characteristically, the eye will show a ciliary flush with the redness showing most intensely in the middle and fading as it radiates outward. Examination findings also include conjunctival injection and corneal clouding.
## Treatment
Episcleritis often parallels intestinal activity and resolves with treatment of the underlying bowel disease. Patients occasionally require cool compresses or topical steroids for relief of symptoms.Scleritis, being more severe than episcleritis, requires more aggressive treatment with systemic corticosteroids or immunosuppressants, adequate control of the underlying intestinal disease, and prompt referral to an ophthalmologist to avoid loss of vision.Uveitis occurs independently of disease activity. Prompt treatment with topical and systemic steroids is essential to prevent progression to permanent visual loss. In patients with disease refractory to steroids, cyclosporine A and infliximab can be used. 7
## Screening recommendations
It is recommended that patients with IBD, especially those on immunosuppressive therapy, undergo regular, annual ophthalmologic evaluation and be encouraged to present to their physician's office to discuss any eye or vision changes. 38
## Hepatobiliary manifestations
Up to 50% of patients with IBD experience hepatobiliary manifestations at some point during their disease.These manifestations include PSC, smallduct PSC, fatty liver disease, granulomatous hepatitis, autoimmune hepatitis, and hepatic steatosis. In addition, extraintestinal complications include cholelithiasis, hepatic abscess, drug-induced hepatotoxicity, and hepatosplenic T-cell lymphoma (HSTCL).PSC PSC is the most common biliary manifestation in IBD. It is more prevalent in patients with UC compared with CD with approximately 2.4%-7.5% of patients affected. However, 75% of patients with PSC suffer from IBD, more commonly UC.Therefore, patients found to have PSC should have a colonoscopy to evaluate for the possibility of concomitant IBD. 7 PSC is a chronic cholestatic disease with progressive inflammation, stricturing, and fibrosis of the intrahepatic and extrahepatic bile ducts.Patients with PSC can experience bouts of acute cholangitis and eventually advance to cirrhosis, portal hypertension, and acute decompensation.It is not associated with underlying intestinal disease activity, is more common among patients 30-59 years of age, and is twice as common in males than females. Patients with UC with more extensive involvement of the colon are more likely to develop PSC than patients with involvement of the left colon only.Studies suggest genetic susceptibility, with HLA-B8 found to be positive in up to 70% of patients with PSC 7 in addition to the most recently identified gene UBASH3A that is non-HLA based.Patients with IBD who develop signs of cholestasis should be evaluated for PSC.Patients may present with unspecific findings at first including fatigue, abdominal pain, and weight loss and later pruritis and intermittent jaundice.Laboratory findings show elevated alkaline phosphatase and bilirubin levels whereas aspartate aminotransferase and alanine aminotransferase typically remain normal. Albumin levels and prothrombin time typically remain normal until disease progresses to cirrhosis.Serological workup may reveal positive autoantibodies in approximately 65%-88% of patients including antinuclear, anti-smooth muscle, and/or anti-perinuclear cytoplasmic antibodies.The diagnostic test of choice is magnetic resonance cholangiopancreatography (MRCP) since visualization of the biliary tree is required for diagnosis. The classic finding is the "bead on a string" appearance, which describes the presence of irregular bile ducts with multifocal strictures and segmental dilation. Endoscopic retrograde cholangiopancreatography (ERCP)is reserved for patients requiring interventions including stenting of high-grade strictures because of its associated risk of causing cholangitis due to inadequate biliary drainage.It is interesting to note that PSC appears to influence the course of IBD; patients who have both PSC and UC demonstrate a milder course of their colitis with less inflammation of the colon on histology compared with patients with IBD without PSC.However, PSC is thought to be an independent risk factor for the development of colorectal cancer (CRC) in patients with IBD. 7 PSC is also the greatest risk factor for developing cholangiocarcinoma.
## Hepatobiliary complications
Up to 30% of patients with IBD develop cholelithiasis, especially in patients with CD with ileal involvement or after ileal resection.Some of the factors contributing to this high risk include increased enterohepatic circulation of bile, impaired bile salt absorption, and reduced gallbladder motility. Hepatic abscess is a possible complication of IBD that can potentially be life-threatening. The patient may present with fever and abdominal pain and be found to have leukocytosis, which is a similar clinical presentation to an IBD flare. Fluid drained from the abscess may culture oral or intestinal flora or be sterile and predominantly composed of neutrophils. Both usually respond to drainage and antibiotics, with sterile abscesses also responding to corticosteroids.In general, medications that have been found to be associated with hepatotoxicity comprise of thiopurines, methotrexate, sulfasalazine, cyclosporine, and biologic agents.Specific drugs that might play a role in the development of non-alcoholic fatty liver disease (NAFLD), which, along with non-alcoholic steatohepatitis (NASH), affect approximately 9.4% of patients with UC and 19.3% of patients with CD, include corticosteroids, methotrexate, and azathioprine. Also, several cases have been reported of patients with IBD who went on to develop HSTCL after being treated with a combination of anti-TNF therapy, corticosteroids, and thiopurines. 7
## Treatment
Unfortunately, no medical therapy has been found to prevent the progression of PSC as bile duct damage is irreversible and does not seem to respond to medication. Ursodeoxycholic acid (UDCA) has been found to improve liver enzymes without having any real effect on the disease course.In addition, one study on the long-term use of UDCA in high doses reported an increased risk of CRC in patients with PSC and UC.ERCP can be used to dilate dominant strictures in some patients. Nevertheless, the majority of patients with PSC will ultimately require a liver transplantation. 4
## Screening recommendations
Patients who have PSC and IBD have a 10%-15% lifetime risk of developing cholangiocarcinoma. Consequently, some centers recommend annual screening using ultrasound or MRCP and measurement of tumor marker cancer antigen 19-9 (CA 19-9) in these patients.PSC is also an independent risk factor for the development of CRC in patients with IBD. Therefore, it is recommended that these patients undergo annual surveillance colonoscopies.In addition, it is always crucial to screen patients for hepatitis B, hepatitis C, and TB infection prior to initiating biologic therapy since these agents could potentially promote hepatic viral reactivation.
## Pancreatic manifestations
Pancreatic manifestations found in patients with IBD include acute pancreatitis, autoimmune pancreatitis, and exocrine and endocrine pancreatic insufficiency, whereas the main pancreatic complication seen is acute drug-induced pancreatitis.
## Acute pancreatitis
Patients with IBD develop acute pancreatitis with a prevalence of 1%-1.5% independent of medical therapy.This increased risk is thought to be secondary to gallstones, CD of the duodenum, or CD-associated granulomatous inflammation of the pancreas.Drug-induced pancreatitis has been linked to azathioprine, 5-aminosalicylic acid, 6-mercaptopurine, metronidazole, and, less commonly, corticosteroids. Based on a genome wide association study, it was found that patients with the HLA-DQA1-HLA-DRB1 haplotype have a genetic susceptibility to pancreatitis after administration of thiopurine immunosuppressants.Heterozygous patients have a 9% risk of developing pancreatitis induced by thiopurine, while homozygous patients have a 17% risk.Patients usually present with abdominal pain associated with nausea and vomiting starting shortly after beginning therapy and usually resolving quickly following discontinuation of the offending agent. 7,16 Autoimmune pancreatitis IBD-associated autoimmune pancreatitis (AIP) carries a worse prognosis due to the greater degree of severity, with an increased incidence in UC.IBD has a stronger association with type 2 AIP, which has normal IgG4 levels, in contrast to type 1 AIP which has elevated IgG4 levels.Treatment Treatment of acute pancreatitis in IBD is the same as for the general population including early adequate aggressive intravenous fluid resuscitation, electrolyte replacement, bowel rest, analgesia, and treatment of underlying cause of acute pancreatitis.
Treatment of AIP in IBD is similar to the standard therapy of type 2 AIP which is oral steroids with similar recurrence rate of 9%.
## Screening recommendations
To date, there are no established guidelines on screening recommendations aimed at pancreatic manifestations in patients with IBD; especially that autoimmune pancreatitis is a relatively newly recognized disease with only several cases reporting the diagnosis of AIP in association with IBD.
## Pulmonary manifestations
Pulmonary manifestations in patients with IBD are rare. However, lung involvement is probably more common than reported since many patients remain asymptomatic and imaging may be normal even when pulmonary function tests are not.Patients with UC are more likely to develop pulmonary EIMs than patients with CD. Pulmonary manifestations include large and small airway, upper airway, and parenchymal disease. Complications include drug-induced pulmonary disease.
Bronchiectasis, a manifestation of the large airways, is the most common pulmonary disease found in patients with IBD followed by chronic bronchitis.Diagnosis is made with a high-resolution computed tomography (CT) that shows dilated airways and bronchial wall thickening.On the contrary, upper airway manifestations are very rare and include subglottic stenosis diffuse tracheitis. The most frequent parenchymal lung disease in patients with IBD is cryptogenic organizing pneumonia (COP). Clinical presentation involves fever, cough, and dyspnea, therefore, requiring exclusion of infectious causes. Chest X-ray reveals opacities that range from patchy to diffuse and CT scan demonstrates scattered foci of consolidations that are either unilateral or bilateral in addition to centrilobular nodules.The use of methotrexate in patients with IBD has been linked to the pulmonary complications of hypersensitivity pneumonitis and pulmonary fibrosis.Also, sulfasalazine and mesalamine have been found, on rare occasions, to induce interstitial lung disease.
## Treatment
Pulmonary symptoms caused by manifestations of IBD or complications of the treatment show good response to inhaled corticosteroids and, in severe cases, oral or intravenous corticosteroids. 7
## Screening recommendations
To date, there are no established guidelines on screening recommendations for pulmonary manifestations in IBD.
## Renal and genitourinary manifestations
Approximately 4%-23% of patients with IBD are affected by renal manifestations or complications.Reported renal manifestations include glomerulonephritis, tubulointerstitial nephritis, and amyloidosis. Renal complications that impact patients with IBD include nephrolithiasis, enterovesicular fistula, perivesicular abscess, non-calculous obstructive uropathy, and drug-induced renal complications.Both glomerulonephritis and tubulointerstitial nephritis have been found to follow the intestinal course of the underlying bowel disease.The most common glomerulopathy that was found to be reported in patients with IBD is IgA nephropathy with HLA-DR1 being the investigated associated gene.Tubulointersititial nephritis may also be a drug-induced complication of IBD caused by the use of 5-aminosalicylic acid.Amyloidosis is a rare but serious EIM that predominantly affects patients with CD with a 10-fold increased risk compared with patients with UC. It is also three times more common in males than females. Amyloidosis is characterized by extracellular deposition of serum amyloid A and can be diagnosed with liver, rectal, or renal biopsy.Patients may present with proteinuria and uremia, which may proceed, if left untreated, to nephrotic syndrome and renal failure with poor outcomes.
## Renal and genitourinary complications
Nephrolithiasis is the most common renal complication affecting 5%-15% of patients with IBD, predominantly patients with CD with ileocolonic involvement.Calcium oxalate and uric acid renal stones are the most common. The increased risk of calcium oxalate stones in IBD is explained by the malabsorption of bile acids causing free fatty acids to reach the colon, bind to calcium, and increase the amount of oxalate that is free to be reabsorbed. Uric acid stones form due to decreased urine pH and decreased urine volumes that result from diarrhea.
An additional complication seen in patients with IBD includes non-calculous obstructive uropathy, which usually occurs due to retroperitoneal inflammation, fibrosis, and/or scarring in patients with CD and surgical complications in patients with UC.
The use of certain drugs has been found to induce renal complications. Cyclosporine and tacrolimus can lead to renal vasoconstriction causing acute renal dysfunction and interstitial fibrosis causing chronic renal impairment.Anti-TNF therapy has been found to be associated with several cases of severe glomerulonephritis.There also exists a possible risk of cervical dysplasia and cancer in patients with IBD. A recent Danish cohort study found that women with UC had an increased risk of both low-grade and highgrade lesions when compared with healthy controls, whereas women with CD had an increased risk of low-grade and high-grade lesions in addition to cervical cancer. They also noted an 8% increased risk of dysplasia in women with a history of azathioprine use.Treatment Therapy for glomerulonephritis, tubulointerstitial nephritis, and amyloidosis focuses on controlling and treating the underlying inflammatory process in IBD.Therapy for nephrolithiasis depends on the composition of the stone. For calcium oxalate stones, calcium supplementation is recommended in the amount of 1-2 g per day to help prevent oxalate stone formation.Therapy for uric acid stones includes fluid replacement, a purine-reduced diet, and alkalization of the urine.
## Screening recommendations
Conflicting data exist regarding the risk of cervical dysplasia and cancer in patients with IBD; however, studies have consistently reported an increased risk in patients using immunosuppressants.Therefore, the American College of Obstetricians and Gynecologists (ACOG) and the CDC recommend annual screening for women with a history of chronic immunosuppression starting at the age of 21.Unfortunately, some studies show that women with IBD are screened even less frequently than recommended for healthy women, which is once every 3 years, particularly those on immunosuppressive therapy.
Even though there are no specific cervical screening guidelines, it is important, as the European Crohn's and Colitis Organization (ECCO) states, for all women with IBD to strictly adhere to a screening program of cervical surveillance and undergo human papilloma virus (HPV) vaccination according to recommendations. 20 Papanicolaou (Pap) smear is the standard screening test and the HPV vaccine is recommended for all men and women 9-26 years of age. It is crucial to note that the HPV vaccine does not include all high-risk types and therefore does not eliminate the continued need for regular screening. 36
## Hematologic manifestations
Hematologic manifestations that have been found to occur in patients with IBD include venous and arterial thromboembolisms and anemia. Some studies have revealed an increased risk of venous thrombosis by 2 to 4-fold in patients with IBD, whereas the risk of arterial thrombosis increased to a lesser degree.Chronic inflammation plays a large role in activating coagulation and fibrinolysis.Both patients with active disease and those in remission have been found to experience thromboembolic events more frequently than seen in the general population. Studies have not been able to find increased rates of common genetic factors linked to hypercoagulability in patients with IBD including factor V Leiden and methylenetetrahydrofolate reductase (MTHFR) gene mutation.However, increased levels of homocysteine in the blood have been reported in patients with IBD compared with controls, but it is unclear whether this contributes to the increased risk of thromboembolism.
Anemia occurs in 19%-32% of patients with IBD. The different types of anemia encountered in IBD include anemia of chronic disease, iron deficiency anemia, and megaloblastic anemia from vitamin B12 or folate deficiency.Because vitamin B12 and folate are absorbed in the ileum, patients with CD are more likely to be affected than those with UC, especially patients with CD who have undergone bowel resection.
Drug toxicity is another cause of anemia in patients with IBD using azathioprine, 6-mercaptopurine, and methotrexate due to myelosuppression.There is also an association between immunosuppressive agents, particularly thiopurines, and increased risk of lymphoproliferative disorders. This was studied in the French Cancers Et Surrisque Associé aux Maladies inflammatoires intestinales En France (CESAME) cohort study, which found that patients who received thiopurines were five times more likely to develop lymphoproliferative disorders. Similar to previous studies, the incidence of lymphoma was found to be low and patients who discontinued thiopurine therapy went back to having a similar incidence of lymphoma as the general population. The CESAME study also revealed that it is possible for HSTCL to occur in patients exposed to thiopurine therapy alone or in combination with anti-TNF therapy.
## Treatment
Given the increased risk of thromboembolism among patients with IBD, it is recommended that all patients with IBD who are hospitalized or immobilized be treated with low-dose heparin for prophylaxis.Supplementation of iron is recommended for patients who present with iron deficiency anemia by the intravenous route, especially in active disease.If patients do not respond to iron supplementation or are not found to have iron deficiency, anemia of chronic disease is likely and treatment with erythropoietin is required. Oral supplementation of folate of at least 1 mg per day and intramuscular vitamin B12 of 1000 μg per month should be administered if the patient is found to be deficient.
## Screening recommendations
There is no current effective screening method available for the timely diagnosis of lymphoproliferative disorders in patients with IBD exposed to thiopurine therapy. Presenting symptoms that should alert physicians to investigate for lymphoproliferative disorders include headaches that are unexplained, fatigue, fever, hepatosplenomegaly, or lymphadenopathy not attributable to intestinal inflammation. Given the possible increased risk of HSTCL in patients on a combination of thiopurine and anti-TNF therapy, it is reasonable to avoid using this combination for greater than 2 years in men less than 35 years old. Physicians should provide clear information about the risks associated with certain therapies to their patients, especially since no specific screening recommendations are available. 36
## Neurologic and psychiatric manifestations
Intracerebral focal white matter lesions have been found on MRI in up to 42% of patients with CD and 46% of patients with UC who are asymptomatic. Demyelinating diseases including multiple sclerosis and ischemic optic neuropathy have been found among patients with IBD. Recurrent facial nerve palsy associated with Melkersson-Rosenthal syndrome has been described in some patients with CD specifically. 7 However, the most common neurological manifestations found in patients with IBD are peripheral neuropathies. These have not been found to follow intestinal activity of the underlying bowel disease and therefore do not respond to IBD-specific treatments.A typical neurological complication encountered among patients with IBD, particularly patients with CD who have undergone ileocecal resection, is peripheral polyneuropathy due to vitamin B12 deficiency. Drug-induced neurotoxicity is also described for various therapies used in patients with IBD. Metronidazole, sulfasalazine, and calcineurin inhibitors may potentially induce peripheral neuropathy. Calcineurin inhibitors are also associated with tremor, psychosis, parasthesia, ataxia, and motor deficit in up to 25% of patients. Anti-TNF therapy has been found to be associated with Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and multifocal motor neuropathy.Depression is a common problem affecting up to 35% of patients with IBD.In a recent systemic review, anxiety was found to be present in 19% of patients with IBD compared with 9.6% in the control group, while depression was found in 21.2% of patients with IBD compared with 13.4% in non-IBD controls.Predisposing risk factors for the development of depression in patients with IBD include frequent relapses and use of specific medications including steroids.Treatment Therapy for each of the above mentioned neurologic and psychiatric manifestations will be similar to that of the general population.
## Screening recommendations
The American College of Preventive Medicine (ACPM) has provided evidence that effective screening for depression can be accomplished with just two questions: A positive response to either of these questions should prompt further investigation into the symptoms. Appropriate patient counseling can be offered during the office visit and continued at coming visits for mild symptoms. However, moderate to severe symptoms should be treated with pharmacological intervention and/or referral to primary care or a mental health specialist. With the availability of this quick and efficient method, screening for depression and anxiety in patients with IBD should be accomplished at every office visit.Addressing this issue in patients with IBD may be crucial for disease management and enhancing the chance of good outcomes. 20
## Rare eims
Myocarditis, which is an inflammation of cardiac myocytes, is a rare cardiac manifestation of IBD which occurs as either a result of exposure to autoantigens leading to an autoimmune response or as a result of drug toxicity of 5-aminosalicylic acid or its derivatives.Treatment is supportive with immunosuppressive therapy including immunoglobulins and corticosteroids for a duration ranging from 3 to 6 months.Chronic recurrent multifocal osteomyelitis, a rare disease characterized by aseptic inflammation of long bones, is a rare skeletal manifestation of IBD which is mainly seen among children and adolescents with less than 30 cases reported worldwide.Treatment is mainly with corticosteroids and if that fails, reports have shown success of with anti-TNF agents and bisphosphonates. 62
## Additional general screening recommendations
## Crc screening
Patients with IBD have a significantly increased risk of developing colorectal cancer (CRC). The precursor to CRC is colonic dysplasia and, therefore, it is crucial to promptly identify and treat all IBD associated dysplasia.
Risk factors for CRC that are unique to IBD include the extent, severity, and duration of inflammation as well as previous history of colonic dysplasia. Patients that have extensive colitis (>50% involvement) that has been diagnosed for more than 10 years have a 7-fold increased risk of developing CRC compared with those without longstanding extensive colitis.Additional independent risk factors for the development of CRC in patients with IBD include active inflammation, and endoscopic findings of strictures, inflammatory polyps, and foreshortened colons.Also, patients with concomitant UC and PSC were found in a metaanalysis to have a 4-fold increased risk of CRC compared with patients with UC without PSC.Current guidelines in the United States recommend CRC surveillance every 1-2 years after disease has been present for 8-10 years. Exceptions involve certain conditions that warrant yearly surveillance starting at the time of diagnosis including patients with a family history of CRC in a firstdegree relative, active inflammation, or PSC.European guidelines including the British Society of Gastroenterology (BSG) and the European ECCO similarly recommend initiation of surveillance 8-10 years after diagnosis. They go on to offer detailed surveillance guidance based on risk stratification. Annual surveillance is recommended for patients with moderate to severe active inflammation, family history of CRC in a first-degree relative less than 50 years of age, or history of stricture, dysplasia or PSC. Surveillance is recommended every 2-3 years in patients with mild active inflammation, family history of CRC in a relative who is not less than 50 years of age, or history of post-inflammatory polyps. Finally, surveillance every 5 years is recommended for patients with only left-sided colitis, Crohn's colitis with involvement of less than 50% of the colon, or extensive colitis but no active endoscopic and histologic inflammation.Furthermore, Surveillance for Colorectal Endoscopic Neoplasia Detection and Management in IBD Patients International Consensus (SCENIC) also proposed evidence-based recommendations for the surveillance and management of colorectal dysplasia in IBD patients. In summary, these recommendations included the routine use of chromoendoscopy for the detection of dysplasia, the endoscopic removal of visible dysplasia, and, for patients with endoscopically invisible dysplasia that is confirmed by a gastrointestinal pathologist, referral to an endoscopist who is an expert in IBD surveillance using chromoendoscopy with high-definition colonoscopy.Chromoendoscopy with targeted biopsies has been found to have a higher sensitivity for detecting dysplasia and neoplasia than white light endoscopy with random biopsies. 21
## Smoking cessation
All patients with IBD should be encouraged to stop smoking.Smoking is associated with the development of CD.Also, patients with CD who are smokers have more disease progression, more frequent flares, higher rates of surgery, poorer medical and surgical outcomes, and an increased need for steroids and immunomodulators.These negative effects have been found to be dose dependent with higher tobacco load shown to be an independent predictor of stenosing phenotype.Therefore, any reduction in cigarette smoking is beneficial in improving disease course.Smoking cessation has been shown to decrease all of these risks stressing the importance of inquiring about tobacco use and discussing smoking cessation with IBD patients who are active smokers at every visit. 38
# Conclusion
IBD should be considered a systemic disease due to the high prevalence of EIMs and complications. Almost any organ system in the body may be affected, including those beyond the gastrointestinal tract, and in some cases, EIMs may be even more debilitating than the intestinal disease itself. Therefore, it is imperative that all measures be taken to quickly identify and adequately treat EIMs to minimize morbidity and mortality in affected patients. A thorough understanding of the current recommendations for screening and surveillance of EIMs in these patients is crucial and has the potential to greatly improve quality of life.summarizes the EIMs in IBD, associated genes, treatment, and screening recommendations.
## Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
# Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
## Orcid id
Inaya Hajj Hussein https://orcid.org/0000-0001-5497 -5688 |
Operative Techniques to Prevent Dialysis Access-associated Steal Syndrome in High-risk Patients Undergoing Surgery for Hemodialysis Access: A Systematic Review
# Introduction and background
End stage renal disease (ESRD) affects more than 1500 people per million population per year. Approximately two-third of these undergo hemodialysis [bib_ref] End-stage renal disease, Abbasi [/bib_ref]. Hemodialysis access-related complications are the most common cause of hospital admissions in ESRD patients on dialysis [bib_ref] Hemodialysis vascular access morbidity, Feldman [/bib_ref]. Dialysis access-associated steal syndrome (DASS) is one of those and the most dreadful [bib_ref] End-stage renal disease, Abbasi [/bib_ref] 1 2 1 1 complication which affects up to 10% of high risk patients [bib_ref] Incidence and characteristics of patients with hand ischemia after a hemodialysis access..., Morsy [/bib_ref] [bib_ref] Understanding strategies for the treatment of ischemic steal syndrome after hemodialysis access, Wixon [/bib_ref]. Traditionally, after arteriovenous (AV) access surgery if someone develops DASS, remedial surgery is offered which ranges from salvage procedures usually "distal revascularization and interval ligation -DRIL Procedure" to reciting of AV access. Certain features have been identified that put patients at high risk for developing DASS. These include age more than 60 years, diabetes mellitus, female gender, peripheral arterial disease, and history of previous DASS [bib_ref] Incidence and characteristics of patients with hand ischemia after a hemodialysis access..., Morsy [/bib_ref] [bib_ref] Understanding strategies for the treatment of ischemic steal syndrome after hemodialysis access, Wixon [/bib_ref] [bib_ref] Steal syndrome complicating hemodialysis access, Decaprio [/bib_ref] [bib_ref] Distal arterial flow in patients undergoing upper extremity dialysis shunting: a prospective..., Goldfeld [/bib_ref]. In these high-risk patients simultaneous prophylactic procedure to prevent DASS along with index AV access surgery for haemodialysis has been reported to reduce the morbidity [bib_ref] Extension technique': a modified technique for brachio-cephalic fistula to prevent dialysis access-associated..., Ehsan [/bib_ref] [bib_ref] Primary arteriovenous fistula inflow proximalization for patients at high risk for dialysis..., Jennings [/bib_ref] [bib_ref] Prophylactic distal revascularization with interval ligation and simultaneous arteriovenous fistula creation in..., Leake [/bib_ref] [bib_ref] Prophylactic distal revascularization and interval ligation procedure during femoral vein transposition fistula..., Nader [/bib_ref]. These prophylactic surgical techniques include "Proximalization of arterial inflow", "Extension technique" and "Prophylactic DRIL" [bib_ref] Extension technique': a modified technique for brachio-cephalic fistula to prevent dialysis access-associated..., Ehsan [/bib_ref] [bib_ref] Primary arteriovenous fistula inflow proximalization for patients at high risk for dialysis..., Jennings [/bib_ref] [bib_ref] Prophylactic distal revascularization with interval ligation and simultaneous arteriovenous fistula creation in..., Leake [/bib_ref].
Proximalization of arterial inflow as shown in [fig_ref] 1: Characteristics of the studies included in the systematic review [/fig_ref] works by taking inflow of the fistula from proximal larger diameter artery (Axillary artery) hence providing adequate blood to both the fistula and distal limb by virtue of higher blood flow [bib_ref] Prevention of vascular access hand ischemia using the axillary artery as inflow, Jennings [/bib_ref] [bib_ref] Proximalization of the arterial inflow: a new technique to treat access-related ischemia, Zanow [/bib_ref]. Moreover it also adds to the length of outflow segment and the needling site when reversed basilic vein is used [bib_ref] Primary arteriovenous fistula inflow proximalization for patients at high risk for dialysis..., Jennings [/bib_ref]. On the other hand, PTFE graft can also be used, but only at the cost of higher risk of infection and thrombosis [bib_ref] Alternative prosthetic vascular access creation using subscapular artery as inflow to prevent..., Song [/bib_ref]. Song and Yun described inflow from sub-scapular artery, which has advantage of proximalization as well as avoiding the main arterial inflow to the limb [bib_ref] Alternative prosthetic vascular access creation using subscapular artery as inflow to prevent..., Song [/bib_ref].
In the extension technique, instead of taking inflow from brachial artery, one of the forearm arteries is used 2-3 cm distal to bifurcation of brachial artery to anastomose with ante-cubital vein as given in [bib_ref] Extension technique': a modified technique for brachio-cephalic fistula to prevent dialysis access-associated..., Ehsan [/bib_ref]. The perfusion of distal limb is kept intact via the other artery of forearm. It takes 10-15 minutes more than simple brachio-cephalic AV fistula. In addition, it has the additional benefit of maturing both the cephalic and basilic veins simultaneously [bib_ref] Extension technique': a modified technique for brachio-cephalic fistula to prevent dialysis access-associated..., Ehsan [/bib_ref].
## Figure 2: extension technique.
The principle of prophylactic DRIL (Distal Revascularization and Interval Ligation) remains same when it is applied for treatment of DASS, i.e., both the fistula and distal limb are perfused simultaneously and reversal of flow is prevented by interval ligation, as depicted in schematic diagram [bib_ref] Treatment of hemodialysis access steal syndrome by distal revascularization arterial ligature: report..., Polimanti [/bib_ref]. It was first described by Schanzer et al. in 1988 with a technical success of 90% and bypass patency of 80% at four years [bib_ref] Treatment of ischemia due to "steal" by arteriovenous fistula with distal artery..., Schanzer [/bib_ref]. Despite all this, one of the major limitations of DRIL procedure is that the native artery is ligated and the limb perfusion is dependent upon bypass graft, which itself is at risk of complications [bib_ref] Prophylactic distal revascularization and interval ligation procedure during femoral vein transposition fistula..., Nader [/bib_ref]. However, Nader et al. and Leake et al. did not show additional morbidity related to procedure.
## Figure 3: distal revascularization and interval ligation.
Comparative efficacy of these techniques is not known [bib_ref] Extension technique': a modified technique for brachio-cephalic fistula to prevent dialysis access-associated..., Ehsan [/bib_ref] [bib_ref] Primary arteriovenous fistula inflow proximalization for patients at high risk for dialysis..., Jennings [/bib_ref] [bib_ref] Prophylactic distal revascularization with interval ligation and simultaneous arteriovenous fistula creation in..., Leake [/bib_ref].
## Objective
This systematic review was conducted to review and compare outcome of various prophylactic surgical techniques performed along with primary AV access surgery for hemodialysis to prevent dialysis access steal syndrome (DASS) in high-risk patients.
## Review
This systematic review is registered with PROSPERO (2017:CRD42017060804). We searched for all types of studies including case report, observational studies and interventional trials that reported prophylactic intra-operative techniques to prevent DASS in high-risk patients undergoing hemodialysis access procedure. We considered studies published from January 1990 till March 2017. Studies published in languages other than English, performed on animals or un-published reports were excluded. A thorough systematic search for relevant studies was done on Google scholar, PubMed and Cochrane Database and the last date of search was April 30, 2019.
In order to identify relevant studies, the search strategy was based upon concepts of population, intervention and outcome. Population was identified as adults with end stage renal disease (ESRD), high risk for DASS, and requiring permanent hemodialysis access. Search terms used to look for population of interest were "high risk" OR "prone to" OR "females more than 60 years of age" OR "diabetics" OR "peripheral arterial disease" AND "Arterio-venous fistula" OR "AVF" OR "Hemodialysis Access" OR "AVBG" OR "Brachiocephalic fistula" OR "Basilic transposition fistula" OR "Brachio-basilic fistula". Intervention of interest was prophylactic intra-operative techniques to prevent DASS for which we used "Prophylactic" OR "Pre-emptive" OR "Preventive" OR "Intra-operative" OR "Per-operative" AND "Techniques" OR "Steps" OR "procedure" OR "Extension technique" OR "DRIL" OR "Proximalization of inflow" OR "RUDI" as relevant terms. Outcome was identified using "Dialysis access steal syndrome" OR "steal syndrome" Or "distal ischemia".
Using relevant search terms for individual concepts, two investigators separately searched and reviewed the literature. If there was any disagreement between two, the third investigator was involved. Initial screening for inclusion was done reading the title of the studies and identifying duplicates. Further eligibility according to selection criteria was done through stages of abstract and full manuscript evaluation. References of included studies were also searched to identify any missing reports. Specifically designed data extraction sheet was used to collect the data from individual studies. Information such as first author's name, journal, year of publication, study design, country of origin, target population, prophylactive intra-operative technique used, sample size/number of cases, site of arteriovenous fistula, mean follow-up in months, dialysis access steal syndrome were extracted from studies.
# Results
A total of 125 studies were retrieved after applying search strategy, out of those six met the inclusion criteria as summarized in [fig_ref] FIGURE 4: Flow diagram for selection of studies [/fig_ref]. Majority of studies were retrospective case series while only one was a case report. The largest study sample size was 32 in one of the case series by Ehsan et al. [bib_ref] Extension technique': a modified technique for brachio-cephalic fistula to prevent dialysis access-associated..., Ehsan [/bib_ref]. Five studies involve AV access creation on arm whereas one by Nader et al. has described AV access in thigh [bib_ref] Prophylactic distal revascularization and interval ligation procedure during femoral vein transposition fistula..., Nader [/bib_ref]. Intraoperative techniques described were variable. "Proximalization of arterial inflow" was described in three, "prophylactic DRIL" in two whereas "Extension technique" was reported in one study to prevent DASS ( Our review found that majority of the studies favor "Proximalization of Arterial Inflow" as a useful technique to prevent DASS in high-risk patients undergoing surgery for permanent hemodialysis access, though both "Extension technique" and "Prophylactic DRIL" have also been shown to be effective in preventing DASS. These results can help vascular surgeons in decision making regarding AV access in high risk patients.
Due to lack of large sample studies and retrospective nature of studies, quality of evidence available is low.
# Conclusions
Proximalization of inflow has been reported as the most common procedure performed to prevent DASS followed by extension technique and DRIL procedure. All three procedures have satisfactory outcome with no clear superiority of one over the other.
# Additional information disclosures
## Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
[fig] FIGURE 4: Flow diagram for selection of studies (PRISMA Diagram). [/fig]
[fig] 1: Characteristics of the studies included in the systematic review. DASS: Dialysis access-associated steal syndrome; DRIL: Distal revascularization and interval ligation.Follow-up ranged from seven to 42 months as shown in Figure 5. Only one patient in a case series by Ehsan et al. developed DASS which required correction, while none of the patients in other studies developed this complication after prophylactic procedure (Table 2)[7]. [/fig]
[fig] FIGURE 5 FIGURE 5: Mean follow-up of patients in included studies. [/fig]
[table] TABLE 2: Dialysis access-associated steal syndrome (DASS) in included studies. [/table]
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Potential involvement of the extracranial venous system in central nervous system disorders and aging
Background: The role of the extracranial venous system in the pathology of central nervous system (CNS) disorders and aging is largely unknown. It is acknowledged that the development of the venous system is subject to many variations and that these variations do not necessarily represent pathological findings. The idea has been changing with regards to the extracranial venous system.Discussion: A range of extracranial venous abnormalities have recently been reported, which could be classified as structural/morphological, hemodynamic/functional and those determined only by the composite criteria and use of multimodal imaging. The presence of these abnormalities usually disrupts normal blood flow and is associated with the development of prominent collateral circulation. The etiology of these abnormalities may be related to embryologic developmental arrest, aging or other comorbidities. Several CNS disorders have been linked to the presence and severity of jugular venous reflux. Another composite criteria-based vascular condition named chronic cerebrospinal venous insufficiency (CCSVI) was recently introduced. CCSVI is characterized by abnormalities of the main extracranial cerebrospinal venous outflow routes that may interfere with normal venous outflow.Summary: Additional research is needed to better define the role of the extracranial venous system in relation to CNS disorders and aging. The use of endovascular treatment for the correction of these extracranial venous abnormalities should be discouraged, until potential benefit is demonstrated in properly-designed, blinded, randomized and controlled clinical trials.Please see related editorial: http://www.biomedcentral.com/1741-7015/11/259.
# Background
Mounting evidence suggests that a number of inflammatory and neurodegenerative central nervous system (CNS) disorders may be related to vascular factors [bib_ref] The blood-brain barrier in health and chronic neurodegenerative disorders, Zlokovic [/bib_ref]. While the role of arterial supply abnormalities in relation to the pathology of CNS disorders is well-defined, the role of venous drainage impairment, for example, extracranial venous abnormalities, is largely unknown [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] The chronic cerebrospinal venous insufficiency syndrome, Zamboni [/bib_ref] [bib_ref] Chronic cerebral spinal venous insufficiency in multiple sclerosis, Haacke [/bib_ref] [bib_ref] What went wrong? The flawed concept of cerebrospinal venous insufficiency, Valdueza [/bib_ref]. The complexity, inter-individual variability and frequent asymmetry of the extracranial venous system, compared to the peripheral venous and arterial systems make exploration of the link between intracranial and extracranial pathology extremely difficult [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] The vertebral venous plexus as a major cerebral venous outflow tract, Epstein [/bib_ref]. Moreover, additional factors, including postural change, cardiac function, respiration, frequent change in lumen diameter, hydration status, hypovolemia and the presence of nearby structures, may influence correct assessments of the veins in regards to the presence of structural or hemodynamic extracranial venous abnormalities [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] The chronic cerebrospinal venous insufficiency syndrome, Zamboni [/bib_ref] [bib_ref] Chronic cerebral spinal venous insufficiency in multiple sclerosis, Haacke [/bib_ref] [bib_ref] What went wrong? The flawed concept of cerebrospinal venous insufficiency, Valdueza [/bib_ref].
Compared with the arterial system, the development of the extracranial venous system is subject to many variations. Therefore, in the past, these variations were acknowledged as non-pathological findings [bib_ref] Direct venous spinal reabsorption of cerebrospinal fluid: a new concept with serial..., Biceroglu [/bib_ref] [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref] [bib_ref] Diagnosis and treatment of venous malformations Consensus Document of the International Union..., Lee [/bib_ref] [bib_ref] The association of extracranial and intracranial vascular malformations in children, Pascual-Castroviejo [/bib_ref]. A variety of congenital extracranial venous abnormalities/ developmental variants has been described [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref] [bib_ref] Diagnosis and treatment of venous malformations Consensus Document of the International Union..., Lee [/bib_ref]. However, investigations aimed to define the nature of these venous abnormalities/developmental variants and their clinical significances are lacking [bib_ref] Altered collagen expression in jugular veins in multiple sclerosis, Coen [/bib_ref] [bib_ref] A technical approach to dissecting and assessing cadaveric veins pertinent to chronic..., Diaconu [/bib_ref].
Several CNS disorders, such as transient global amnesia, transient monocular blindness, cough headache and primary exertional headache, have been linked to the presence and severity of uni-or bi-lateral jugular venous reflux (JVR) in the last two decades [bib_ref] Cough headache and thoracic inlet valvular competence in uremia, Chuang [/bib_ref] [bib_ref] Jugular venous reflux and plasma endothelin-1 are associated with cough syncope: a..., Chung [/bib_ref] [bib_ref] Transient global amnesia: cerebral venous outflow impairment-insight from the abnormal flow patterns..., Chung [/bib_ref] [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref] [bib_ref] Incompetence of internal jugular valve in patients with primary exertional headache: a..., Doepp [/bib_ref] [bib_ref] Reflux of jugular and retrobulbar venous flow in transient monocular blindness, Hsu [/bib_ref]. More recently, an intense interest in better understanding the role of the extracranial venous system in the pathophysiology of CNS disorders has been generated by the introduction of a composite criteria-based vascular condition named chronic cerebrospinal venous insufficiency (CCSVI). CCSVI is characterized by abnormalities of the main extracranial cerebrospinal venous outflow routes that interfere with normal venous drainage, as evidenced by Doppler sonography (DS) [bib_ref] A prospective open-label study of endovascular treatment of chronic cerebrospinal venous insufficiency, Zamboni [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref]. It was originally hypothesized that CCSVI implies a pathological condition that leads to abnormal venous drainage of the brain parenchyma and increased susceptibility to multiple sclerosis (MS) [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref]. While, the condition was originally described in MS patients, it became immediately clear from the independent results of the first controlled studies that patients with other CNS disorders and healthy individuals may also show a high prevalence of this condition [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref]. However, because healthy individuals do not suffer from CNS disorders, its clinical relevance as a nosological entity was immediately questioned [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency and multiple sclerosis, Khan [/bib_ref]. Indeed, as more research studies have become available, the very concept of CCSVI, its diagnostic utility and clinical impact for MS have all been questioned, as no causal relationship between CCSVI and MS has been confirmed [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref]. In addition, the controversy and debate around CCSVI has been fueled by the postulated therapeutic effect of venous insufficiency correction using endovascular procedures [bib_ref] A prospective open-label study of endovascular treatment of chronic cerebrospinal venous insufficiency, Zamboni [/bib_ref] , without first determining a real need for the procedure itself and testing its safety and efficacy in properly designed randomized, controlled and blinded trials [bib_ref] Chronic cerebrospinal venous insufficiency and multiple sclerosis, Khan [/bib_ref] [bib_ref] CCSVI and MS: no meaning, no fact, Baracchini [/bib_ref] [bib_ref] Role of venoplasty for treatment of multiple sclerosis: value of open-label studies..., Zivadinov [/bib_ref].
Given that substantial resources by various governments and funding agencies have been devoted to studying the concept of CCSVI, it was recently proposed that funding of CCSVI research should be immediately abandoned because it is a waste of valuable time, money and intellectual energy [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Ghezzi [/bib_ref] [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Hutchinson [/bib_ref] [bib_ref] Media, politics and science policy: MS and evidence from the CCSVI trenches, Pullman [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: the final curtain, Paul [/bib_ref] [bib_ref] Multiple sclerosis: CCSVI deconstructed and discarded, Barkhof [/bib_ref]. Nevertheless, the concept of CCSVI has triggered an intense and rapid accumulation of knowledge over the past four years and has stimulated the need for further research to better understand the function and potential role of the extracranial venous system in CNS disorders and aging [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Zivadinov [/bib_ref]. This review article highlights the need for better classification of extracranial venous abnormalities/developmental variants that is independent of any single diagnostic imaging modality. It also examines the anatomy, etiology and pathophysiology associated with venous abnormalities, as well as the clinical correlates in relation to various CNS disorders and aging.
## Anatomy of the extracranial venous system
In order to understand the potential role of the extracranial venous system in diseases of the CNS and aging, it is important to first appreciate the structure and function of the cerebral venous drainage system. Because this system is complex and poorly understood, in this section, a brief overview of the relevant anatomy is presented to assist the reader.
Cerebral circulation encompasses both the arterial and venous systems. The venous system contains approximately 70% of the blood volume, with approximately three-quarters of it within small veins and venules [bib_ref] Autonomic control of the venous system in health and disease: effects of..., Pang [/bib_ref] [bib_ref] Compartmental analysis of compliance and outflow resistance of the cerebrospinal fluid system, Marmarou [/bib_ref] [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Venous territories of the brain, Meder [/bib_ref] [bib_ref] The cerebral venous system, Schmidek [/bib_ref] [bib_ref] Variations of the basal vein: identification using three-dimensional CT angiography, Suzuki [/bib_ref] [bib_ref] Jugular venous reflux, Chung [/bib_ref]. It is a system that is often asymmetric and considerably represents a more variable pattern than the arterial system [bib_ref] The chronic cerebrospinal venous insufficiency syndrome, Zamboni [/bib_ref].
## Cerebral venous system; superficial and deep veins
The venous drainage from cerebral hemispheres consists of two systems; the superficial and the deep venous system [fig_ref] Figure 1: Intracranial venous system anatomy of dural sinuses, cortical veins, deep intracerebral veins... [/fig_ref] [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Venous territories of the brain, Meder [/bib_ref] [bib_ref] The cerebral venous system, Schmidek [/bib_ref] [bib_ref] Variations of the basal vein: identification using three-dimensional CT angiography, Suzuki [/bib_ref] [bib_ref] Jugular venous reflux, Chung [/bib_ref]. The superficial system drains blood from the cortex and superficial white matter (WM) by cortical veins, collected by dural sinuses. There are two important dural sinuses: the superior sagittal sinus (SSS) draining dorso-laterally and the cavernous sinus draining anteroventrally. The transverse sinus then drains the SSS equally on both sides in only 20% of cases and asymmetrically in more than 50% of cases, depending on the configuration of torcular Herophili [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Variations of the basal vein: identification using three-dimensional CT angiography, Suzuki [/bib_ref]. In 20% of cases, one transverse sinus drains the SSS in total (most often on the right side) and the other one drains the straight sinus, which collects blood from the deep venous system [bib_ref] Variations of the basal vein: identification using three-dimensional CT angiography, Suzuki [/bib_ref]. The cavernous sinus extends from the superior orbital fissure to the petrous apex, which receives orbital venous and middle cranial fossa drainage. From the cavernous sinus, blood drains posterolaterally along the superior petrosal sinus into the transverse sinus and inferior-laterally along the inferior petrosal sinus into the sigmoid sinus.
The deep cerebral venous system drains the deep WM and the regions surrounding the lateral and third ventricles or the basal cistern [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Venous territories of the brain, Meder [/bib_ref] [bib_ref] The cerebral venous system, Schmidek [/bib_ref]. Three veins unite just behind the interventricular foramen of the Monro to form the internal cerebral vein(s). These include the choroid vein, septal vein and thalamostriate vein. The vein of Galen is a short (1 to 2 cm long), thick vein that passes posterosuperiorly behind the splenium of corpus callosum in the quadrigeminal cistern. The vein of Galen receives the internal cerebral vein, the basal veins of Rosenthal and the posterior fossa veins and then drains to the anterior end of the straight sinus where this unites with the inferior sagittal sinus. The main collecting vein for the deep venous system is the straight sinus, which receives the venous blood from the vein of Galen and flows into the transverse sinus (most often into the left side). The basal vein of Rosenthal is an important collateral pathway for the internal cerebral veins and the vein of Galen. By connecting with the superficial Sylvian vein via the deep Sylvian vein, venous blood flow can bypass the straight sinus.
Venous drainage of the posterior fossa mainly depends on the galenic system and the petrosal system and to a lesser extent, the tentorial veins and the transverse sinuses [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Venous territories of the brain, Meder [/bib_ref] [bib_ref] The cerebral venous system, Schmidek [/bib_ref] [bib_ref] Variations of the basal vein: identification using three-dimensional CT angiography, Suzuki [/bib_ref]. Therefore, factors influencing galenic system drainage would lead to venous congestion in both posterior fossa and brain regions drained by the deep venous system.
## Extracranial cerebral venous drainage pathway -neck veins
Most of the cerebral venous drainage is via neck veins; mainly the internal jugular vein (IJV), vertebral venous system and deep cervical veins (veins in cervical soft tissue) [fig_ref] Figure 2: Illustration depicting the predominant veins and sinuses involved in the craniocervical venous... [/fig_ref] [bib_ref] Craniocervical junction venous anatomy on enhanced MR images: the suboccipital cavernous sinus, Caruso [/bib_ref] [bib_ref] Postural dependency of the cerebral venous outflow, Valdueza [/bib_ref] [bib_ref] The craniocervical venous system in relation to cerebral venous drainage, San Millan Ruiz [/bib_ref] [bib_ref] Extrajugular pathways of human cerebral venous blood drainage assessed by duplex ultrasound, Schreiber [/bib_ref] [bib_ref] How does the blood leave the brain? A systematic ultrasound analysis of..., Doepp [/bib_ref]. Consequently, there is a good reason to believe that impaired extracranial venous drainage functions or structures might cause cerebral venous drainage insufficiency and consequent neurological deficits.
The IJVs are the largest veins in the neck and are generally considered to be the most important cerebral venous outflow pathways. Venous drainage of the superficial and deep cerebral venous system is via the transverse sinuses to the sigmoid sinuses, which then drain into the IJV. The inferior petrosal sinus, a major drainage route collecting blood from the cavernous sinus, communicates with the basilar plexus, anterior and lateral condylar veins, anterior condylar confluence and vertebral venous plexus before draining into the IJVs [bib_ref] The craniocervical venous system in relation to cerebral venous drainage, San Millan Ruiz [/bib_ref] [bib_ref] Venous structures at the craniocervical junction: anatomical variations evaluated by multidetector row..., Tanoue [/bib_ref] [bib_ref] Study on inferior petrosal sinus and its confluence pattern with relevant veins..., Zhang [/bib_ref]. The IJVs then join with the subclavian veins to form the brachiocephalic vein (BV). The confluence of the bilateral BV is the superior vena cava, which ultimately drains venous blood into the heart. Several tributaries in the neck also drain into the IJVs [bib_ref] Selective superior and inferior thyroid vein catheterization, Doppman [/bib_ref] [bib_ref] Anatomy of microvascular anastomosis in the neck, Shima [/bib_ref]. These bilateral IJV branches will interconnect with each other at the midline to form anastomosing plexi that can serve as collateral channels to maintain adequate venous drainage when the principal pathways are obstructed [bib_ref] Selective superior and inferior thyroid vein catheterization, Doppman [/bib_ref]. The vertebral venous system consists of two components; one is the vertebral venous plexus and the other is the vertebral vein (VV) [bib_ref] The vertebral venous plexus as a major cerebral venous outflow tract, Epstein [/bib_ref] [bib_ref] The craniocervical venous system in relation to cerebral venous drainage, San Millan Ruiz [/bib_ref] [bib_ref] The anatomy of collateral venous flow from the brain and its value..., Andeweg [/bib_ref] [bib_ref] The physiologic significance of the vertebral venous plexus, Eckenhoff [/bib_ref]. The vertebral venous plexus can be subdivided as internal (posterior and anterior internal vertebral plexus) and external (posterior and anterior external vertebral plexus) [bib_ref] The vertebral venous plexus as a major cerebral venous outflow tract, Epstein [/bib_ref] [bib_ref] The craniocervical venous system in relation to cerebral venous drainage, San Millan Ruiz [/bib_ref] [bib_ref] The anatomy of collateral venous flow from the brain and its value..., Andeweg [/bib_ref] [bib_ref] The physiologic significance of the vertebral venous plexus, Eckenhoff [/bib_ref].
Complex connections of cerebral venous outflow with the vertebral venous system over the craniocervical junction have been displayed by several human cadavers and angiographic studies [bib_ref] Craniocervical junction venous anatomy on enhanced MR images: the suboccipital cavernous sinus, Caruso [/bib_ref] [bib_ref] The craniocervical venous system in relation to cerebral venous drainage, San Millan Ruiz [/bib_ref] [bib_ref] Venous structures at the craniocervical junction: anatomical variations evaluated by multidetector row..., Tanoue [/bib_ref] [bib_ref] The suboccipital cavernous sinus, Arnautovic [/bib_ref] [bib_ref] The posterior condylar canal, Ginsberg [/bib_ref] [bib_ref] Condylar canal vein: unfamiliar normal structure as seen at CT and MR..., Weissman [/bib_ref]. The IJVs can also exhibit anastomosis with the other extracranial venous drainage system within the craniocervical junction region, which includes the anterior condylar confluent (ACC) and its tributes. Numerous anastomoses of the ACC make it a crossroad between the cavernous sinus, dural sinuses of the posterior fossa, IJVs and posterior cervical outflow tract (vertebral venous system and deep cervical veins).
## Ijv valves
The IJV valves make IJV a buffer zone between large central veins and the cerebral venous system. Although there are anatomical variations, the valves are generally located about 0.5 cm above the union of the subclavian vein and IJVs at the lower limit of the jugular bulb [bib_ref] Doppler ultrasonography of normal neck veins, Pucheu [/bib_ref] [bib_ref] The internal jugular vein valve may have a significant role in the..., Silva [/bib_ref] [bib_ref] Ultrasonic evaluation of jugular venous valve competence, Brownlow [/bib_ref] [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref] , which are shown in 96.8% of the general population [bib_ref] Doppler ultrasonography of normal neck veins, Pucheu [/bib_ref] [bib_ref] Ultrasonic evaluation of jugular venous valve competence, Brownlow [/bib_ref]. The IJV valves are generally thought to prevent the backflow of venous blood and backward venous pressure into the cerebral venous system during conditions where the central venous pressure or intrathoracic pressure is increased, such as chest compression during external cardiopulmonary resuscitation, severe or repetitive cough and straining [bib_ref] The internal jugular vein valve may have a significant role in the..., Silva [/bib_ref] [bib_ref] Ultrasonic evaluation of jugular venous valve competence, Brownlow [/bib_ref] [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref] [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref]. The pressure gradient across competent IJV valves can be as high as 100 mmHg [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref]. Without competent IJV valves, a sustained or prolonged retrograde-transmitted venous pressure via IJVs might impair cerebral venous drainage and lead to neurological deficits. For example, IJV valve incompetence has been associated with encephalopathy after cardiopulmonary resuscitation [bib_ref] The internal jugular vein valve may have a significant role in the..., Silva [/bib_ref] [bib_ref] Ultrasonic evaluation of jugular venous valve competence, Brownlow [/bib_ref] [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref].
## Other neck veins serving as collaterals for cerebral venous drainage
Collateral veins probably represent physiological variations of the venous system that may play a compensatory role when there is narrowing of the principal pathways of the extracranial venous system [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] The chronic cerebrospinal venous insufficiency syndrome, Zamboni [/bib_ref]. The extra-jugular cerebral venous drainage system for cerebral venous drainage mainly consists of the vertebral venous system and deep cervical veins [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] Craniocervical junction venous anatomy on enhanced MR images: the suboccipital cavernous sinus, Caruso [/bib_ref] [bib_ref] Postural dependency of the cerebral venous outflow, Valdueza [/bib_ref] [bib_ref] The craniocervical venous system in relation to cerebral venous drainage, San Millan Ruiz [/bib_ref] [bib_ref] Extrajugular pathways of human cerebral venous blood drainage assessed by duplex ultrasound, Schreiber [/bib_ref] [bib_ref] How does the blood leave the brain? A systematic ultrasound analysis of..., Doepp [/bib_ref] [bib_ref] Venous adaptation following bilateral radical neck dissection with excision of the jugular..., Gius [/bib_ref] [bib_ref] Intracranial-pressure studies incident to resection of the internal jugular veins, Sugarbaker [/bib_ref] [bib_ref] Venous collateral blood flow assessed by Doppler ultrasound after unilateral radical neck..., Doepp [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref]. The external jugular vein (EJV) and anterior jugular vein (AJV), compared with the IJV, are located superficially in the neck. They serve as collaterals and become prominent (enlarged lumen) when the main cerebral venous drainage pathways (IJV and VV) are compromised [bib_ref] It's not a cervical lymph node, it's a vein: CT and MR..., Escott [/bib_ref] [bib_ref] Prediction of a small internal jugular vein by external jugular vein diameter, Stickle [/bib_ref]. EJV is formed by the confluence of the posterior branch of the posterior facial vein and the posterior auricular vein. It usually terminates into the confluence of the subclavian and IJV [bib_ref] Contribution to the study of the tributaries and the termination of the..., Deslaugiers [/bib_ref]. The AJV receives blood from superficial veins, such as EJVs, facial veins or IJVs. They usually end in the subclavian vein or EJV [bib_ref] Contribution to the study of the tributaries and the termination of the..., Deslaugiers [/bib_ref]. Bilateral AJVs may communicate via the jugular venous arch (JVA), which is located just above the sternum. The JVA receives tributaries from the thyroid gland via inferior thyroid veins [bib_ref] The anterior jugular venous system: variability and clinical impact, Schummer [/bib_ref] [bib_ref] Venous chest anatomy: clinical implications, Chasen [/bib_ref]. In summary, venous collaterals in the neck include the anterior (jugular venous system) and the posterior (vertebral and other deep neck venous system) and different patterns of collateral establishment may reflect the location and severity of venous outflow obstruction.
## Extracranial cerebral venous drainage pathway -abdominal and thoracic veins
The vertebral venous system, which is a rich plexus, communicates with the deep thoracic and lumbar veins, intercostal veins as well as the hemiazygos and azygos veins [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref]. Abnormalities in these abdominal and thoracic veins may impair venous drainage from the vertebral venous system, which serves as an important collateral for cerebral venous drainage. The hemiazygos arch is connected with the left renal vein that represents a major outflow route for shunting blood into the inferior vena cava [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref]. Ultimately, the azygos vein serves as the final venous blood collector and drains into the superior vena cava. The anatomy and developmental stages of the abdominal/thoracic blood vessels can be quite variable [fig_ref] Figure 3: Paired anterior cardinal veins form common cardinal veins with paired posterior cardinal... [/fig_ref]. For example, in some rare variations, the azygos vein also drains thoracic veins, bronchial veins and even gonadal veins. The vein is so named because it has no symmetrically equivalent vein on the left side of the body.
## Extracranial venous abnormalities and their etiology
A range of abnormalities have been reported in the extracranial veins that drain cerebral venous blood flow. These can be classified as structural/morphological, hemodynamic/functional and those determined only by the composite criteria and use of multimodal imaging. For example, structural/morphological venous abnormalities can be divided into those creating narrowing or occlusion and those causing abnormal distensibility. On the other hand, hemodynamic functional venous abnormalities represent an abnormal cerebral venous outflow in the presence or absence of a structural venous anomaly in the extracranial veins. Finally, because it is almost impossible to determine the relevance of a single structural/morphologic or hemodynamic/functional venous abnormality, regardless of the imaging modality or methodology utilized, the need for use of composite criteria by uni-or multi-modal imaging modalities of the extracranial venous system is emerging [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref].
The etiology of these extracranial venous abnormalities is not well-defined. However, it has been hypothesized that these abnormalities may be related to embryologic developmental arrest, aging or other comorbidities [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref].
## Structural/morphological venous abnormalities
The reason for the narrowing of extracranial veins can be intra-luminal or extra-luminal [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref] [bib_ref] Diagnosis and treatment of venous malformations Consensus Document of the International Union..., Lee [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref].
The venous system develops through stages that may be associated with a number of developmental variants that do not necessarily represent pathological findings [bib_ref] Direct venous spinal reabsorption of cerebrospinal fluid: a new concept with serial..., Biceroglu [/bib_ref] [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref] [bib_ref] Diagnosis and treatment of venous malformations Consensus Document of the International Union..., Lee [/bib_ref] [bib_ref] The association of extracranial and intracranial vascular malformations in children, Pascual-Castroviejo [/bib_ref]. Lee et al. [bib_ref] Diagnosis and treatment of venous malformations Consensus Document of the International Union..., Lee [/bib_ref] recently published a consensus document in which they proposed that most of the extracranial venous abnormalities are a result of congenital truncular venous malformations, which represent an embryologically defective vein where developmental arrest has occurred during the vascular trunk formation period in the 'later stage' of embryonic development [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref]. For example, a truncular venous malformation lesion, such as a venous web at the hepatic venous outlet, causes portal hypertension, giving a profound damage/ impact to the liver [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref]. Similar truncular venous malformations involving the abdominal, thoracic and neck venous system may cause venous drainage impairment of the CNS. These truncular malformations are mostly represented by intra-luminal abnormalities.
Different extra-luminal etiologies lead to IJV narrowing/ occlusion at different levels [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Kantarci [/bib_ref] [bib_ref] Extracranial Doppler sonographic criteria of chronic cerebrospinal venous insufficiency in the patients..., Simka [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref]. The sigmoid sinus exits the skull and becomes the upper jugular bulb, where it is directed anteriorly to sweep over the lateral arch of the first cervical vertebra. IJV narrowing at this level is commonly associated with displacement and compression of the vein as it courses over the anterior aspect of the lateral mass of the C1 vertebral body. At the mid-cervical level, IJV has been observed to be compressed by adjacent tissues, including carotid arteries and the sternocleidomastoid muscle group. The severity of these compressions could be dynamic, depending on the individual's posture, neck flexion or extension and ipsilateral or contralateral rotation of the head position [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] [bib_ref] Compression of the internal jugular vein by the transverse process of the..., Seoane [/bib_ref] [bib_ref] Anatomic relationship of the internal jugular vein and the common carotid artery..., Turba [/bib_ref]. Most recently, the omohyoid muscle anatomic variants were reported as a possible reversible cause of IJV extrinsic compression [bib_ref] The omohyoid muscle entrapment of the internal jugular vein. A still unclear..., Gianesini [/bib_ref].
It has also been proposed that the origin of these extracranial intra-and extra-luminal venous structural abnormalities could be acquired, due to inflammatory, viral, bacterial, cardiovascular, degenerative and aging processes [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Multiple sclerosis: a chronic infective cerebrospinal venulitis?, Thibault [/bib_ref] [bib_ref] Risk factors for chronic cerebrospinal venous insufficiency (CCSVI) in a large cohort..., Dolic [/bib_ref] [bib_ref] Heart disease, overweight, and cigarette smoking are associated with increased prevalence of..., Dolic [/bib_ref]. In particular, it can be hypothesized that a lack of exercise, which is associated with higher disability present in numerous CNS diseases as well as in aging, may further contribute to the impairment of structural/morphological extracranial venous drainage pathways.
Pathological studies aimed to define the nature of these venous abnormalities or developmental variants are lacking [bib_ref] Altered collagen expression in jugular veins in multiple sclerosis, Coen [/bib_ref] [bib_ref] A technical approach to dissecting and assessing cadaveric veins pertinent to chronic..., Diaconu [/bib_ref]. Most recently, Diaconu et al. examined the IJVs, the BV and the azygos vein from 20 cadavers (10 controls and 10 MS patients) and concluded that the anatomy of the extracranial venous system has significant variability, including a differing number of valves in different regions and variable characteristics of the valves [bib_ref] A technical approach to dissecting and assessing cadaveric veins pertinent to chronic..., Diaconu [/bib_ref]. Coen et al. examined specimens from the IJVs of MS patients who underwent surgical reconstruction of the IJV, specimens of the great saphenous vein used for surgical reconstruction and specimens from patients without MS [bib_ref] Altered collagen expression in jugular veins in multiple sclerosis, Coen [/bib_ref]. Focal thickenings of the wall associated with a higher expression of type III collagen in the adventitia was detected in specimens of MS patients. It could be hypothesized that this focal thickening of the venous wall is associated with the vein wall not reacting to a given change in transmural pressure. This phenomenon can be detected with various imaging modalities, as reduced distensibility/pulsatility/paradox.
## Narrowing or occlusion of the venous drainage pathways
Restriction of the extracranial venous lumen may lead to abnormal narrowing, which represents a stenosis or even complete occlusion. The definition of "significant narrowing leading to stenosis of the major extracranial veins" is still arbitrary as no consensus guidelines are available at this time [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref]. The lumen of the extracranial veins is not constant and may exhibit considerable variability, depending on anatomical location. Usually, the presence of significant narrowing or stenosis is defined as venous lumen reduction ≥50% respect to the proximal adjacent vein segment, on magnetic resonance venography (MRV), catheter venography (CV) and intravascular ultrasound (IVUS) [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Assessment of jugular endovascular malformations in chronic cerebrospinal venous insufficiency: colour-Doppler scanning..., Scalise [/bib_ref] [bib_ref] The hypothesis of patho-physiological correlation between chronic cerebrospinal venous insufficiency and multiple..., Lugli [/bib_ref] [bib_ref] Using magnetic resonance imaging as a means to study chronic cerebral spinal..., Utriainen [/bib_ref]. However, the concept of a significant obstruction being when the vessel has been reduced to 50% of its diameter (which corresponds to a 75% reduction in cross-sectional area (CSA)) is derived mainly from observations in the arterial system [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref]. Therefore, these criteria may not be applicable to the venous system as there are some fundamental differences between the two. In addition, the diameter of the veins varies with the anatomical level of the vein, particularly in the IJVs. Therefore, more sophisticated qualitative and quantitative criteria are needed to adequately assess the significant narrowing of the extracranial veins. Finally, further research is needed to determine whether the concept of significant narrowing corresponds to the hemodynamic consequences for the intra-cranial venous drainage, as recently reported [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Magnetic resonance imaging signatures of vascular pathology in multiple sclerosis, Utriainen [/bib_ref]. For example, Traboloulsee et al.recently proposed that a hemodynamically significant narrowing of the extracranial vein on CV is present, if at least one of the following criteria is recorded: 1) reflux (persistent retrograde flow of most of the contrast bolus after injection is completed); 2) stasis (contrast present 4 s after the injection); or 3) abnormal collaterals (one or more vessels >50% the size of the adjacent primary vessel or two or more collateral vessels present at <50% the size of the adjacent primary vessel).
Narrowing or occlusion of the extracranial veins can be observed at any level and the presence of multiple stenotic lesions is frequently observed [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Kantarci [/bib_ref] [bib_ref] Extracranial Doppler sonographic criteria of chronic cerebrospinal venous insufficiency in the patients..., Simka [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref]. By far, the most frequently identified site of IJV venous structural/morphological abnormalities is at the region of the jugular valve just cephalad to the internal jugular confluence with the BV [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Kantarci [/bib_ref] [bib_ref] Extracranial Doppler sonographic criteria of chronic cerebrospinal venous insufficiency in the patients..., Simka [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref]. In the azygos vein, the most common location of narrowing is at the level of the azygos arch [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref].
Extracranial cerebral venous drainage pathway narrowing or occlusion is most frequently detected by single imaging modalities, including DS, MRV, CV or IVUS [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Using magnetic resonance imaging as a means to study chronic cerebral spinal..., Utriainen [/bib_ref] [bib_ref] Intravascular ultrasound in the diagnosis and treatment of chronic cerebrospinal venous insufficiency, Sclafani [/bib_ref] , although other non-invasive diagnostic techniques such as computed tomography venography and plethysmoghy are emerging as useful tools to study these abnormalities in a research setting [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Cerebral veins: comparative study of CT venography with intraarterial digital subtraction angiography, Wetzel [/bib_ref] [bib_ref] Cerebral venous outflow resistance and interpretation of cervical plethysmography data with respect..., Beggs [/bib_ref] [bib_ref] Assessment of cerebral venous return by a novel plethysmography method, Zamboni [/bib_ref].
Intra-luminal abnormalities A intra-luminal structural/morphological abnormality is defined on DS as an echogenic structure extending from the endothelial lining of the vein wall with or without associated hemodynamic changes (reflux, decreased/no flow and so on) [bib_ref] The chronic cerebrospinal venous insufficiency syndrome, Zamboni [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. These include abnormal valves, web, multiple septa and/or flaps located in a cluster. Flaps can be defined as thin linear echogenic structures extending from the endothelial lining of a vein wall, while septum is a thin linear echogenic structure extending from the endothelial lining of a vein wall and attached to it at both ends. The septum may extend across a vein to attach on opposing sides or attach on the same side and the membrane shows as membranous structure almost occluding the entire diameter of the vein [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. Webs represent multiple septae and/or flaps located in a cluster. In addition, Karmon et al. [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] described these on IVUS as intra-luminal hyperechoic filling defects and double parallel lumen of the veins. Various subtypes of malformed IJV valves have been reported, including fused leaflets, transverse leaflets, long leaflet, ectopic leaflet, accessory leaflet, inverted valves, sigmoid valves and double valves [bib_ref] A technical approach to dissecting and assessing cadaveric veins pertinent to chronic..., Diaconu [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref].
Intra-luminal venous abnormalities are found at proximal IJV just cephalad to the junction with BV by B mode of DS and IVUS [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Kantarci [/bib_ref] [bib_ref] Extracranial Doppler sonographic criteria of chronic cerebrospinal venous insufficiency in the patients..., Simka [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref] , while IVUS emerges as the most useful technique to detect intra-luminal abnormalities in the azygos vein [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Assessment of jugular endovascular malformations in chronic cerebrospinal venous insufficiency: colour-Doppler scanning..., Scalise [/bib_ref] [bib_ref] The hypothesis of patho-physiological correlation between chronic cerebrospinal venous insufficiency and multiple..., Lugli [/bib_ref] [bib_ref] Multimodal noninvasive and invasive imaging of extracranial venous abnormalities indicative of CCSVI:..., Zivadinov [/bib_ref]. There are no consensus guidelines with respect to the usefulness of CV for the detection of intra-luminal abnormalities. The recent position statement of The International Society for Neurovascular Disease (ISNVD) on the use of CV did not provide clear guidelines on this issue. However, it has been reported that the use of diluted angiographic contrast may allow a better visualization of these intra-luminal structures (valve leaflets, webs and so on), while the nondiluted contrast allows a better opacification of epidural and other collaterals, as well as a better estimation of overall features of the veins.
In addition, it is very difficult to estimate the real contribution of intra-luminal abnormalities to significant narrowing, as they can be easily displaced by the catheter or by an inflated balloon and upon deflation, fall right back in to their original position and continue to obstruct flow. It is also unknown at this time what the variations of these abnormalities are with respiratory, positional and activity changes.
The role of intra-luminal abnormalities in venous drainage impairment has to be defined according to the temporal evidence of altered brain drainage due to these abnormalities. For example, Dolic et al. reported that the presence and number of intra-luminal IJV malformations were related to a higher number of collateral veins and functional abnormalities [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. Of all intra-luminal abnormalities examined, the malformed valve (impaired mobility or thickened fibrotic valve), the septum and flap occurred most frequently in MS patients, as well as in healthy subjects [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref].
The prevalence of intra-luminal abnormalities is not firmly established in the general population. Dolic et al.
reported that a substantial number of MS patients (68%) and healthy subjects (49.2%) presented with at least one intra-luminal venous abnormality in their IJVs, as evidenced by DS [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. In the Prospective Randomized Endovascular therapy in Multiple Sclerosis (PREMiSe) study, Karmon et al. found that intra-luminal abnormalities can be even more frequent in the azygos vein of MS patients (85%), as evidenced by IVUS. Further invasive studies are, therefore, required to investigate the prevalence of intra-luminal abnormalities in a variety of CNS diseases and the general population as well as their impact on the hemodynamic consequences of intra-cranial venous drainage.
## Extra-luminal abnormalities
The extra-luminal structural/morphological abnormalities include narrowing and annulus [bib_ref] The chronic cerebrospinal venous insufficiency syndrome, Zamboni [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. As previously stated, the significant extra-luminal narrowing is considered a vessel that has been reduced to 50% of its diameter and that corresponds to a ≤0.3 cm 2 of CSA proximal IJV measurement on DS in the supine position [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. Annulus, a circumferential thickened vein wall that is restricting the vein from fully expanding with respiratory or positional changes, is another extra-luminal type of narrowing [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref].
The prevalence of extra-luminal abnormalities has only been anecdotally investigated. Dolic et al. reported that 22% of MS patients and 11.1% of healthy subjects presented with narrowing ≤0.3 cm 2 of CSA proximal IJV on DS in the supine position [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. In another recent invasive study, Traboulsee et al. performed a CV in 79 MS patients and 98 healthy controls in which they investigated >50% narrowing of the IJVs (valvular or non-valvular vein segment) in comparison with a normal reference segment (widest vein segment below the mandible) in Classification of the venous drainage pathways due to the extracranial structural/morphological, venous abnormalities Types [bib_ref] Embryological background of truncular venous malformation in the extracranial venous pathways as..., Lee [/bib_ref] [bib_ref] Diagnosis and treatment of venous malformations Consensus Document of the International Union..., Lee [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Assessment of jugular endovascular malformations in chronic cerebrospinal venous insufficiency: colour-Doppler scanning..., Scalise [/bib_ref] [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref] Definition Intra-luminal:
- Web: multiple septae and/or flaps located in a cluster.
This is an echogenic structure detected by DS or by IVUS extending from the endothelial lining of the vein wall with/without the presence of functional abnormality. Use of a diluted angiographic contrast may help identification of these abnormalities on CV. These abnormalities include web, flap, septum, membrane, hyperechoic filling defect, double parallel lumen and malformed valve.
- Flap: thin linear echogenic structure extending from the endothelial lining of a vein wall.
- Septum: thin linear echogenic structure extending from the endothelial lining of a vein wall and attached to it at both ends. The septum may extend across a vein to attach on opposing sides or attach on the same side.
- Membrane: membranous structure almost occluding the entire diameter of the vein.
- Hyperechoic filling defect: an eccentric hyperechoic crescent with a distinct sonographic signal, reminiscent of chronic organized thrombus.
- Double parallel lumen: multiple small channels in the venous wall.
- Malformed valve: dysdynamic or fibrous valve.
## Extra-luminal:
- Narrowing: presence of significant narrowing (defined as venous lumen reduction ≥50% respect to the proximal adjacent vein segment on CV or CSA measurement of proximal IJV ≤0.3 cm 2 on DS.
This is a restriction of the venous wall or narrowing detected on DS, CV, IVUS or MRV. These abnormalities include narrowing and annulus.
- Annulus: circumferential thickened vein wall that is restricting the vein from fully expanding with respiratory or positional changes.
- Vein wall not reacting to a given change in transmural pressure on CV, IVUS or DS; non-compliant.
Abnormal IJV distensibility/pulsatility/paradox: the supine position, and >50% narrowing of the azygos vein relative to the largest normal segment in the supine position. Therefore, the >50% narrowing on the CV was not assessed respect to the proximal adjacent vein segment measurement. Using these criteria, they found that 74% of MS patients, 70% of healthy controls and 66% of unaffected siblings of MS patients had >50% narrowing on the CV in at least one of these three extracranial veins. In addition, they reported that 51%, 54% and 45% of these narrowing, respectively, created hemodynamically abnormal flow, as defined by the CV criteria. Although this high rate of narrowing was described for the first time in healthy controls and while the authors concluded that venous narrowing is a common anatomical variant in healthy subjects, these data have to be interpreted with caution because of the narrowing criteria definition applied. Only longitudinal studies will be able to discern the real prevalence of extra-luminal abnormalities based on the demographic characteristics in different populations.
## Abnormal ijv distensibility/pulsatility/paradox
Vessel compliance describes the extent to which volume changes in response to a given change in transmural pressure. A venous wall not reacting to a given change in transmural pressure on CV, IVUS or DS is considered to be non-compliant . Venous compliance was studied in vitro and in vivo by plethysmography [bib_ref] The pressure/volume relationship of the calf: a measurement of vein compliance?, Neglen [/bib_ref] , DS [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] The effect of position and different manoeuvres on internal jugular vein diameter..., Armstrong [/bib_ref] [bib_ref] Venous myogenic tone: studies in human and canine vessels, Berczi [/bib_ref] [bib_ref] Non-invasive assessment of human large vein diameter, capacity, distensibility and ellipticity in..., Berczi [/bib_ref] [bib_ref] Effects of trendelenburg position and positive intrathoracic pressure on internal jugular vein..., Botero [/bib_ref] [bib_ref] Measurement of anterior-posterior diameter of inferior vena cava by ultrasonography: a new..., Duvekot [/bib_ref] [bib_ref] Cross-sectional area and intravascular pressure of the right internal jugular vein during..., Lobato [/bib_ref] and IVUS [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Assessment of jugular endovascular malformations in chronic cerebrospinal venous insufficiency: colour-Doppler scanning..., Scalise [/bib_ref] [bib_ref] The hypothesis of patho-physiological correlation between chronic cerebrospinal venous insufficiency and multiple..., Lugli [/bib_ref] [bib_ref] Intravascular ultrasound in the diagnosis and treatment of chronic cerebrospinal venous insufficiency, Sclafani [/bib_ref]. Those studies showed that large veins, compared with arteries, have a greater volume increment in response to increased transmural pressure, for example, a greater distensibility, within a wide-range of physiologic pressures. Chung [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref] used DS to measure the change in the vessel-lumen area of IJV during different grades of Valsalva maneuver (VM), which increases transmural pressure in IJV [bib_ref] Venous changes occurring during the Valsalva maneuver: evaluation by intravascular ultrasound, Attubato [/bib_ref] in patients with migraine and in healthy individuals. The venodilatation of IJV in response to each level of VM pressure in patients with migraine was significantly less than that in healthy individuals. The reproducibility of this method appears acceptable [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref]. Dolic et al. measured frequency and the number of paradox (vein wall not reacting to respiratory phase, non-compliant) using DS between healthy individuals and MS patients and found a relatively low prevalence (<1%) of these venous abnormalities in both groups [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref].
Karmon et al. [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] used IVUS to examine reduced respiratory pulsatility or normal pulsatility (presence or absence of expansion movements of the vein wall according to respiratory frequency (10 to 20/minute during deep inspiration and during VM)) to confirm the pathologic versus the physiologic nature of the vein narrowing. They found reduced pulsatility in 35% of right IJVs, 55% of left IJVs and 35% of the azygos vein in MS patients.
## Hemodynamic/functional venous abnormalities
The hemodynamic/functional abnormalities include venous reflux/bidirectional flow, abnormal flow, no flow and abnormal posture control of IJV flow.
The etiologies of continuous JVR include central venous obstruction, such as mediastinal goiter, mediastinal masses, aortic aneurysm or venous thrombosis (SVC syndrome) [bib_ref] Superior vena caval system obstruction caused by benign endothoracic goiter, Silverstein [/bib_ref] [bib_ref] Effect of obstructed mediastinal venous return on dynamic brain blood flow studies:..., Peart [/bib_ref] [bib_ref] Transient compression of the left innominate vein, Fred [/bib_ref] [bib_ref] Interpretation of 99 m Tc superior vena cavograms and results of studies..., Miyamae [/bib_ref] and one special anatomic factor occurring on the left side. Left BV has a more obtuse angle and a longer length before joining the superior vena cava than the right BV. Additionally, the left BV goes through the narrow space between the sternum and the thoracic outlet arteries before entering the superior vena cava. It may be that this narrow space can compress the left BV, causing narrowing of the lumen or even occlusion, resulting in left spontaneous JVR [bib_ref] Alteration of the cerebral bloodflow study due to reflux in internal jugular..., Steinbach [/bib_ref] [bib_ref] Compression of the left brachiocephalic vein: cause of high signal intensity of..., Tanaka [/bib_ref] [bib_ref] Cervical venous reflux: a normal variant of radionuclide cerebral blood flow study..., Lamoureux [/bib_ref]. A higher frequency of JVR in the elderly may be due to the more-frequent engorged thoracic outlet arteries in this population [bib_ref] Compression of the left brachiocephalic vein: cause of high signal intensity of..., Tanaka [/bib_ref].
VM-induced JVR, for example, IJV valve incompetence, is frequently seen in situations which have an elevated central venous pressure, such as congestive heart disease, tricuspid valve regurgitation, primary pulmonary hypertension and chronic obstructive pulmonary disease [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref] [bib_ref] Anatomic and pathophysiologic studies of the human internal jugular valve, Dresser [/bib_ref] [bib_ref] Internal jugular vein valve incompetence in COPD and primary pulmonary hypertension, Doepp [/bib_ref]. These conditions with chronic elevated venous pressure may damage the IJV valve gradually and make them incompetent. As with spontaneous JVR, VM-induced JVR is found more frequently at an older age [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref] [bib_ref] Jugular venous hemodynamic changes with aging, Chung [/bib_ref].
## Venous reflux/bidirectional flow
Venous reflux has been observed in the IJV, JV branches, VV, the azygos vein and in the intracerebral veins (basal veins of Rosenthal, superior and inferior petrosal sinus, and cavernous sinus, superior ophthalmic vein) by use of DS [bib_ref] Incompetence of internal jugular valve in patients with primary exertional headache: a..., Doepp [/bib_ref] [bib_ref] Reflux of jugular and retrobulbar venous flow in transient monocular blindness, Hsu [/bib_ref] [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Proposed chronic cerebrospinal venous insufficiency criteria do not predict multiple sclerosis risk..., Centonze [/bib_ref] [bib_ref] Multiple sclerosis: cerebral circulation time, Mancini [/bib_ref] [bib_ref] Jugular venous reflux, Chung [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Detection of intracranial venous reflux in patients of transient global amnesia, Chung [/bib_ref] [bib_ref] Flow volume in the jugular vein and related hemodynamics in the branches..., Chung [/bib_ref].
Valsalva maneuver induced jugular venous reflux Venous reflux in IJV (JVR) is the most commonly found venous hemodynamic abnormality which has been associated with certain CNS disorders. The pressure gradient determines the direction of flow in the veins [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] ; therefore, JVR indicates an abnormal (reversed) pressure gradient resulting from increased venous pressure proximally [bib_ref] Jugular venous reflux, Chung [/bib_ref]. When JVR results from elevated venous pressure proximal to the IJV valve, it is also known as IJV valve incompetence [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref]. In physiological situations, the most frequently encountered reversed pressure gradient is due to VM-like activities which increase intrathoracic pressure. These activities include coughing, defecating, sexual intercourse and heavy lifting, and so on. During these activities, JVR will happen if the IJV valve is incompetent. This kind of JVR could be detected by DS and IVUS during VM [bib_ref] Jugular venous reflux, Chung [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref]. Generally, VMinduced JVR is found more in the right IJV than in the left one [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref] [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref]. In a large IJV hemodynamic registry with a wide age range from a healthy population, the mean prevalence of VM-induced JVR is 26% and 12% in the right and left IJV, respectively [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref]. There is a higher frequency of VM-induced JVR in the elderly [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref] [bib_ref] Jugular venous hemodynamic changes with aging, Chung [/bib_ref]. In people younger than 40 and older than 70 years old, the prevalence of VM-induced JVR is 18% and 30%, respectively, in the right IJV, and 6% and 26%, respectively, in the left IJV [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref]. Patients with a chronic elevated central venous pressure, such as congestive heart disease [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref] [bib_ref] Anatomic and pathophysiologic studies of the human internal jugular valve, Dresser [/bib_ref] , tricuspid valve regurgitation [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref] [bib_ref] Cervical venous reflux: a normal variant of radionuclide cerebral blood flow study..., Lamoureux [/bib_ref] , primary pulmonary hypertension [bib_ref] Anatomic and pathophysiologic studies of the human internal jugular valve, Dresser [/bib_ref] and chronic obstructive pulmonary disease [bib_ref] Internal jugular vein valve incompetence in COPD and primary pulmonary hypertension, Doepp [/bib_ref] , also have higher frequency of VM-induced JVR.
Spontaneous venous reflux Besides VM-induced JVR, there is another kind of JVR, spontaneous JVR, which is detected spontaneously at rest. Central venous obstruction and dural arterio-venous fistula (AVF) should be considered in individuals with a continuous JVR. The causes of central venous obstruction producing continuous JVR include goiter, mediastinal masses, aortic aneurysm and venous thrombosis (superior vena cava syndrome) [bib_ref] Superior vena caval system obstruction caused by benign endothoracic goiter, Silverstein [/bib_ref] [bib_ref] Effect of obstructed mediastinal venous return on dynamic brain blood flow studies:..., Peart [/bib_ref] [bib_ref] Transient compression of the left innominate vein, Fred [/bib_ref] [bib_ref] Interpretation of 99 m Tc superior vena cavograms and results of studies..., Miyamae [/bib_ref]. Furthermore, continuous JVR is mostly reported on the left side because of the anatomic characteristics of the left BV that drains the left IJV [bib_ref] Alteration of the cerebral bloodflow study due to reflux in internal jugular..., Steinbach [/bib_ref] [bib_ref] Compression of the left brachiocephalic vein: cause of high signal intensity of..., Tanaka [/bib_ref] [bib_ref] Detection of intracranial venous reflux in patients of transient global amnesia, Chung [/bib_ref]. This phenomenon is reported in normal individuals with a frequency of 0.2 to 0.4% [bib_ref] Alteration of the cerebral bloodflow study due to reflux in internal jugular..., Steinbach [/bib_ref] [bib_ref] Cervical venous reflux: a normal variant of radionuclide cerebral blood flow study..., Lamoureux [/bib_ref]. Left JVR caused by this anatomic factor could reflux into the cerebral venous system as high as the level of basilar plexus via sigmoid sinus, transverse sinus and inferior petrosal sinus [bib_ref] Detection of intracranial venous reflux in patients of transient global amnesia, Chung [/bib_ref]. If there is another etiology for spontaneous, continuous JVR and for spontaneous intermittent JVR, it would need further evaluation.
Recently, Zamboni et al. introduced a quantitative definition of spontaneous venous reflux/bidirectional flow in the IJVs and/or in the VVs in sitting and in supine positions, as flow directed towards the brain for a duration of >0.88 s and incorporated it as one of the five venous hemodynamic (VH) criteria for the diagnosis of CCSVI. Using these criteria, Zamboni et al. investigated 65 MS patients and 235 controls composed, respectively, of healthy subjects, healthy subjects older than MS patients, patients affected by other neurological diseases and older controls not affected by neurological diseases but scheduled for CV by means of DS. They reported that 77% of MS patients and 0% of healthy controls (odds ratio 1,123) presented with spontaneous venous reflux/bidirectional flow in the IJVs [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref]. Using the same DS criteria, Zivadinov et al. reported that out of 289 MS patients and 163 healthy controls, 45% of MS patients and 20.2% of healthy controls presented with spontaneous venous reflux/bidirectional flow in the IJVs [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref]. However, Doeep et al., using the same DS criteria in a study involving 56 MS patients and 20 healthy controls, found that nobody presented with spontaneous venous reflux/bidirectional flow in the IJVs. The controversy regarding the methodological validity of the quantitative definition ofClassification for the extracranial hemodynamic/functional venous abnormalities Types [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Al-Omari [/bib_ref] [bib_ref] Multiple sclerosis: cerebral circulation time, Mancini [/bib_ref] [bib_ref] Extracranial venous stenosis is an unlikely cause of multiple sclerosis, Yamout [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Jugular venous reflux, Chung [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Assessment of jugular endovascular malformations in chronic cerebrospinal venous insufficiency: colour-Doppler scanning..., Scalise [/bib_ref] [bib_ref] The hypothesis of patho-physiological correlation between chronic cerebrospinal venous insufficiency and multiple..., Lugli [/bib_ref] [bib_ref] Magnetic resonance imaging signatures of vascular pathology in multiple sclerosis, Utriainen [/bib_ref] [bib_ref] Intravascular ultrasound in the diagnosis and treatment of chronic cerebrospinal venous insufficiency, Sclafani [/bib_ref] [bib_ref] Cerebral venous outflow resistance and interpretation of cervical plethysmography data with respect..., Beggs [/bib_ref] [bib_ref] Assessment of cerebral venous return by a novel plethysmography method, Zamboni [/bib_ref] Definition
## Venous reflux/bidirectional flow:
Valsalva maneuver induced jugular venous reflux:
- Valsalva maneuver-like activities which increase intrathoracic pressure may lead to IJV incompetence, known as jugular vein reflux and measured on DS or IVUS. spontaneous venous reflux has resulted in position statements from the ISNVD [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] , the European Society of Neurosonology and Cerebral Hemodynamics (ESNCH) [bib_ref] CCSVI and MS: a statement from the European Society of neurosonology and..., Baracchini [/bib_ref] and review studies [bib_ref] What went wrong? The flawed concept of cerebrospinal venous insufficiency, Valdueza [/bib_ref] ; all of which expressed considerable concerns regarding the accuracy of the proposed criterion. Zamboni et al. argued that the value of >0.88 s allows operators to differentiate between a physiologic and pathologic reflux, adopting this threshold value from a study that examined IJV valve insufficiency during a VM [bib_ref] Functional and morphological criteria of internal jugular valve insufficiency as assessed by..., Nedelmann [/bib_ref]. Valdueza et al. [bib_ref] What went wrong? The flawed concept of cerebrospinal venous insufficiency, Valdueza [/bib_ref] questioned the validity of this approach because the reference values gained during a VM do likely not apply to situations where the flow measurements take place in resting conditions. Nevertheless, this criterion has been widely-applied in recent studies aimed at determining the prevalence of CCSVI in patients with MS ( One of the important limits of DS for the detection of venous hemodynamic functional abnormalities is that the azygos vein cannot be directly imaged. While the specificity for detecting VV reflux on DS is high, the sensitivity is relatively low [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref]. In our opinion, there are currently no available noninvasive imaging methods that can depict venous reflux in the azygos vein. Therefore, further development of imaging techniques is needed in relation to the accurate detection of venous reflux in the azygos vein [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref].
In addition, using CV, Trabolusee et al. showed that >50% of MS patients and healthy controls showed hemodynamically abnormal flow in their IJVs and azygos vein, although they did not specify what was the exact prevalence of spontaneous reflux. Based on this conflicting information from invasive and non-invasive studies, there is a need to further investigate the real prevalence of spontaneous reflux according to the demographic characteristics in different populations, using both invasive and non-invasive imaging methods. The assessment of this criterion is particularly controversial because the quantification and direction of the blood flow in veins connecting the cortical veins with deep veins may vary considerably as a consequence of the physiologic inter-individual variation of the cerebral venous anatomy and methodological issues related to the use of DS [bib_ref] What went wrong? The flawed concept of cerebrospinal venous insufficiency, Valdueza [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] CCSVI and MS: a statement from the European Society of neurosonology and..., Baracchini [/bib_ref]. To avoid this issue, more sophisticated imaging techniques like fusion imaging technologyand quality Doppler profiles (QDP) were recently proposed; however, validation and applicability of those approaches remain unclear at this time.
## Venous reflux in the intracerebral veins
## Abnormal venous flow distribution in the extracranial veins
The measurement of blood flow, as well as velocity and blood volume, could be potentially more reliable in assessing the degree of venous outflow obstruction in the extracranial venous system.
IJV drains most of the cerebral venous blood flow during supine position [bib_ref] The vertebral venous plexus as a major cerebral venous outflow tract, Epstein [/bib_ref] [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Postural dependency of the cerebral venous outflow, Valdueza [/bib_ref] [bib_ref] Extrajugular pathways of human cerebral venous blood drainage assessed by duplex ultrasound, Schreiber [/bib_ref]. A DS study showed that a total jugular flow volume of more than two-thirds of the global cerebral arterial inflow volume is present in 72% of healthy individuals and that less than one-third of the global cerebral arterial inflow volume is found in only 6% of healthy individuals [bib_ref] How does the blood leave the brain? A systematic ultrasound analysis of..., Doepp [/bib_ref]. Mancini et al. used contrast-enhanced DS to assess cerebral circulation times (CCT) in MS patients and healthy subjects which showed that MS patients had a significantly prolonged CCT and more frequent retrograde flow in IJVs [bib_ref] Multiple sclerosis: cerebral circulation time, Mancini [/bib_ref]. Doepp et al. [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] reported that the decrease of total jugular blood volume flow on switching to the upright position was significantly less pronounced in MS patients, leading to significantly higher blood volume flow in the latter position. The meaning of these findings needs to be further explored but they were interpreted as an important sign of cerebral venous abnormality [bib_ref] Multiple sclerosis appears to be associated with cerebral venous abnormalities, Beggs [/bib_ref].
Another way to determine abnormal flow in the extracranial veins is to use phase-contrast MR angiography (PC-MRI) in order to measure blood flow and velocity [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Magnetic resonance imaging signatures of vascular pathology in multiple sclerosis, Utriainen [/bib_ref] [bib_ref] Venous and cerebrospinal fluid flow in multiple sclerosis: a case-control study, Sundstrom [/bib_ref]. Haacke et al. reported an abnormal flow distribution of IJV in patients with MS [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref]. A total jugular flow volume of less than two-thirds of the global cerebral arterial inflow (arterial/venous flow mismatch) was found more frequently than in the healthy individuals. Furthermore, in these MS patients, the arterial/ venous flow mismatch in the IJV stenotic group was significantly greater than the nonstenotic group. Therefore, this phenomenon of arterial/venous flow mismatch could be indicative of structural abnormalities in the main extracranial venous drainage pathway.
Karmon et al. used CV to estimate emptying time in MS patients [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref]. They found prolonged emptying time in MS patients with stenotic IJVs.
## No flow in the extracranial veins
The absence of flow in the IJV or/and VV in both the supine and sitting positions is mostly demonstrated by DS studies [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Screening for chronic cerebrospinal venous insufficiency (CCSVI) using ultrasound: recommendations for a..., Nicolaides [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Kantarci [/bib_ref] [bib_ref] Extracranial Doppler sonographic criteria of chronic cerebrospinal venous insufficiency in the patients..., Simka [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref]. MRV, IVUS and CV also have played an increasingly important role in diagnosing a lack of flow in the IJVs, VVs and azygos vein [bib_ref] A prospective open-label study of endovascular treatment of chronic cerebrospinal venous insufficiency, Zamboni [/bib_ref] [bib_ref] Progressive multiple sclerosis is not associated with chronic cerebrospinal venous insufficiency, Baracchini [/bib_ref] [bib_ref] Venous drainage in multiple sclerosis: a combined MRI and ultrasound study, Doepp [/bib_ref] [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Extracranial venous stenosis is an unlikely cause of multiple sclerosis, Yamout [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Value of MR venography for detection of internal jugular vein anomalies in..., Zivadinov [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Using magnetic resonance imaging as a means to study chronic cerebral spinal..., Utriainen [/bib_ref] [bib_ref] Magnetic resonance imaging signatures of vascular pathology in multiple sclerosis, Utriainen [/bib_ref] [bib_ref] Catheter venography and endovascular treatment of chronic cerebrospinal venous insufficiency, Mandato [/bib_ref].
## Abnormal posture control of ijv flow
Extracranial venous drainage is position-dependent [bib_ref] The vertebral venous plexus as a major cerebral venous outflow tract, Epstein [/bib_ref] [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Postural dependency of the cerebral venous outflow, Valdueza [/bib_ref] [bib_ref] Extrajugular pathways of human cerebral venous blood drainage assessed by duplex ultrasound, Schreiber [/bib_ref]. Extra-jugular venous pathways are responsible for cerebral venous outflow in the upright position when an IJV is collapsed due to both increased external pressure and decreased IJV venous pressure when upright [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] The effects of 10 degrees reverse trendelenburg position on ICP and CPP..., Tankisi [/bib_ref]. A negative ΔCSA represents the loss of the normal postural control, denoting a positive finding. Zamboni et al.
proposed an assessment of reverted postural control of the main cerebral venous outflow pathway by measuring the difference in the CSA of the IJVs in the supine and upright positions and reported a prevalence of 51% in MS patients and 11% in healthy controls [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref]. A number of other studies showed a substantially lower prevalence of this phenomenon in MS patients and healthy controls [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: clinical correlates from a multicentre..., Bastianello [/bib_ref] [bib_ref] Quantitative ColourDopplerSonography evaluation of cerebral venous outflow: a comparative study between patients..., Monti [/bib_ref] [bib_ref] Multiple sclerosis and CCSVI: a population-based case control study, Patti [/bib_ref]. Other techniques, like plethysmography have been proposed as methods for the assessment of venous obstruction based on an estimation of changes in venous capacitance and venous resistance by posture change [bib_ref] Cerebral venous outflow resistance and interpretation of cervical plethysmography data with respect..., Beggs [/bib_ref] [bib_ref] Assessment of cerebral venous return by a novel plethysmography method, Zamboni [/bib_ref].
## Venous abnormalities determined by composite criteria and multimodal imaging modalities
The venous system is a complex, low-pressure, freely communicating network of vessels that is often asymmetric and represents significantly more variability than extracranial arterial anatomy. Because of this, it is almost impossible to determine the relevance of any single reported finding or imaging modality criteria, when considered in isolation, regardless of the imaging modality or methodology utilized. Therefore, the use of composite criteria using uni-modal and multi-modal imaging modalities are emerging as potentially useful tools to identify and evaluate possible pathologies of the extracranial venous system [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Multimodal noninvasive and invasive imaging of extracranial venous abnormalities indicative of CCSVI:..., Zivadinov [/bib_ref].
## Chronic cerebrospinal venous insufficiency
In 2009, Zamboni et al. coined the term CCSVI introducing four extracranial and one intracranial VH criteria [bib_ref] A prospective open-label study of endovascular treatment of chronic cerebrospinal venous insufficiency, Zamboni [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref]. The VH DS criteria include: (1) reflux present in an outflow pathway (IJV and/or VV) with the head at 0°and 90°; (2) reflux in the intracranial veins/deep cerebral veins; (3) high resolution B-mode evidence of proximal IJV narrowing and/or other B-mode anomalies; (4) flow not detectable in the IJVs and/or VVs despite numerous deep inspirations; and (5) abnormal posture control of IJV flow. CCSVI was described as a vascular condition characterized by anomalies of the main extracranial veins, mainly in IJVs and azygos veins that interfere with normal venous outflow from the brain to the periphery, being specifically associated with MS [bib_ref] A prospective open-label study of endovascular treatment of chronic cerebrospinal venous insufficiency, Zamboni [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref].
CCSVI implies a pathological condition or disorder which is diagnosed using color DS of the extracranial (neck) -and intracerebral (deep cerebral) veins. A cutoff for CCSVI diagnosis classification consists of two or more abnormal DS VH criteria [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref]. The construct of the CCSVI cut-off is based on an arbitrary decision biased toward characteristics of the originally studied population and on the obtained results without further testing and validation of the datasets [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref]. The categorical variable construct of the CCSVI diagnosis may contribute to explaining major inconsistencies in the prevalence of findings of CCSVI between different studies [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref] [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Internal jugular vein morphology and hemodynamics in patients with multiple sclerosis, Al-Omari [/bib_ref] [bib_ref] Progressive multiple sclerosis is not associated with chronic cerebrospinal venous insufficiency, Baracchini [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: clinical correlates from a multicentre..., Bastianello [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency and venous stenoses in multiple sclerosis, Blinkenberg [/bib_ref] [bib_ref] Proposed chronic cerebrospinal venous insufficiency criteria do not predict multiple sclerosis risk..., Centonze [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency is not more prevalent in patients with mild..., Chambers [/bib_ref] [bib_ref] Multiple sclerosis: cerebral circulation time, Mancini [/bib_ref] [bib_ref] No cerebral or cervical venous insufficiency in US veterans with multiple sclerosis, Marder [/bib_ref] [bib_ref] The perfect crime? CCSVI not leaving a trace in MS, Mayer [/bib_ref] [bib_ref] Morphological and haemodynamic abnormalities in the jugular veins of patients with multiple..., Radak [/bib_ref] [bib_ref] Neck duplex Doppler ultrasound evaluation for assessing chronic cerebrospinal venous insufficiency in..., Zaniewski [/bib_ref] [bib_ref] Extracranial Doppler sonographic criteria of chronic cerebrospinal venous insufficiency in the patients..., Simka [/bib_ref] [bib_ref] Occurrence of CCSVI in patients with MS and its relationship with iron..., Van Den Berg [/bib_ref] [bib_ref] CoSMo Collaborative Study Group: Observational case-control study of the prevalence of chronic..., Comi [/bib_ref] [bib_ref] CoSMo Collaborative Study Group: Observational case-control study of the prevalence of chronic..., Comi [/bib_ref]. The prevalence of central CCSVI reading by three DS experts was 3.26% in MS patients, 3.1% in other neurological diseases and 2.13% in healthy controls. The overall CCSVI prevalence in the local readings was significantly higher, as compared to the first centralized reading (14.9% versus 3.2%; P<0.001) but there was no difference in the prevalence among the three study groups. Therefore, it can be concluded from these and other DS CCSVI studies [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] that given that multiple VH criteria are acquired, the reproducibility of the categorical CCSVI diagnosis depends on the training level, skills of the operator and reading criteria. Also to note, it is not easy to be blinded and standardized in either a research or clinical setting [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] CoSMo Collaborative Study Group: Observational case-control study of the prevalence of chronic..., Comi [/bib_ref] [bib_ref] The reproducibility of colour Doppler in chronic cerebrospinal venous insufficiency associated with..., Menegatti [/bib_ref]. Because of this, usefulness and applicability of these criteria in clinical research and practice is limited.
While the CCSVI diagnosis construct is based only on the DS criteria, Zamboni et al. performed CV in their original study and confirmed their DS findings in 65 MS patients and 48 healthy controls [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref]. They created the four patterns of venous obstruction, highly indicative of CCSVI, including narrowing of the proximal azygos vein and complete occlusion of one IJV (type A), narrowing of both IJVs and the proximal azygos vein (type B), bilateral narrowing IJVs only (type C) and azygos vein narrowing (type D). By using these CV patterns indicative of CCSVI, they were able to classify all MS patients into the particular CV patterns and none of the healthy controls [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref]. Most recently, Traboulsee et al. performed a study that investigated the same CV patterns in 79 MS patients and 98 healthy controls and found that only 2% of MS patients, 2% of unaffected siblings and 3% of unrelated healthy controls presented with these CV CCSVI patterns.
Based on this and other evidence [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] , the DS composite criteria-based diagnosis of CCSVI should be used with caution and cannot imply a pathological condition that requires an endovascular intervention. Screening and monitoring of the extracranial venous abnormalities using a combined non-invasive and invasive imaging approach should help establish the actual incidences and prevalence of this condition in various populations.
## Venous hemodynamic insufficiency severity score
To create a more comprehensive quantitative measure indicative of the severity of extracranial venous system drainage impairment that is not biased by categorical construct, Zamboni et al. introduced the venous hemodynamic insufficiency severity score (VHISS). VHISS is based on the sum of extracranial structural and hemodynamic venous abnormality VH criteria based parameters measured for each of the five CCSVI DS criteria examined [bib_ref] The severity of chronic cerebrospinal venous insufficiency in patients with multiple sclerosis..., Zamboni [/bib_ref]. VHISS ranges from 0 to 16. In a number of recent studies, VHISS showed a better relationship with other clinical and MRI outcomes, than did the diagnosis of CCSVI [bib_ref] The severity of chronic cerebrospinal venous insufficiency in patients with multiple sclerosis..., Zamboni [/bib_ref] [bib_ref] Clinical correlates of chronic cerebrospinal venous insufficiency in multiple sclerosis, Weinstock-Guttman [/bib_ref] [bib_ref] Vascular aspects of multiple sclerosis, D'haeseleer [/bib_ref] [bib_ref] Venous hemodynamics in neurological disorders: an analytical review with hydrodynamic analysis, Beggs [/bib_ref] [bib_ref] Cerebral blood perfusion changes in multiple sclerosis, Wuerfel [/bib_ref] [bib_ref] Decreased brain venous vasculature visibility on susceptibility-weighted imaging venography in patients with..., Zivadinov [/bib_ref]. For example, Weinstock-Guttman et al. showed that a CCSVI DS diagnosis was not associated with disability, as measured by the Expanded Disability Status Scale (EDSS) in MS patients, while the VHISS was related to the EDSS subscores [bib_ref] Clinical correlates of chronic cerebrospinal venous insufficiency in multiple sclerosis, Weinstock-Guttman [/bib_ref]. Therefore, quantitative composite criteria which reflect the total amount of extracranial venous abnormalities may be more useful in predicting clinical and other imaging outcomes in CNS disorders and aging than the categorical ones.
## Multimodal imaging application for detection of venous abnormalities
The discrepancy in the prevalence of extracranial venous abnormalities between different studies using non-invasive and invasive imaging techniques emphasizes the urgent need for the use of a multimodal imaging approach for better understanding of these venous abnormalities and developmental variants [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref]. The prevalence of venous abnormalities of the extracranial venous system is even higher, when investigated with sophisticated invasive imaging techniques [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Assessment of jugular endovascular malformations in chronic cerebrospinal venous insufficiency: colour-Doppler scanning..., Scalise [/bib_ref] [bib_ref] The hypothesis of patho-physiological correlation between chronic cerebrospinal venous insufficiency and multiple..., Lugli [/bib_ref] [bib_ref] Intravascular ultrasound in the diagnosis and treatment of chronic cerebrospinal venous insufficiency, Sclafani [/bib_ref]. A multi-modal imaging approach is recommended to determine the range of venous abnormalities and anatomic variants and to what extent they are present in various healthy and disease groups as well as disease conditions [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref]. Creation of multimodal imaging quantitative criteria that will incorporate structural and hemodynamic findings to describe extracranial abnormalities is the most important step toward understanding what is physiological and what is pathological.
## Compensatory mechanisms for venous abnormalities
From a biomechanical point of view, the presence of collateral flows is the strongest evidence for constricted principal venous pathways and venous hypertension. This is because increased up-stream blood pressure is required to open up (inflate) the collateral veins, by overcoming the elastic forces in the endothelia which would normally mean that the lumen of these vessels remains narrow. In subjects with IJV narrowing, prominent extra-jugular veins serving as collaterals have been demonstrated in many studies [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Venous adaptation following bilateral radical neck dissection with excision of the jugular..., Gius [/bib_ref] [bib_ref] Intracranial-pressure studies incident to resection of the internal jugular veins, Sugarbaker [/bib_ref] [bib_ref] Venous collateral blood flow assessed by Doppler ultrasound after unilateral radical neck..., Doepp [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Multimodal noninvasive and invasive imaging of extracranial venous abnormalities indicative of CCSVI:..., Zivadinov [/bib_ref]. While healthy individuals regularly present with extracranial venous collateral circulation, the presence of two or more collateral neck veins most likely represents a compensatory mechanism for impaired venous outflow Classification for the extracranial venous abnormalities determined by composite criteria or use of multimodal imaging with relative compensatory mechanisms Types [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Comparison of MR and contrast venography of the cervical venous system in..., Zaharchuk [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] The severity of chronic cerebrospinal venous insufficiency in patients with multiple sclerosis..., Zamboni [/bib_ref] Definition
Venous abnormalities determined by composite criteria and multimodal imaging modalities:
CCSVI:
- A cutoff for CCSVI diagnosis classification consists of two or more abnormal DS VH criteria.
## Vhiss:
- VHISS is based on the sum of extracranial venous abnormality VH criteria based parameters measured for each of the five CCSVI criteria examined and is ranging from 0 to 16.
Multimodal imaging application for detection of extracranial venous abnormalities
- Use of multimodal imaging criteria on DS, MRV, CV and IVUS to determine a significant narrowing of extracranial venous system with hemodynamic consequences for the intracranial venous drainage.
Compensatory mechanisms for venous abnormalities:
Collateral veins:
- The presence of two or more extracranial collateral veins and of epidural collateral veins may serve as an indirect sign of impaired venous outflow.
Legend: CCSVI, chronic cerebrospinal venous insufficiency; CV, catheter venography; DS, Doppler sonography; IVUS, intravascular ultrasound; MRV, magnetic resonance venography; VH, venous hemodynamic criteria; VHISS, venous hemodynamic insufficiency score.
because it bypasses blocked veins and thereby reduces resistance to drainage [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. The use of CV and MRV represents an excellent way for the assessment of the possible prominence or collateralization of the extracranial neck veins [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref]. Thoracic epidural collateralization was observed in MS patients with a narrowing (detected by IVUS or CV) [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Multimodal noninvasive and invasive imaging of extracranial venous abnormalities indicative of CCSVI:..., Zivadinov [/bib_ref]. The existence of collaterals in cases with no observed azygos vein narrowing may stem from the presence of intra-luminal abnormalities that are evident on IVUS but not on CV [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Multimodal noninvasive and invasive imaging of extracranial venous abnormalities indicative of CCSVI:..., Zivadinov [/bib_ref]. The presence of venous abnormalities may disrupt anterograde flow long enough that collaterals are recruited to compensate. Moreover, the presence of these extensive epidural collaterals may reflect venous hypertension in the cervical and thoracic spinal cord, a hallmark of the CCSVI hypothesis [bib_ref] Comparison of intravascular ultrasound with conventional venography for detection of extracranial venous..., Karmon [/bib_ref] [bib_ref] Multimodal noninvasive and invasive imaging of extracranial venous abnormalities indicative of CCSVI:..., Zivadinov [/bib_ref].
## Pathophysiology of extracranial venous abnormalities (theories and current evidence)
Studies and observations of diseases with inadequate cerebral arterial supply are extensive compared with those related to cerebral venous drainage disorders. The poor understanding of the pathophysiology may consequently underestimate the impact of cerebral venous drainage abnormalities in a variety of CNS disorders [bib_ref] What went wrong? The flawed concept of cerebrospinal venous insufficiency, Valdueza [/bib_ref] [bib_ref] Physiology of cerebral venous blood flow: from experimental data in animals to..., Schaller [/bib_ref] [bib_ref] Vascular aspects of multiple sclerosis, D'haeseleer [/bib_ref]. Consequently, there is a need for more basic science and clinical studies to increase our knowledge and understanding of the clinical association and pathophysiologies of cerebral venous drainage abnormalities. Here below, we report some of the presumed theories and current available evidence regarding the pathophysiologies of extracranial venous abnormalities.
## Decreased cerebral perfusion by increased cerebral venous pressure
An obstruction of the extracranial venous drainage pathways may reduce the supply of brain nutrients and potentially result in hypoxia. A hypoxia-like condition has been evidenced in patients with many neurodegenerative diseases, including MS. Therefore, local blood congestion and secondary hyperemia of the brain parenchyma may be related to extracranial venous hemodynamic abnormalities that result in increased cerebral venous pressure [bib_ref] Venous hemodynamics in neurological disorders: an analytical review with hydrodynamic analysis, Beggs [/bib_ref]. Nevertheless, it is not clear at this time whether reduced perfusion of the brain parenchyma in MS patients is a sign of vascular pathology, decreased metabolic demand [bib_ref] Cerebral blood perfusion changes in multiple sclerosis, Wuerfel [/bib_ref] or precipitated hemodynamic changes in the extracranial venous pathways [bib_ref] Decreased brain venous vasculature visibility on susceptibility-weighted imaging venography in patients with..., Zivadinov [/bib_ref] [bib_ref] Periventricular venous density in multiple sclerosis is inversely associated with T2 lesion..., Sinnecker [/bib_ref].
## Jugular venous reflux
Retrograde flow detected in IJV, for example, JVR, might cause cerebral venous drainage impairment. Without a competent IJV valve or with venous pressure higher than IJV valve's competence, JVR will occur [bib_ref] Jugular venous reflux, Chung [/bib_ref] [bib_ref] Venous hemodynamics in neurological disorders: an analytical review with hydrodynamic analysis, Beggs [/bib_ref]. The elevated venous pressure would cause retrograde transmission through IJVs into the cerebral venous system, which may increase cerebral venous pressure and then decrease cerebral perfusion pressure and cerebral blood flow (CBF), leading to cerebral venous ischemia [bib_ref] Brain hemodynamic changes associated with chronic cerebrospinal venous insufficiency are not specific..., Garaci [/bib_ref] [bib_ref] Jugular venous reflux, Chung [/bib_ref] [bib_ref] Determinants and clinical significance of jugular venous valve competence, Fisher [/bib_ref] [bib_ref] Venous hemodynamics in neurological disorders: an analytical review with hydrodynamic analysis, Beggs [/bib_ref] [bib_ref] Hypoperfusion of brain parenchyma is associated with the severity of chronic cerebrospinal..., Zamboni [/bib_ref] [bib_ref] Pathogenesis of leukoaraiosis: role of jugular venous reflux, Chung [/bib_ref]. The exact magnitude of increased cerebral venous pressure that would lead to altered CBF is unknown at this time. For example, Meyer-Schwickerath et al. investigated intracranial venous pressure by using ophthalmodynamometry in 29 MS patients, 28 healthy subjects and 19 cases with elevated intracranial pressure and found no evidence of increased intracranial pressure in MS patients or healthy controls [bib_ref] Intracranial venous pressure is normal in patients with multiple sclerosis, Meyer-Schwickerath [/bib_ref]. On the other hand, Beggs et al. reported that rapid discharging of the contents of the cortical veins might lead to a transient increase in pressure in the SSS of patients with MS [bib_ref] Cerebral venous outflow resistance and interpretation of cervical plethysmography data with respect..., Beggs [/bib_ref]. More research is needed to elucidate whether extracranial venous abnormalities may lead to increased venous pressure in the SSS.
After several clinical observations concerning JVR, Chung and Hu [bib_ref] Transient global amnesia: cerebral venous outflow impairment-insight from the abnormal flow patterns..., Chung [/bib_ref] [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref] [bib_ref] Reflux of jugular and retrobulbar venous flow in transient monocular blindness, Hsu [/bib_ref] [bib_ref] Jugular venous reflux, Chung [/bib_ref] [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref] [bib_ref] Jugular venous hemodynamic changes with aging, Chung [/bib_ref] [bib_ref] Detection of intracranial venous reflux in patients of transient global amnesia, Chung [/bib_ref] [bib_ref] Flow volume in the jugular vein and related hemodynamics in the branches..., Chung [/bib_ref] [bib_ref] Pathogenesis of leukoaraiosis: role of jugular venous reflux, Chung [/bib_ref] [bib_ref] Jugular venous reflux affects ocular venous system in transient monocular blindness, Chung [/bib_ref] [bib_ref] Jugular venous reflux could influence cerebral blood flow: a transcranial Doppler study, Wu [/bib_ref] have made efforts to provide more evidence supporting the theory that retrograde transmission of venous pressure by JVR has an impact on cerebral circulation. They studied healthy individuals and found that subjects with VMinduced JVR have wider retinal venular diameters and higher CBF decrement during VM compared to subjects without JVR [bib_ref] Jugular venous reflux affects ocular venous system in transient monocular blindness, Chung [/bib_ref] [bib_ref] Jugular venous reflux could influence cerebral blood flow: a transcranial Doppler study, Wu [/bib_ref]. These results imply that retrograde transmission of venous pressure by JVR could reach the cerebral venous system and decrease CBF respectively. They have also established an animal model of JVR to elucidate a more detailed pathophysiology of JVR [bib_ref] Validation of the jugular venous reflux animal model by threedimensional time-of-flight MRA..., Shieh [/bib_ref].
There is other evidence supporting the theory that JVR can cause harm to cerebral structures, especially to the WM [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref] [bib_ref] MRI and SPECT studies of dural arteriovenous fistulas presenting as pure progressive..., Waragai [/bib_ref] [bib_ref] Diffuse white matter changes caused by dural arteriovenous fistula, Yamakami [/bib_ref] [bib_ref] Dementia resulting from dural arteriovenous fistulas: the pathologic findings of venous hypertensive..., Hurst [/bib_ref]. Clinical reports of unilateral dural AVF with venous reflux from sigmoid sinus could produce bilateral diffuse cerebral WM abnormalities on MRI and hypoperfusion in these WM abnormalities on single-photon emission computed tomography [bib_ref] MRI and SPECT studies of dural arteriovenous fistulas presenting as pure progressive..., Waragai [/bib_ref] [bib_ref] Diffuse white matter changes caused by dural arteriovenous fistula, Yamakami [/bib_ref] [bib_ref] Dementia resulting from dural arteriovenous fistulas: the pathologic findings of venous hypertensive..., Hurst [/bib_ref]. Another clinical study of aged people also showed that the severity of age-related WM abnormalities (leukoraiosis) is associated with the severity of JVR which is not caused by AVF [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref].
Even in dural AVF, an additional precipitating factor, such as contralateral venous outflow obstruction, would be needed to exacerbate the severity of cerebral venous congestion and neurological deficits [bib_ref] Ligation of the jugular veins does not result in brain inflammation or..., Atkinson [/bib_ref] [bib_ref] Ultrasound study of the cranial venous system in the human new-born infant..., Cowan [/bib_ref] [bib_ref] Effects of bilateral jugular vein ligation on local cerebral blood flow, Sakata [/bib_ref]. For example, JVR needs other precipitating factors, which would cause cerebral vascular abnormalities, to be able to correlate with the severity of age-related WM abnormalities [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref]. The association between the presence of JVR and cough syncope is strengthened when there is an elevated level of circulatory endothelin 1, on which a strong vasoconstrictor may synergistically act on cerebral vessels and perfusion [bib_ref] Jugular venous reflux and plasma endothelin-1 are associated with cough syncope: a..., Chung [/bib_ref].
## Extracranial venous drainage obstruction
There are only a few clinical studies to evaluate the impact of extracranial venous drainage obstruction on cerebral circulation. Bilateral occlusion of IJV in infants has shown a decrease of extracranial artery inflow, most likely due to increased cerebral venous pressure and decreased perfusion pressure [bib_ref] Ultrasound study of the cranial venous system in the human new-born infant..., Cowan [/bib_ref]. Rat models with bilateral jugular vein occlusion showed a reversible decrease of CBF and no histopathological changes in the brain; however, this study only observed the effects within one week [bib_ref] Effects of bilateral jugular vein ligation on local cerebral blood flow, Sakata [/bib_ref]. A recent study used SJL mice with bilateral jugular vein ligation and the mice were observed for up to six months after ligation [bib_ref] Ligation of the jugular veins does not result in brain inflammation or..., Atkinson [/bib_ref]. Sham-operated mice and mice induced with experimental autoimmune encephalomyelitis were used as negative and positive controls, respectively. The authors did not identify changes in the brain-blood barrier (BBB) permeability, neuroinflammation, demyelination or clinical signs in the jugular vein ligation group compared to the sham group. Whether or not it does and how cerebral extracranial venous drainage pathway obstructions, such as narrowing/occlusion, influent cerebral circulation and structures contribute to the problem need more study.
Since prominent venous collaterals appear after occlusion of the principal venous drainage pathways in human and animal studies [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref] [bib_ref] Extrajugular pathways of human cerebral venous blood drainage assessed by duplex ultrasound, Schreiber [/bib_ref] [bib_ref] The anatomy of collateral venous flow from the brain and its value..., Andeweg [/bib_ref] [bib_ref] The physiologic significance of the vertebral venous plexus, Eckenhoff [/bib_ref] [bib_ref] Venous adaptation following bilateral radical neck dissection with excision of the jugular..., Gius [/bib_ref] [bib_ref] Intracranial-pressure studies incident to resection of the internal jugular veins, Sugarbaker [/bib_ref] [bib_ref] Venous collateral blood flow assessed by Doppler ultrasound after unilateral radical neck..., Doepp [/bib_ref] [bib_ref] Use of MR venography for characterization of the extracranial venous system in..., Zivadinov [/bib_ref] [bib_ref] Extracranial venous drainage patterns in patients with multiple sclerosis and healthy controls, Mctaggart [/bib_ref] [bib_ref] Patients with multiple sclerosis with structural venous abnormalities on MR imaging exhibit..., Haacke [/bib_ref] , it is reasonable to postulate that the capacity for the establishment of collaterals might play an important role in determining the impacts of extracranial venous drainage obstruction on cerebral circulation and structures.
As in JVR, additional precipitating factors may be needed in addition to extracranial venous drainage obstruction, in order for pathological effects to occur. For example, IJV compression by the lateral arch of C1 vertebra would cause cerebellar venous congestion and hemorrhage only under a long-term posture (head rotation to contralateral side with neck extension) for unilateral supratentorial craniotomy [bib_ref] Compression of the internal jugular vein by the transverse process of the..., Seoane [/bib_ref].
## Cerebral microvascular damage by cerebral venous hypertension
Cerebral venous hypertension would cause microvascular abnormalities, such as impaired arteriolar autoregulation and endothelial function, BBB damage, venular wall thickening, hyalinosis and possibly iron deposition [bib_ref] Dementia resulting from dural arteriovenous fistulas: the pathologic findings of venous hypertensive..., Hurst [/bib_ref] [bib_ref] Microcirculation after cerebral venous occlusions as assessed by laser Doppler scanning, Nakase [/bib_ref] [bib_ref] Local cerebral blood flow autoregulation following "asymptomatic" cerebral venous occlusion in the..., Nakase [/bib_ref] [bib_ref] Cerebral arteriovenous malformations, steal, and the hypertensive breakthrough threshold. An experimental study..., Morgan [/bib_ref] [bib_ref] Cerebral venous infarction: the pathophysiological concept, Schaller [/bib_ref] [bib_ref] Chronic superior sagittal sinus thrombosis with phlebosclerotic changes of the subarachnoid and..., Shintaku [/bib_ref]. To demonstrate whether extracranial venous drainage obstruction may elevate cerebral venous hypertension and lead to these microvascular abnormalities would need further studies. However, Beggs [bib_ref] Venous hemodynamics in neurological disorders: an analytical review with hydrodynamic analysis, Beggs [/bib_ref] and Dake et al. [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: a historical perspective, Dake [/bib_ref] postulated that extracranial venous drainage abnormalities may increase cerebral venous pressure and consequently cause microvascular endothelial activation as well as BBB damage, which might favor autoimmune leukocyte accumulation in cerebral vasculatures and invasion into the brain. This presumption would support that extracranial venous abnormalities may play a potential role in the pathophysiology of CNS disorders.
Altered cerebrospinal fluid flow dynamics, as consequence of impaired extracranial venous drainage Normal cerebrospinal fluid (CSF) circulation, in which homeostasis is maintained between the ultra-filtration of CSF (in the veins of the lateral ventricles) and clearance into the venous system at the level of the dural sinuses, depends on efficient extracranial venous drainage. Any occlusion of the extracranial venous drainage pathways is likely to induce hypertension in the venous sinuses [bib_ref] Multiple sclerosis: cerebral circulation time, Mancini [/bib_ref]. Increased pressure in the SSS can inhibit the absorption of CSF through the arachnoid villi, decrease CSF brain parenchyma drainage and induce hypoxic stress in the endothelia [bib_ref] Cine cerebrospinal fluid imaging in multiple sclerosis, Magnano [/bib_ref]. Moreover, after reopening of the extracranial veins drainage pathways by means of venous angioplasty in MS patients, significant improvement in the CSF flow were detected [bib_ref] Changes of cine cerebrospinal fluid dynamics in patients with multiple sclerosis treated..., Zivadinov [/bib_ref].
A recent hydrodynamic analysis by Beggs summarizes the relationship between extracranial venous abnormalities and increased CSF pulsatility dynamics and decreased CBF changes intracranially, which are commonly observed in conditions like leukoraiosis, normal-pressure hydrocephalus (NPH) and MS [bib_ref] Venous hemodynamics in neurological disorders: an analytical review with hydrodynamic analysis, Beggs [/bib_ref]. Given that NPH is associated with venous hypertension in the dural sinuses [bib_ref] Vascular compliance in normal pressure hydrocephalus, Bateman [/bib_ref] , it may be that impaired cerebral venous outflow alters the dynamics of the intracranial CSF system, irrespective of any pathology. In order to evaluate whether or not CCSVI is associated with changes in the dynamics of the intracranial CSF system, Beggs et al. undertook a study involving 51 age-matched healthy individuals (32 CCSVI negative and 19 CCSVI positive subjects) with no family history of MS [bib_ref] Aqueductal cerebrospinal fluid pulsatility in healthy individuals is affected by impaired cerebral..., Beggs [/bib_ref]. They found that net positive CSF flow was 32% greater in the CCSVI positive group compared with the CCSVI negative group indicating that CSF dynamics are altered in CCSVI positive healthy individuals, as demonstrated by increased pulsatility. This finding was accompanied by enlargement of the CSF spaces, suggesting that structural changes may be occurring in the brain parenchyma of CCSVI positive healthy individuals.
A recent article reported that natural sleep or anesthesia is associated with an increased flushing of the toxic material from the CNS, suggesting a new biological purpose for sleep. The authors found a 60% increase in the interstitial space during sleep, resulting in a striking increase in convective exchange of CSF with interstitial fluid. Alzheimer's disease (AD), the most common form of dementia in the elderly, is thought to be caused by an imbalance between amyloid-β (Aβ) production and clearance leading to Aβ accumulation in the CNS, which then causes neuronal damage and death manifesting as progressive clinical dementia [bib_ref] Decreased clearance of CNS beta-amyloid in Alzheimer's disease, Mawuenyega [/bib_ref]. Patients with AD have a 30% slower clearance of Aβ [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref]. One of the possible etiologies of decreased Aβ clearance may be related to decreased CSF flow due to narrowing of the extracranial venous system pathways, as recently suggested [bib_ref] Aqueductal cerebrospinal fluid pulsatility in healthy individuals is affected by impaired cerebral..., Beggs [/bib_ref] [bib_ref] Jugular venous reflux and white matter abnormalities in Alzheimer's disease: a pilot..., Chung [/bib_ref].
Because the venous drainage of the CNS is mostly driven by the IJVs in the supine position, the relationship between CSF flow clearance and the presence of extracranial venous abnormalities should be further explored in aging and neurodegenerative disorders.
## The role of precipitating risk factors for the extracranial venous abnormalities
Exploring the role of precipitating risk factors for extracranial venous abnormalities may help elucidate their pathophysiology [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Risk factors for chronic cerebrospinal venous insufficiency (CCSVI) in a large cohort..., Dolic [/bib_ref] [bib_ref] Heart disease, overweight, and cigarette smoking are associated with increased prevalence of..., Dolic [/bib_ref]. Dolic et al. studied 240 healthy individuals and found that the presence of heart disease, especially heart murmurs, obesity and cigarette smoking were associated with an increased prevalence of extracranial venous abnormalities. In another study, including 252 healthy individuals, they reported that a history of infectious mononucleosis and irritable bowel syndrome was associated with a diagnosis of CCSVI [bib_ref] Risk factors for chronic cerebrospinal venous insufficiency (CCSVI) in a large cohort..., Dolic [/bib_ref]. While, these results may imply that acquired cardiac valvular disease-related hemodynamic changes and inflammation (autoimmune or infection) may be involved in the pathophysiology of venous structural and hemodynamic venous abnormalities; no causality can be established without conducting prospective longitudinal observational studies [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref].
Evidence is mounting that the prevalence of extracranial venous abnormalities increases with aging [bib_ref] The role of noninvasive and invasive diagnostic imaging techniques for detection of..., Dolic [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref]. However, at this time, it is not clear whether an incidence of these abnormalities may differ over the lifetime or in relation to the disease states. For example, Dolic et al. used DS and MRV to study extracranial venous abnormalities in the IJVs of 150 MS patients and 63 healthy individuals. They reported that different structural and hemodynamic venous abnormalities were observed at different stages of MS disease [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. Based on these findings, they proposed a chronological development of venous abnormalities in which intra-luminal structure abnormalities develop first, followed by hemodynamic functional abnormalities and the development of venous compensatory response mechanisms (collaterals establishment). When this compensatory ability is overcome, extra-luminal abnormalities begin to form [bib_ref] Intra-and extraluminal structural and functional venous anomalies in multiple sclerosis, as evidenced..., Dolic [/bib_ref]. This theory is supported by a number of recent studies which found that extra-luminal venous abnormalities are very rare at MS disease onset but become more frequent in subjects with a longer MS duration [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] sensitivity, and specificity of chronic cerebrospinal venous insufficiency in MS, Zivadinov [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: clinical correlates from a multicentre..., Bastianello [/bib_ref] [bib_ref] Venous drainage in multiple sclerosis: a combined MRI and ultrasound study, Doepp [/bib_ref] [bib_ref] Sensitivity and specificity for screening of chronic cerebrospinal venous insufficiency using a..., Dolic [/bib_ref] [bib_ref] Extracranial venous stenosis is an unlikely cause of multiple sclerosis, Yamout [/bib_ref]. However, longitudinal observational studies will need to be conducted in order to prove or disprove the dynamic of extracranial venous system changes over time.
## Decreased ijv distensibility in migraine
Large veins have a great distensibility in response to increased transmural pressure, which helps keep venous pressure within a normal physiologic pressure [bib_ref] The effect of position and different manoeuvres on internal jugular vein diameter..., Armstrong [/bib_ref] [bib_ref] Venous myogenic tone: studies in human and canine vessels, Berczi [/bib_ref] [bib_ref] Non-invasive assessment of human large vein diameter, capacity, distensibility and ellipticity in..., Berczi [/bib_ref] [bib_ref] Effects of trendelenburg position and positive intrathoracic pressure on internal jugular vein..., Botero [/bib_ref] [bib_ref] Measurement of anterior-posterior diameter of inferior vena cava by ultrasonography: a new..., Duvekot [/bib_ref] [bib_ref] Cross-sectional area and intravascular pressure of the right internal jugular vein during..., Lobato [/bib_ref]. When IJV loses this compensation ability, it becomes prone to IJV venous hypertension, which might impair cerebral venous drainage or retrogradely transmit venous hypertension into cerebral circulation.
A decreased IJV distensibility in subjects with migraine was found compared to healthy individuals [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref]. Trigger factors, such as stress, sleep deprivation and menstrual cycle, are frequently found in patients with migraine [bib_ref] Migraine and tension-type headache in a general population: precipitating factors, female hormones,..., Rasmussen [/bib_ref] [bib_ref] Precipitating and aggravating factors of migraine versus tension-type headache, Spierings [/bib_ref] [bib_ref] Trigger factors of migraine and tension-type headache: experience and knowledge of the..., Wöber [/bib_ref]. Certain triggering factors of migraine would increase the sympathetic tone which could increase the venous tone and pressure [bib_ref] Influence of the menstrual cycle on sympathetic activity, baroreflex sensitivity, and vascular..., Minson [/bib_ref] [bib_ref] Impact of psychological factors on the pathogenesis of cardiovascular disease and implications..., Rozanski [/bib_ref] [bib_ref] Increased sympathetic and decreased parasympathetic cardiovascular modulation in normal humans with acute..., Zhong [/bib_ref]. It has been postulated that less compliant IJVs in subjects with migraine have less ability to compensate and alleviate increased IJV pressure by these triggering factors and, therefore, increased IJV pressure might transmit into cerebral venous structures and lead to a headache attack [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref].
## Associated central nervous system disorders and aging
A link between the presence and severity of extracranial venous abnormalities and several CNS disorders as well as aging are emerging. The described associations are mainly reported with JVR, CCSVI and abnormal distensibility vein conditions.
The central issue to be determined is whether structural/functional abnormalities and their developmental variations may play a potential role, as precipitating factors, in increased susceptibility for a number of CNS diseases.
## Associations with jugular venous reflux
Studies finding clinical associations between JVR and neurological disorders are emerging [bib_ref] Jugular venous reflux, Chung [/bib_ref].
## Inducible central nervous system disorders
CNS disorders induced by VM-like activities (for example, cough, straining and certain physical exercises, and so on) are found to be associated with VM-induced JVR (for example, IJV valve incompetence). These CNS disorders include transient global amnesia [bib_ref] Transient global amnesia: cerebral venous outflow impairment-insight from the abnormal flow patterns..., Chung [/bib_ref] [bib_ref] Detection of intracranial venous reflux in patients of transient global amnesia, Chung [/bib_ref] [bib_ref] Aetiology of transient global amnesia, Lewis [/bib_ref] [bib_ref] Disturbance of venous flow patterns in patients with transient global amnesia, Sander [/bib_ref] [bib_ref] Transient global amnesia: a clinical and sonographic study, Maalikjy Akkawi [/bib_ref] [bib_ref] Internal jugular vein valve incompetence and intracranial venous anatomy in transient global..., Schreiber [/bib_ref] , transient monocular blindness [bib_ref] Reflux of jugular and retrobulbar venous flow in transient monocular blindness, Hsu [/bib_ref] , cough, headache [bib_ref] Cough headache and thoracic inlet valvular competence in uremia, Chuang [/bib_ref] , exertional headache [bib_ref] Incompetence of internal jugular valve in patients with primary exertional headache: a..., Doepp [/bib_ref] and cough syncope [bib_ref] Jugular venous reflux and plasma endothelin-1 are associated with cough syncope: a..., Chung [/bib_ref] [bib_ref] Internal jugular vein valve insufficiency in cough syncope, Styczynski [/bib_ref]. JVR during VM-like activities causes retrograde transmission of pressure into cerebral venous circulation and causes transient cerebral venous hypertension and decreased CBF in certain brain regions and relevant neurological deficits.
## Age-related central nervous system disorders
Compared with inducible JVR, sustained JVR may cause sustained, elevated cerebral venous pressure and CBF decrement. Besides chronic hypoperfusion, chronic venous hypertension would cause venular wall thickening and activate inflammation in venular walls and perivenular tissues [bib_ref] Chronic superior sagittal sinus thrombosis with phlebosclerotic changes of the subarachnoid and..., Shintaku [/bib_ref] [bib_ref] Molecular basis of venous insufficiency, Schmid-Schonbein [/bib_ref]. In image and autopsy studies of chronic cerebral venous hypertension, diffuse WM changes, BBB damage and perivenular demyelinating were noted [bib_ref] Jugular venous reflux could influence cerebral blood flow: a transcranial Doppler study, Wu [/bib_ref] [bib_ref] Validation of the jugular venous reflux animal model by threedimensional time-of-flight MRA..., Shieh [/bib_ref] [bib_ref] MRI and SPECT studies of dural arteriovenous fistulas presenting as pure progressive..., Waragai [/bib_ref] [bib_ref] Diffuse white matter changes caused by dural arteriovenous fistula, Yamakami [/bib_ref] [bib_ref] Dementia resulting from dural arteriovenous fistulas: the pathologic findings of venous hypertensive..., Hurst [/bib_ref] [bib_ref] Neurological manifestations of intracranial dural arteriovenous malformations, Lasjaunias [/bib_ref] [bib_ref] Intracranial venous hypertension and the effects of venous outflow obstruction in a..., Bederson [/bib_ref] [bib_ref] Hemodynamic aspects of cerebral arteriovenous malformations, Nornes [/bib_ref].
Recently, it has been found that the severity of agerelated WM changes (leukoraiosis) is related to the severity of JVR, especially lesions in caudal brain regions (the occipital, basal ganglia and infratentorial regions) [bib_ref] More severe white matter changes in the elderly with jugular venous reflux, Chung [/bib_ref]. As mentioned above, the frequencies of both spontaneous and VM-induced JVR does increase with age [bib_ref] Jugular valve incompetence: a study using air contrast ultrasonography on a general..., Akkawi [/bib_ref] [bib_ref] Compression of the left brachiocephalic vein: cause of high signal intensity of..., Tanaka [/bib_ref] [bib_ref] Jugular venous hemodynamic changes with aging, Chung [/bib_ref]. JVR with a sustained (in spontaneous JVR) or long-term repetitive (in VM-induced JVR) retrogradetransmitted venous pressure into cerebral venous system would cause harm to cerebral vasculatures and tissues, which may accumulate with aging and lead to agerelated chronic cerebral hypoperfusion and consequently WM abnormalities [bib_ref] Pathogenesis of leukoaraiosis: role of jugular venous reflux, Chung [/bib_ref] [bib_ref] Jugular venous reflux affects ocular venous system in transient monocular blindness, Chung [/bib_ref] [bib_ref] Jugular venous reflux could influence cerebral blood flow: a transcranial Doppler study, Wu [/bib_ref]. Most recently, Chung et al. investigated whether JVR is associated with cerebral WM changes in 12 individuals with AD, 24 with mild cognitive impairment (MCI) and in 17 elderly ageand sex-matched controls [bib_ref] Jugular venous reflux and white matter abnormalities in Alzheimer's disease: a pilot..., Chung [/bib_ref]. The results of this study suggested that there may be an association between JVR and WM in AD patients, implying that cerebral venous outflow impairment may play a role in the dynamics of WM changes/formation in AD patients, particularly in the periventricular regions. Whether or not JVR plays a role in other neurological diseases associated with agerelated cerebral circulatory insufficiency, is a question to be answered in future longitudinal studies.
## Associations with chronic cerebrospinal venous insufficiency (ccsvi)
CCSVI was initially described in the context of MS [bib_ref] Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, Zamboni [/bib_ref] [bib_ref] The value of cerebral Doppler venous haemodynamics in the assessment of multiple..., Zamboni [/bib_ref]. It gained quick popularity among MS patients because of the postulated possibility of venous insufficiency correction using endovascular procedures [bib_ref] Assessment of cerebral venous return by a novel plethysmography method, Zamboni [/bib_ref]. However, it became clear with the first controlled studies that CCSVI is not the cause of MS and can be present in healthy individuals and patients with other neurologic diseases [bib_ref] No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset, Baracchini [/bib_ref] [bib_ref] No cerebrocervical venous congestion in patients with multiple sclerosis, Doepp [/bib_ref] [bib_ref] Proposed chronic cerebrospinal venous insufficiency criteria do not predict multiple sclerosis risk..., Centonze [/bib_ref] [bib_ref] No cerebral or cervical venous insufficiency in US veterans with multiple sclerosis, Marder [/bib_ref] [bib_ref] The perfect crime? CCSVI not leaving a trace in MS, Mayer [/bib_ref] [bib_ref] Decreased brain venous vasculature visibility on susceptibility-weighted imaging venography in patients with..., Zivadinov [/bib_ref]. The major amount of knowledge regarding MS points toward immune etiopathogenesis [bib_ref] Multiple sclerosis-the plaque and its pathogenesis, Frohman [/bib_ref]. A number of recent studies examined a cause-and-effect relationship between MS and CCSVI by applying the so-called Bradford Hill criteria [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Ghezzi [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency and multiple sclerosis, Bagert [/bib_ref]. The Bradford Hill criteria examines the strength of the association, the consistency, the specificity, the temporality, the biological gradient and plausibility, the coherence, the experiment and the analogy between the two phenomena [bib_ref] The environment and disease: association or causation, Hill [/bib_ref]. In the case of MS and CCSVI, all of these criteria are partially or not fulfilled [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: diagnostic, pathogenetic, clinical and treatment..., Zivadinov [/bib_ref] [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Ghezzi [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency and multiple sclerosis, Bagert [/bib_ref]. However, the precipitating role of extracranial venous abnormalities in facilitating immune attack, mediated by host-viruses in genetically predetermined individuals, cannot be excluded, in our opinion at this time completely, and should be further investigated. However, it could also be that reduced perfusion in MS patients may exert a precipitating role in inducing structural/functional changes of the extracranial venous system.
The CCSVI hypothesis has provoked great controversy and debate in the MS research community since it was first presented [bib_ref] Chronic cerebrospinal venous insufficiency and multiple sclerosis, Khan [/bib_ref] [bib_ref] CCSVI and MS: no meaning, no fact, Baracchini [/bib_ref] [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Ghezzi [/bib_ref] [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Hutchinson [/bib_ref] [bib_ref] Media, politics and science policy: MS and evidence from the CCSVI trenches, Pullman [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: the final curtain, Paul [/bib_ref] [bib_ref] Multiple sclerosis: CCSVI deconstructed and discarded, Barkhof [/bib_ref] [bib_ref] Funding CCSVI research is/was a waste of valuable time, money and intellectual..., Zivadinov [/bib_ref]. Whether CCSVI is a syndrome or condition that is primarily characterized by symptoms, such as headache, fatigue, sleep disturbances, autonomic dysfunctions and so on, that can be improved using endovascular treatment and possibly independent from the other underlying disease process is unclear at this time [bib_ref] Role of venoplasty for treatment of multiple sclerosis: value of open-label studies..., Zivadinov [/bib_ref].
Many MS patients have undergone endovascular treatment for CCSVI procedures in either an open-label or private care setting [bib_ref] Role of venoplasty for treatment of multiple sclerosis: value of open-label studies..., Zivadinov [/bib_ref]. The most important driver of this momentum has been a tremendous patient advocacybased response in support of the widespread availability of venous angioplasty. Many patients with a desire to achieve a cure for this chronic, severely disabling malady have traveled far and wide to receive treatments from surgeons specializing in the venous angioplasty procedure. As with many yet unproven therapies, safety and efficacy concerns have been raised [bib_ref] Media, politics and science policy: MS and evidence from the CCSVI trenches, Pullman [/bib_ref] [bib_ref] Chronic cerebrospinal venous insufficiency in multiple sclerosis: the final curtain, Paul [/bib_ref] [bib_ref] Multiple sclerosis: CCSVI deconstructed and discarded, Barkhof [/bib_ref] [bib_ref] Complications in MS patients after CCSVI procedures abroad (Calgary, AB), Burton [/bib_ref] [bib_ref] Serious complication of percutaneous angioplasty with stent implantation in so called "chronic..., Dolezal [/bib_ref] [bib_ref] Endovascular treatment for chronic cerebrospinal venous insufficiency: is the procedure safe?, Ludyga [/bib_ref] [bib_ref] Safety of endovascular treatment of chronic cerebrospinal venous insufficiency: a report of..., Mandato [/bib_ref] [bib_ref] Safety profile of endovascular treatment for chronic cerebrospinal venous insufficiency in patients..., Petrov [/bib_ref] [bib_ref] Endovascular treatment of chronic cerebrospinal venous insufficiency in multiple sclerosis: a retrospective..., Alroughani [/bib_ref] [bib_ref] MS Study Group-Italian Society of Neurology: Adverse events after endovascular treatment of..., Ghezzi [/bib_ref] [bib_ref] MS Study Group-Italian Society of Neurology: Endovascular treatment of CCSVI in patients..., Ghezzi [/bib_ref] [bib_ref] Clinical improvement after extracranial venoplasty in multiple sclerosis, Hubbard [/bib_ref] [bib_ref] Disability caused by multiple sclerosis is associated with the number of extra..., Denislic [/bib_ref] without properly designed clinical trials [bib_ref] Role of venoplasty for treatment of multiple sclerosis: value of open-label studies..., Zivadinov [/bib_ref] [bib_ref] Clinical improvement after extracranial venoplasty in multiple sclerosis, Hubbard [/bib_ref] [bib_ref] Disability caused by multiple sclerosis is associated with the number of extra..., Denislic [/bib_ref]. An unknown number of MS patients have reportedly suffered serious adverse events, including stroke and death. For example, Ghezzi et al. reported in a retrospective study severe adverse events after endovascular treatment in 15 of 462 subjects (3.3%) at a variable interval after the procedure [bib_ref] MS Study Group-Italian Society of Neurology: Adverse events after endovascular treatment of..., Ghezzi [/bib_ref]. These included jugular thrombosis in seven patients, and tetraventricular hydrocephalus, stroke, paroxysmal atrial fibrillation, status epilepticus, aspiration pneumonia, hypertension with tachycardia or bleeding of bedsores in the remaining seven cases. One patient died because of myocardial infarction 10 weeks after the procedure. Therefore, the risk of severe adverse events related to endovascular treatment for CCSVI must be carefully considered.
A number of uncontrolled endovascular studies reported subjective physical and quality of life improvements in MS patients after endovascular treatment for CCSVI [bib_ref] Clinical improvement after extracranial venoplasty in multiple sclerosis, Hubbard [/bib_ref] [bib_ref] Disability caused by multiple sclerosis is associated with the number of extra..., Denislic [/bib_ref] [bib_ref] Is chronic fatigue the symptom of venous insufficiency associated with multiple sclerosis?..., Malagoni [/bib_ref]. However, no objective evidence of improvement is available at this time [bib_ref] Multiple sclerosis: CCSVI deconstructed and discarded, Barkhof [/bib_ref]. It is our view that the association between CCSVI and MS can only be studied in blinded, randomized, controlled clinical trials that will assess the benefits of endovascular interventions according to established clinical (annualized relapse rate, sustained disability progression), MRI (lesion activity and brain atrophy) and quality-of-life treatment outcomes. However, only safe and ethical approaches should be encouraged in designing new clinical trials.
## Associations with abnormal extracranial vein distensibility
Chung and Hu [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref] found that patients with common migraine have decreased venodilatation of IJV in response to each level of VM pressure compared with healthy individuals, which may play a role in the pathophysiology of migraine [bib_ref] Decreased jugular venous distensibility in migraine, Chung [/bib_ref].
# Conclusions and perspectives
The classification of the presence and severity of extracranial venous abnormalities/developmental variants by imaging and pathology findings should be the first step in the determination of their role in the pathology of CNS disorders and aging. The extracranial venous abnormalities could be classified as structural/morphological, hemodynamic/functional and those determined only by the composite criteria and use of multimodal imaging.
One of the central issues to be further investigated is the definition of significant narrowing leading to stenosis of the major extracranial veins. The current definition (narrowing of >50%) respect to the proximal adjacent vein segment is mainly derived from observations in the arterial system. Even more important is to establish what constitutes a significant narrowing of extracranial venous system with hemodynamic consequences for the intracranial venous drainage. More sophisticated and validated quantitative single or composite multimodal imaging criteria are needed to adequately assess the clinical impact of significant narrowing with hemodynamic consequences of the extracranial veins for the CNS pathology. Because disruption of normal flow is associated with prominent collateral circulation as the main compensatory mechanism, this has to be taken into account when determining the impact of significant narrowing.
The etiology and pathophysiology of extracranial venous abnormalities in relation to aging or the development of other CNS comorbidities should be further investigated. Pathological and imaging approaches need to investigate the origin of extracranial venous abnormalities. It is necessary to determine the incidence and prevalence of extracranial venous abnormalities in relation to embryologic/developmental arrest factors, demographic factors (such as age, sex, race), cardiovascular risk factors (smoking, obesity, hypertension, diabetes, hyperlipidemia), inflammatory comorbidities and other possible precipitating risk factors, such as one's level of exercise and diet. Only properly designed, safe and ethical studies should be encouraged in collecting this longitudinal observational information.
While some CNS disorders have been linked to the presence and severity of JVR and CCSVI, the ultimate cause-consequence relationship has not been firmly established. CCSVI triggered great interest and debate, highlighting the need for a better understanding of the role of extracranial venous abnormalities but many questions remain unanswered at this time. The use of endovascular treatment for the correction of these extracranial venous abnormalities should be discouraged until the potential benefit is demonstrated in properly-designed blinded, randomized and controlled clinical trials. Competing interest Robert Zivadinov received personal compensation from Teva Neuroscience, Biogen Idec, EMD Serono, Bayer, Genzyme-Sanofi, Novartis, Claret and General Electric for speaking and consultant fees. He received financial support for research activities from Biogen Idec, Teva Neuroscience, Genzyme-Sanofi, Novartis and EMD Serono. Chih-Ping Chung has no conflict of interest to report.
Authors' contributions RZ and C-PC conceptualized and designed the study, performed literature research, drafted the manuscript and revised it critically. They approved the final version of the manuscript and both serve as guarantors of the study.
[fig] Figure 1: Intracranial venous system anatomy of dural sinuses, cortical veins, deep intracerebral veins and cavernous sinus. The figure was reproduced with permission from the Radiology Assistant website: (http://www.radiologyassistant.nl/en/p4befacb3e4691/cerebral-venousthrombosis.html). [/fig]
[fig] Figure 2: Illustration depicting the predominant veins and sinuses involved in the craniocervical venous outflow. Venous narrowing is depicted at locations of interest in chronic cerebrospinal venous insufficiency. The figure was reproduced with permission from Lazzaro MA, Zaidat OO, Mueller-Kronast N, Taqi MA, Woo D. Endovascular therapy for chronic cerebrospinal venous insufficiency in multiple sclerosis. Front Neurol 2011, 2:44. [/fig]
[fig] Figure 3: Paired anterior cardinal veins form common cardinal veins with paired posterior cardinal veins, draining centrally into the sinus venosus (sinus horns) as depicted (top). Paired anterior cardinals soon form an anastomosis between them; the connection grows from the left to the right anterior cardinal vein to form the left brachiocephalic (innominate) vein (bottom). The left anterior cardinal vein distal (cranial) to the anastomosis becomes the 'left internal jugular vein,' while the left anterior cardinal vein proximal to the brachiocephalic anastomosis regresses/atrophies to become the base of the 'coronary sinus' of the heart as displayed. The right anterior cardinal (precardinal) vein proximal to the right brachiocephalic vein forms the superior vena cava (SVC) with the common cardinal, and terminal/proximal segment of the posterior cardinal (postcardinal) vein. The figure was reproduced with permission from Lee BB: Venous embryology: the key to understanding anomalous venous conditions. Phlebolymphology 2012, 4:170-181. [/fig]
[fig] *: Spontaneous venous reflux: • Present on DS examination in the IJV and vertebral veins and for more than 0.88 seconds with the head at 90°and 0°; delayed emptying time on CV. Venous reflux in the intracerebral veins: • Reflux/bidirectional flow on DS in the deep cerebral veins is defined as reverse flow for a duration of 0.5 s in one of the intra-cranial veins. Abnormal venous flow distribution in extracranial veins: • Measurement of blood flow, blood volume and blood velocity by using DS, MR phase contrast imaging, CV or IVUS. No flow in extracranial veins: • No flow on DS or IVUS or contrast noted in the vein on CV and MRV, despite deep breaths. Abnormal posture control of IJV flow: • A negative ΔCSA on DS represents the loss of the normal postural control; altered estimation of changes in venous capacitance and venous resistance by posture change on plethysmography.Legend: CSA. cross sectional area; CV, catheter venography; DS, Doppler sonography; IJV, internal jugular vein; IVUS, intravascular ultrasound; MRV, magnetic resonance venography. The controversy regarding the methodological validity of these quantitative definitions for spontaneous and intracerebral venous reflux included recent position statements from the ISNVD[97], the European Society of Neurosonology and Cerebral Hemodynamics (ESNCH)[132] and review studies[7] that expressed considerable concerns regarding the accuracy of the proposed criterion. [/fig]
[table] Table 2: [22].Zivadinov et al. reported a prevalence of 46.8% in MS patients and 12.7% in healthy controls [26], while Doepp et al. showed that no healthy controls and only one of 56 MS patients presented with this DS criterion. [/table]
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Elevated risk of early reoperation in total hip replacement during the stage of unit closure
A population-based registry study of total hip and knee replacements in Finland,
Background and purpose -The effects of launch or closure of an entire arthroplasty unit on the first or last patients treated in these units have not been studied. Using a 3-year follow-up, we investigated whether patients who were treated at the launch or closure stage of an arthroplasty unit of a hospital would have a higher risk of reoperation than patients treated in-between at the same units.
Patients and methods -From the Finnish Arthroplasty Register, we identified all the units that had performed total joint arthroplasty and the units that were launched or closed in Finland between 1998 and 2011. The risks of reoperation within 3 years for the 41,748 total hip and knee replacements performed due to osteoarthritis in these units were modeled with Cox proportional-hazards regression, separately for hip and knee and for the launch and the closure stage.
Results -The unadjusted and adjusted hazard ratios (HRs) for total hip and knee replacements performed in the initial stage of activity of the units that were launched were similar to the reoperation risks in patients who were operated in these units after the early stage of activity. The unadjusted and risk-adjusted HRs for early reoperation after total hip replacement (THR) were increased at the closure stage (adjusted HR = 1.8, 95% CI: 1.2-2.8). The reoperation risk at the closure stage after total knee replacement (TKR) was not increased.
Interpretation -The results indicate that closure of units performing total hip replacements poses an increased risk of reoperation. Closures need to be managed carefully to prevent the quality from deteriorating when performing the final arthroplasties. How good are hospitals at maintaining the quality of a treatment they offer over time? Quality of care may be perceived as a relatively stable characteristic, but at some stage the quality of care could deviate from the hospital's average. For example, variations in the quality of an activity might appear when a specific treatment is launched or when it is being discontinued in a hospital.
The initiation of total joint replacement surgery in a new setting might entail a learning curve, manifest as an increased risk of early reoperation in the initial stage of the unit's functioning. To initiate replacement surgery in a hospital, the team members must be recruited and the team may have a learning curve [bib_ref] Individual experience and experience working together: predicting learning rates from knowing who..., Reagans [/bib_ref]. It would presumably take time for the team to reach full efficiency. In the event that a hospital decides to close a unit performing replacement surgery, members of the surgical team might lose their motivation or seek other employment opportunities, thus leading to a high turnover of the members of the surgical team. As a consequence of this, the quality of care might be affected [bib_ref] The effects of hospital restrucvturing that included layoffs on individual nurses who..., Cummings [/bib_ref] [bib_ref] A Review of physician turnover: rates, causes, and consequences, Misra-Hebert [/bib_ref] [bib_ref] Reviewing the benefits of health workforce stability, Buchan [/bib_ref].
We investigated whether patients who underwent total joint replacement in the launch or closure phase between 1998 and 2011 had a higher risk of reoperation within 3 years than patients who were operated on in the same units after the launch phase or before the closure phase. In addition, we studied the performance of all the units that carried out total joint replacements in Finland, and evaluated the performance of the units that opened or closed in relation to units with continuous total joint replacement surgery over the period 1998-2011.
# Patients and methods
## Data
We gathered individual-level administrative data on all total hip and knee replacements performed in Finland between 1996 and 2013 from several registers. The Finnish Arthroplasty Register (FAR) was used to identify all total hip and knee replacements, and the patients' hospital discharge records from the beginning of 1987 to the end of 2013 were extracted from the Finnish Hospital Discharge Register (FHDR). The FAR and FHDR covered all units, both public and private, that performed total joint replacements in the study period. In addition, data on the patients' purchases of prescribed medications and special reimbursement decisions were gathered from the administrative databases of the Social Insurance Institution (SII) for the period 1998-2013.
The individual-level data on operations, which were used in analysis of the risk of reoperation in the 3 years after the primary surgery, only included operations performed due to osteoarthritis in the period 1998-2011. We also used the individual-level data collected for the Performance, Effectiveness, and Costs of Treatment Episodes (PERFECT) project (Häkkinen 2011, [bib_ref] Regional and hospital variance in performance of total hip and knee replacements:..., Mäkelä [/bib_ref]. According to the PERFECT study protocol, patients who had previously presented with symptoms indicating possible mechanisms other than primary osteoarthritis as the cause of surgery were excluded (diagnoses are listed in [bib_ref] Regional and hospital variance in performance of total hip and knee replacements:..., Mäkelä [/bib_ref]. Patients who were not Finnish citizens or who were residents of the Åland Islands were also excluded from the individual-level study data, since their use of the hospital service could not be tracked reliably using the Finnish registries.
Reoperations-including revisions of the joint, implant removals, and (in knee cases) the addition of a patellar part-were tracked using both the FAR and the FHDR until December 31, 2013. From the FHDR, the codes NFC* and NGC* of the Finnish version of the NOMESCO classification of surgical procedures (meaning secondary prosthetic replacement of the hip and knee joint) were used to identify the reoperations. Patients suffering from heart failure (WHO International Classification of Diseases, tenth edition (ICD-10) codes I50*), coronary heart disease (I20* to I25*), atrial fibrillation (I48*), hypertension (I10* to I15*, diabetes (E10* to E14*), psychotic disorders (F20* to F31*), cancer (C00* to C99*), or depression (F32* to F34*) were identified based on diagnoses in the hospital discharge records and the special reimbursements for medications for these diseases issued by the SII prior to surgery. For a more detailed description of the registry methodology used, see [bib_ref] A methodological approach for register-based evaluation of cost and outcomes in health..., Peltola [/bib_ref]. For an analysis of the effects of the selected comorbid conditions on reoperation risk, see [bib_ref] Comorbid diseases as predictors of survival of primary total hip and knee..., Jämsen [/bib_ref].
## Definitions of launched and closed units, and the stage of surgery
For each unit performing arthroplasty in Finland, we calcu-lated the number of total hip and knee replacements the units had performed each year between 1997 and 2012, using the FAR. We considered a unit that performed total hip or knee replacements in any year between 1998 and 2011, but that had not performed these surgeries in the previous year, to be a unit that had newly opened. Likewise, a unit that did not perform total hip or knee replacement in the year after any of the years 1998-2011 was considered to have been discontinued.
For the launched and closed units that performed at least 200 total joint replacements between 1998 and 2011, we analyzed reoperation risk within 3 years of the primary surgery (using the individual-level data), which is referred to from here on as risk of early reoperation. For the units that were launched or closed, the total hip and knee replacements performed for any reason were assigned order numbers within each unit, based on the date of surgery.
In the units that were launched, the early (or launch) stage was taken to cover the first 100 total hip or knee replacements of the unit. Similarly, the closure stage in the units that were closed was taken to cover the last 100 surgeries in the unit. Our choice of the first or last 100 surgeries was arbitrary, but as the numbers of units that had been launched or closed were presumably low, the data would not permit the use of a continuous-or more finely classified-launch or closure stage.
For all surgeries in the units that were launched, we compared the risk of early reoperation in patients who were operated on in the early stage of the unit's functioning with that for surgeries performed in these units after the early stage of functioning. In the individual-level analyses of the units that were closed, the risk of early reoperation of the surgeries performed at the closure stage was compared with that for surgeries performed prior to the closure stage.
## Statistics
The total joint replacements performed in the launching and closing units were described by giving frequencies or mean values for patient and operation characteristics. We graphically displayed the risk-adjusted reoperation rates with 95% confidence intervals for all units with more than 100 total hip or knee replacements in the study period, using the individuallevel study data and separately for hip and knee replacements. The 3-year reoperation rates in the hospitals were based on the observed number of reoperations in 3 years, divided by the expected number of reoperations provided by logistic regression analysis for each unit, with the rate of 1.0 as the average reoperation rate in all units. The factors used in the risk adjustment were sex, age (classified as below 65, 65-69, 70-74, and 75 and over), fixation method (cemented, uncemented, or hybrid), year of surgery, and the comorbid diseases mentioned previously.
We calculated Kaplan-Meier survival curves for total hip and knee replacement patients who were operated on in the launching and closing units that had at least 200 TJRs between 1998 and 2011, separately for hip and knee replacements, with grouping according to the stage of functioning of the unit in which the surgery was performed. We performed Cox proportional-hazards regression modeling to study the association between launch and closure on the one hand and reoperation risk on the other in the 3 years after the primary replacement. The modeling was done separately for hip and knee replacements. The adjusted models included the same confounders as the logistic regression models used in the risk adjustment were included in the analysis of the reoperation risk at the closure stage in the units that had closed.
The risk-adjusted 3-year reoperation rates showed that units (with at least 100 surgeries at the individual-level data) varied in their reoperation rates, for both THR and TKR . For THR, the performance of the units that were launched was better than average-or was average-compared to all the units. For TKR, one launched unit had a poorer performance than the units had on average. In both hip and knee replace- of hospital-specific reoperation rates described above. Schoenfeld residuals were used to test that the proportional-hazards assumption was not violated in any model. In addition, as the data might include many observations from the same individual, we performed sensitivity analyses including only the first observed operations of the patients. Only the results that were based on the whole data are shown, as the sensitivity analyses gave similar results.
# Results
Altogether, 83 units reported total hip and knee replacements to the FAR in the period 1998-2011. Between 1998 and 2011, 19 units started total joint replacement in Finland, and 8 of these performed more than 200 surgeries in the period. Performance of total hip and knee replacements was discontinued in 30 units, and 20 of these had performed more than 200 total hip and knee replacements since 1998 before closing. According to the FAR and the FHDR, 201,787 total hip and knee replacements were performed in Finland in the years 1998-2011 [fig_ref] Figure 1: performed due to causes other than primary osteoarthritis [/fig_ref]. After exclusions, 150,038 total hip and knee replacements were included in the individual-level study data. Of these surgeries, 7,678 were total hip replacements (THRs) and 10,674 were total knee replacements (TKRs) performed in the units that had launched surgery [fig_ref] Table 1: Descriptive statistics of total hip and knee replacements in launched and closed... [/fig_ref]. 10,650 THRs and 12,746 TKRs ment, the performance of the closed units was distributed evenly across the whole spectrum of performance. also shows that a unit's risk-adjusted reoperation rate in THR was not on a par with its performance in TKR.
In the newly started units, THRs and TKRs performed at the early stage of functioning did not have different implant survivorship from corresponding surgeries performed after the early stage of functioning in the same units. In the units that were closed, the THRs performed at the closure stage had statistically significantly worse implant survivorship than THRs performed prior to the closure stage (p = 0.004, log-rank test). In TKR, implant survivorship was similar before the closure stage and at the closure stage [fig_ref] Figure 3: Kaplan-Meier survival curves for hip and knee replacements in launched and closed... [/fig_ref].
The unadjusted and risk-adjusted hazard ratios for THRs and TKRs performed at the early stage of functioning in the newly opened units was different from the reoperation risk in patients who were operated on in the newly opened units after the early stage [fig_ref] Table 2: Unadjusted and risk-adjusted hazard ratios [/fig_ref]. The unadjusted and risk-adjusted hazard ratios for early reoperation after THR were statistically significantly higher at the closure stage, but there was no increase in reoperation risk at the closure stage after TKR.
# Discussion
Teamwork is an essential component in providing good-quality care (see, for example, [bib_ref] Teamwork and patient safety in dynamic domains of healthcare: a review of..., Manser [/bib_ref] [bib_ref] Effectiveness of patient care teams and the role of clinical expertise and..., Bosch [/bib_ref] [bib_ref] Does teamwork improve performance in the operating room? A multilevel evaluation, Weaver [/bib_ref] [bib_ref] Role of effective teamwork and communication in delivering safe, high-quality care, Leonard [/bib_ref]. The launch stage involves education and learning-by-doing in the operating team. In orthopedics, operating room teamwork plays a central role in maintaining efficiency, quality of care, and meeting of operating room standards, with deficiencies in . Risk-adjusted reoperation rate for reoperation within 3 years, with 95% CI, for units that performed total joint replacements in Finland between 1998 and 2011. Total hip and knee replacements are shown separately, ordered by risk-adjusted rate of hip replacements. communication being a major factor behind, for example, most wrong-site surgeries [bib_ref] Medical errors in orthopaedics. Results of an AAOS member survey, Wong [/bib_ref] [bib_ref] The WHO checklist is vital, but staff operating room etiquette skills may..., Kellett [/bib_ref] [bib_ref] Efficiency and safety during knee arthroplasty surgery, Van Strien [/bib_ref]. Hospital closures have gained attention in the literature, and the consequences of closures have been investigated from the points of view of nurses, physicians, and patients-and also from the standpoint of neighboring hospitals [bib_ref] Impact of hospital closures on nearby hospitals studied, Dombrosk [/bib_ref] [bib_ref] Coping with downsizing and job loss: lessons from the Shaughnessy Hospital closure, Havlovic [/bib_ref] [bib_ref] The unintended and unexpected impact of downsizing: costly hospitals become more costly, Shanahan [/bib_ref] [bib_ref] A case study of hospital closure and centralization of coronary revascularization procedures, Hemmelgarn [/bib_ref] [bib_ref] Impact of rural hospital closures in Saskatchewan, Liu [/bib_ref] [bib_ref] The effects of hospital restrucvturing that included layoffs on individual nurses who..., Cummings [/bib_ref]. However, the effects of closure on the last patients treated in the units that are to be closed have not been studied.
Of all the units that performed more than 200 total joint replacements between 1998 and 2011, 8 units started and 20 units ended total joint replacement surgery in Finland. The performance of these units, as measured by the risk-adjusted reoperation rate by the end of 2013, appeared to be distributed evenly across the spectrum of performance of all units that had performed total joint replacement over the study period. Our results showed that launch of total joint replacement was not associated with an increased risk of early reoperation for the first 100 patients who were operated on in these units (i.e. during the launch stage) when compared to surgeries after the launch stage. However, the last 100 THRs performed at the closure stage had an increased risk of reoperation compared to THRs in these units before the closure stage. In TKR, such an effect before closure was not found. The reason for an increased risk of reoperation after hip replacement might be luxations, but our data do not allow us to verify this idea.
Our study showed that registry or administrative data can be used in the analysis of the effects of restructuring of hospital services, or of a treatment such as total joint replacement, on the outcomes of treatment. The results suggest that hospital closures should be carefully managed in order to avoid deterioration of the quality of care and patient safety. We believe that the external validity of our results is good, since the effects are mediated through factors related to teamwork and motivation, which are universal phenomena.
The present study had a number of limitations. Most importantly, we did not have data on operating room personnel and personnel turnover at the different stages of operation of a unit. In the data, it was not possible to identify the surgeons, so we were unable to distinguish between experienced and inexperienced surgeons. In Finland, surgeons may operate at several hospitals, and this may have confounded the results. Usually, however, the surgeons who move between hospitals are experienced professionals. If such a surgeon had performed surgery in 1 or more of the units that were to close, when the resident surgeons were not available, this could have had an effect on our results. The possible bias stemming from this could lead to an underestimate of the reoperation risk at the closure stage. Similarly, in the units that started up, the surgeons performing the replacements were more likely to be experienced orthopedists, thus affecting reoperation risk estimates in the launched units.
The low number of patients operated on in the units that were launched or closed did not allow a more detailed specification of the learning effect. For instance, it has been shown that introduction of a new hip or knee implant in a hospital entails a learning effect for the first 15 operations only [bib_ref] Introducing a knee endoprosthesis model increases risk of early revision surgery, Peltola [/bib_ref] , and the implementation of a new technique such as hip resurfacing entails a learning curve of around 25 operations [bib_ref] The learning curve for adopting hip resurfacing among hip specialists, Nunley [/bib_ref] -or for anterior-supine minimally invasive THA, of around 40 operations [bib_ref] Anterior-supine minimally invasive total hip arthroplasty: defining the learning curve, Seng [/bib_ref]. Thus, the 100-patient limit in our study may not have been sufficiently sensitive to effects that would be reflected by only the very first patients who were operated on in the units. In addition, as we did not have information on the dates that closures were announced to the personnel in the units that were to close, we were not able to use calendar time to identify the surgeries performed in the actual closure stage. The coverage, accuracy, and reliability of the Finnish administrative health data used in the study have been shown to be adequate [bib_ref] Low rate of infected knee replacements in a nationwide series--is it an..., Jämsen [/bib_ref] [bib_ref] Quality of the Finnish Hospital Discharge Register: a systematic review, Sund [/bib_ref].
The incidences of THR and TKR have increased (Pabinger and Geissler 2014,, and to meet this increasing demand new units performing total hip and knee replacement may be established. On the other hand, in a public healthcare system with financial pressure to reorganize the supply of services, centralization of surgery and closure of units is a likely scenario. Decision makers need information on the details of the supply of services and on the consequences that reorganization services may have on the quality of care for the patients. Our study improves our understanding of the dynamic features of surgical teamwork and their effect on quality. The data do not allow us to make strong inferences on whether the differences in outcomes between hospitals are primarily related to the characteristics of the patients or to the performance of the centers. The differences in reoperation rates indicate a need for continuous benchmarking of centers undertaking hip and knee arthroplasty, and auditing of those with poor results.
The outcome of total joint replacement is not independent of changes in the production environment. In particular, before closing a unit, decision makers should pay attention to the quality of the healthcare given. Our findings highlight the fact that closures should be managed carefully to prevent the quality from deteriorating when performing the last arthroplasties in a unit.
MPe conceived the study, prepared and analyzed the data, and wrote the initial draft of the manuscript. AM, MPa, and SS took part in designing the study, interpreting the results, and editing the manuscript.
The study was supported by funding from Orton Hospital, Helsinki.
No competing interests declared.
[fig] Figure 1: performed due to causes other than primary osteoarthritis (n = 34,455) -operations with possible secondary osteoarthritis (n = 16,356) -operations performed on Ålandians and foreigners (n = 854) -operations with same-day total hip and knee replacement (Flow chart of data. [/fig]
[fig] Figure 3: Kaplan-Meier survival curves for hip and knee replacements in launched and closed units (with at least 200 TJRs between 1998 and 2011). [/fig]
[table] Table 1: Descriptive statistics of total hip and knee replacements in launched and closed arthroplasty units in Finland between 1998 and 2011 [/table]
[table] Table 2: Unadjusted and risk-adjusted hazard ratios (HRs; with 95% CI) for early revision in the launch stage of units that were opened [/table]
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Accumulation of protein aggregates induces autolytic programmed cell death in hybrid tobacco cells expressing hybrid lethality
Hybrid cells ofNicotiana suaveolens x N. tabacum grow normally at 36 °C, but immediately express lethality due to probable autoimmune response when transferred from 36 to 28 °C. Our recent study showed that the temperature-sensitive lethality of these hybrid cells occurs through autolytic programmed cell death (PCD). However, what happens in hybrid cells following the induction of autoimmune response to autolytic PCD is unclear. We hypothesized that accumulation of protein aggregates in hybrid cells induces autolytic PCD and examined detergent-insoluble protein (protein aggregates) isolated from hybrid cells expressing lethality. The amount of insoluble proteins increased in hybrid cells. Sodium 4-phenylbutyrate, a chemical chaperone, inhibited both the accumulation of insoluble proteins and irreversible progression of cell death. In contrast, E-64, a cysteine protease inhibitor, accelerated both the accumulation of insoluble proteins and cell death. Moreover, proteome analysis revealed that proteasome-component proteins were accumulated specifically in cells treated with E-64, and proteasome activity of hybrid cells decreased after induction of lethality. These findings demonstrate that accumulation of protein aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This suggests a novel process inducing plant PCD by loss of protein homeostasis and provides clues to future approaches for elucidating the whole process.
Hybrid lethality is the phenomenon of death occurring in the hybrids of specific plant crosses. The causative genes of hybrid lethality in plants have been identified and are commonly found to encode Resistance (R) proteins or R protein-interacting proteins, suggesting that an autoimmune response via epistatic interaction of R genes is a common mechanism of hybrid lethality. Studies have shown that many defense-related genes are induced in Nicotiana hybrids and Arabidopsis hybrids exhibiting lethality.
Hybrid seedlings and suspension cultured cells of Nicotiana suaveolens x N. tabacum are grown normally without any lethal symptoms when they cultured at 36 °C, but immediately express hybrid lethality when transferred from 36 to 28 °C, which is the optimal temperature for growth of the parents of the hybrids. Physiological and cell biological features of programmed cell death (PCD) have been observed in these hybrid seedlings and cells expressing temperature-sensitive lethality. Yamada et al.reported that the cell death process in the temperature-sensitive lethality reaches a point of no return between 2 and 3 h after the hybrid cells are transferred from 36 to 28 °C. In our recent study on this lethality, we revealed that the cell death is autolytic PCD, which is characterized by tonoplast rupture and the subsequent rapid clearance of the cytoplasm. However, little is known about what happens in hybrid cells due to induction of autoimmunity triggered by the R gene to autolytic PCD.
In the cells of N. suaveolens x N. tabacum exhibiting hybrid lethality, autophagy-related features such as the increases of monodansylcadaverine-stained structures and autophagy-related gene transcripts have been observed at early periods of autolytic PCD. Autophagy is one of the major pathways for degrading cellularUnited Graduate School of Agricultural Science, tokyo University of Agriculture and technology, tokyo, Japan. 2 faculty of Agricultural Science, Meiji University, Kanagawa, Japan. correspondence and requests for materials should be addressed to t.Y. (email: [email protected]) components and is primarily responsible for the degradation of most long-lived or aggregated proteins and cellular organelles. Several reports show that autophagy decreases protein aggregation in animal cells. In plants, various proteins, such as cytochrome b5-RFP aggregates, insoluble ubiquitinated protein aggregates, and inactive proteasomes, are degraded by autophagy. In addition, protein aggregates are often observed as electron-dense bodies by transmission electron microscopy (TEM) analysis. In hybrid tobacco cells harboring autophagy-related features, electron-dense bodies have frequently been detected in vacuoles.
Protein aggregates are observed following separation from lysate as the detergent-insoluble fraction using low-speed centrifugation. Protein aggregation occurs from oligomeric complexes of non-native conformers that arise from unfolded proteins trapped with partial misfolded states, whose hydrophobic interaction makes them increasingly larger, more stable, and less soluble during severe stress conditions. In animals and yeast, aggregates lack the function of the protein and heavy accumulation of protein aggregates causes the induction of cell death. Accumulation of protein aggregates can be experimentally inhibited by sodium 4-phenylbutyrate (PBA), a well-described chemical chaperone in animal and plant cells, and E-64, a cysteine protease inhibitor that blocks autophagic degradation in vacuoles, causes the accumulation of the degradative protein aggregates. However, little has been reported on the involvement of the accumulation of protein aggregates in cell death in plants. Moreover, it is unclear what impact differing amounts of protein aggregates have on cell death.
Based on these findings, we hypothesized that protein aggregates accumulate in N. suaveolens x N. tabacum hybrid cells and consequently cause autolytic PCD. In this study, we first investigated the amount of proteins in the detergent-insoluble fraction isolated from hybrid cells. Then, we examined the effects of exogenous treatment of PBA and E-64 on the accumulation of insoluble proteins and the progress of cell death in these hybrid cells. Moreover, to clarify which types of proteins are aggregated in hybrid cells, we conducted proteome analysis on insoluble proteins.
# Results
## Accumulation of insoluble proteins in hybrid cells expressing temperature-sensitive lethality.
Insoluble protein as a percentage of total protein in hybrid cells increased significantly in cells incubated at 28 °C starting at 3 h and then plateaued at 4 h. In contrast, cells incubated at 36 °C showed no change in insoluble protein level. The amount of total protein did not differ for cells incubated at 28 °C and at 36 °C (data not shown). To quantify the progression of cell death in hybrid cell cultures grown at 28 °C, the percentage of trypan www.nature.com/scientificreports www.nature.com/scientificreports/ blue-stained cells in individual cultures was determined. The percentage of trypan blue-stained cells was significantly higher in cultures incubated at 28 °C for 5 h than in cultures incubated at 36 °C.
To determine whether the percentage of insoluble proteins plays a role in the lethality in hybrid cells, we treated cultures with PBA, a chemical chaperone that prevents protein aggregation, and incubated the cultures for 6 h at 28 or 36 °C. After 6 h at 36 °C, there was no difference in cultures without or with PBA. Cultures at 28 °C showed that treatment with PBA significantly suppressed the accumulation of insoluble proteinand also the percentage of trypan blue-stained cells.
## Involvement of the accumulation of insoluble proteins in the determination of cell death in hybrid cells after induction of lethality.
The point of no return is the point after which cell fate is irrevocably programmed cell death. In the case of N. suaveolens x N. tabacum, this occurs between 2 and 3 h of incubation at 28 °C following the transition from 36 °C. Specifically, hybrid cells transferred from 36 to 28 °C survived when they were returned to 36 °C up to 2 h after the transfer but died when they were returned to 36 °C at 3 h or later. In this experiment, in order to determine whether the accumulation of insoluble proteins contributes to the determination of cell death in hybrid cells, cells transferred from 36 to 28 °C were treated with PBA and returned to 36 °C at 6 h after the transfer. Up to 24 h after the hybrid cells were returned to 36 °C, cells treated with PBA remained greenand the percentage of trypan blue-stained cells treated with PBA was significantly lower than for untreated cells. In addition, the increase of trypan blue-stained cells was observed only in the without PBA treatment.
## Effects of e-64 on accumulation of insoluble proteins and temperature-sensitive lethality in hybrid cells.
We investigated the effects of E-64, which is an inhibitor of autophagic degradation, on insoluble proteins and the percentage of trypan blue-stained cells. Insoluble protein as a percentage of total protein in E-64-treated cultures increased significantly at 1 h incubation at 28 °C and then plateaued at 3 h. In contrast, for incubation at 36 °C, insoluble protein content did not markedly change. The percentage of trypan blue-stained cells was significantly higher in E-64-treated cultures at 28 °C from 4 to 6 h than in cultures incubated at 36 °C. Moreover, the time to detect a significant increase in insoluble protein and cells stained with trypan blue due to incubation at 28 °C was faster in E-64-treated cultures than in the untreated cultures (Figs 1A,B, 3A,B).
## Effects of e-64 on formation of electron-dense bodies in vacuoles of hybrid cells.
Insoluble protein increased significantly by 3 h with incubation at 28 °C in E-64-treated cultures while only a slight increase was observed at the same time point in untreated cultures (Figs 1A, 3A). Observation of the ultrastructure of hybrid cells cultured at 28 °C for 3 h with and without E-64 via TEM showed that electron-dense bodies were mainly detected in the vacuoles of hybrid cells in both cultures. For more accurate analysis, we measured the area, number and size of electron-dense bodies on TEM image analysis. The area and number of electron-dense bodies in each cell vacuole were significantly higher in E-64-treated cultures than in untreated cultures. The size of electron-dense bodies was also larger in E-64-treated cultures but not significantly greater.
## Proteome analysis of insoluble proteins.
To confirm what kind of insoluble proteins were generated and degraded in hybrid cells, especially with incubation at 28 °C in the presence of E-64 for 3 h, we carried out LC-MS/MS analyses of insoluble proteins extracted from four groups of hybrid cells incubated for 3 h with E-64 or DMSO: group 28D was incubated at 28 °C with DMSO; group 28E was incubated at 28 °C with E-64; group 36D was incubated at 36 °C with DMSO; group 36E was incubated at 36 °C with E-64. A total of 2462 proteins The data at 28 °C for 6 h is from. Different superscript letters indicate significant differences between cultures for each time period as determined by the Tukey-Kramer test at P < 0.05. . Of these proteins, 1347 proteins were detected in 28D, 1673 proteins in 28E, 1247 proteins in 36D and 1500 proteins in 36E. Then, we identified insoluble proteins (i) insoluble proteins which increased in protein abundance with E-64 at 28 °C compared to without E-64 at 28 °C and with E-64 at 36 °C and (ii) insoluble proteins which increased in protein abundance with E-64 at 36 °C compared to without E-64 at 36 °C and with E-64 at 28 °C using the following criteria of %emPAI value: (i) 28E > 28D and 28E > 36E and (ii) 36E > 36D and 36E > 28E. As a result, 848 and 1041 proteins were identified in (i) and (ii), respectively, and were subjected to further analysisand .
GO analysis and pathways of the identified proteins were analyzed and compared between (i) and (ii) (see . GOs associated with translational initiation (GO:0006413), megagametogenesis (GO:0009561), response to chemical stimulus (GO:0042221), macromolecule metabolic process (GO:0043170), establishment of protein localization (GO:0045184) and response to other organisms (GO:0051707) were significantly overrepresented among the insoluble proteins which increased in protein abundance with E-64 at 28 °C (i). From pathway analysis, carbon metabolism, metabolic pathways and proteasome were significantly overrepresented among the proteins (i). Most of the proteasome component proteins (26 out of 34) were identified in (i). In contrast, GOs associated with response to stress (GO:0006950), cellular component assembly (GO:0022607) and macromolecule localization (GO:0033036) were significantly overrepresented among the insoluble proteins which increased in protein abundance with E-64 at 36 °C (ii). In addition, only the spliceosome pathway was significantly overrepresented among the insoluble proteins (ii). www.nature.com/scientificreports www.nature.com/scientificreports/ Proteasome activity in hybrid cells. From the proteome analysis of insoluble proteins, more proteasome component proteins were significantly detected among the insoluble proteins which increased in protein abundance with E-64 at 28 °C than at 36 °C. Then, we measured proteasome activity in hybrid cells incubated at 28 °C. Proteasome activity of hybrid cells was more significantly decreased in cultures exposed to 28 °C for 3 h than in cultures exposed to 36 °C. In addition, proteasome activity was significantly reduced by MG-132, a proteasome inhibitor, so the exact detection of proteasome activity by the method used in this experiment was confirmed.
# Discussion
Insoluble protein as a percentage of total proteins in hybrid cells increased in incubation at 28 °C for 3 h before the progression of cell death as indicated by the increase of trypan-blue stained cells. PBA, a chemical chaperone, suppressed the increase of insoluble proteins at 28 °C for 6 h, but at the same time, no increase in trypan-blue stained cells was observed with PBA. Several reports demonstrate that PBA acts as a chemical chaperone that reduces protein aggregates and suppresses endoplasmic reticulum (ER) stress. The point of no return during PCD is defined as the step beyond which the cell is irreversibly committed to die, and PBA could allow the hybrid cells to survive, even after the point of no return. These results indicate that the increase in insoluble protein causes the irreversible cell death (autolytic PCD) in hybrid cells during hybrid lethality. Moreover, the increase in insoluble protein was considered to be attributable to the accumulation of protein aggregates in hybrid cells.
We used E-64, a cysteine protease inhibitor that blocks autophagic degradation in vacuoles, to inhibit protein degradation in hybrid cells. Inoue et al.also showed that E-64 blocks autophagosome degradation in Arabidopsis www.nature.com/scientificreports www.nature.com/scientificreports/ and barley, which is followed by the accumulation of cytoplasmic inclusions within the central vacuole. Practically, the increase of insoluble proteins occurred 2 h earlier with E-64 than without. For incubation at 28 °C for 3 h, insoluble protein was highly increased in E-64-treated cultures, and then, enlargement and increases in electron-dense bodies were detected in vacuoles of hybrid cells via TEM. These results indicate that as protein aggregates increased, insoluble proteins are accumulated but also degraded by cysteine proteases in vacuoles. Thus, the increase in insoluble proteins is only apparent at 3 h without E-64 treatment. However, the increase of insoluble proteins at 3 h after induction of lethality even without E-64 indicates that accumulation of insoluble proteins exceeded the amount of endogenous degradation for insoluble proteins in hybrid cells. In addition, the increase of dead cells was observed 1 h earlier with E-64 than without E-64. This agrees with the finding that an increase in insoluble proteins induces autolytic PCD in hybrid cells during hybrid lethality.
From proteome analysis, we identified what kind of insoluble proteins showed increased in abundance in hybrid cells, specifically in the presence of E-64 at 28 °C, that were generated but subsequently degraded at the point of no return. Almost all proteasome-component proteins were identified as insoluble proteins from hybrid cells expressing lethality at 28 °C. Proteasome activity actually decreased significantly in hybrid lethality at 28 °C for 3 hat the same time that insoluble proteins were accumulated. The ubiquitin proteasome system (UPS) plays the critical role of recognizing and selectively degrading misfolded and damaged proteins to prevent potentially toxic effects of protein aggregation. Therefore, these proteasome-component proteins were mostly aggregated and detected as insoluble proteins and because they caused impairment of UPS, insoluble proteins consequently increased.
In plants, a putative role for the proteasome in PCD has been suggested. showed that dysfunction of UPS by gene silencing of proteasome subunits activates PCD in plants. However, the reason why impairment of proteasome activity leads to cell death is poorly understood. One possibility is that protein aggregates accumulate by the dysfunction of UPS, leading to cell death as in our study. Generally, the accumulation of protein aggregates in ER lumen activates the ER-stress response, and when it is prolonged, cell death occurs in plants 32 . Thus, it is possible that accumulation of protein aggregates by the impairment of proteasome activity causes persistent ER-stress and eventually leads to autolytic PCD in hybrid cells.
On the other hand, what are the reasons for proteasome activity impairing hybrid cells during hybrid lethality? It is known that protein aggregates can clog up the proteasome and inhibit proteolysis 21 , but why were protein aggregates first generated in hybrid cells during hybrid lethality? Protein aggregates in general comprise proteins that have been damaged by excessive production of reactive oxygen species (ROS). Mino et al.reported that some kinds of ROS are produced in large quantities in tobacco hybrids (N. gossei × N. tabacum) exhibiting lethality and they are involved in the lethality. From these studies, we considered the possibility that ROS generated after the induction of lethality in hybrid cells causes the formation of protein aggregates, which clog the proteasomes to the point that activity is impaired, which in turn leads to excess accumulation of protein aggregates, causing continuous ER-stress, and eventually leads to autolytic PCD in hybrid cells. Additional experiments are needed to further elucidate this hypothesis.
Proteome analysis of insoluble proteins also suggested that more proteins related to carbon metabolism and metabolic pathways aggregated and lost functionality during hybrid lethality at 28 °C than at 36 °C. After induction of lethality, hybrid cells underwent growth arrest and death. Therefore, the high abundance of proteins, which are responsible for normal growth such as metabolic reactions, might be damaged and aggregated, becoming insoluble. Spliceosomes, which are composed of small nuclear ribonucleoproteins that are required for proper RNA splicing, are more likely generated under normal growth conditions than stress conditions and are mainly aggregated and degraded. In our study, the spliceosome pathway was enriched with insoluble proteins in hybrid cells at 36 °C, but we have no mechanistic explanation for why spliceosomal proteins were specifically aggregated in healthy cells.
In conclusion, accumulation of insoluble proteins caused induction of autolytic PCD in hybrid cells. Moreover, we considered insoluble proteins as protein aggregates, and thus proposed that the aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This is the first evidence for the relationships between protein aggregates and autolytic PCD in plants.
# Materials and methods
Cell culture. The suspension cell line was established from cotyledonary segments of hybrid seedlings (N. suaveolens x N. tabacum) as described in Yamada et al.. Hybrid cells were cultured at 36 °C and subcultures were made every 10 days. In all experiments, 3 mL of packed cell volume from cultured cells was transferred to incubation chambers set to 28 °C or 36 °C, as described in Masuda et al., and incubated for 6 h. At 1 h intervals, protein extraction and staining with 0.4% (w/v) trypan blue in phosphate-buffered saline (PBS) were conducted, and cell death was scored under a light microscope. The extent of cell death in hybrid cells was estimated based on the percentage of dead cells among all cells. To evaluate the effects of E-64 and PBA on protein aggregates and cell death of hybrid cells, the hybrid cell cultures were incubated at 28 or 36 °C in the same way in medium containing 10 µM E-64 (Peptide Institute) and 5 mM PBA (Cayman Chemical). As a control, we added the same amount of solvent for E-64 and PBA, dimethyl sulfoxide (DMSO) and distilled water respectively. To inhibit proteasome activity, the hybrid cell cultures were incubated in medium containing 10 µM MG-132 (Peptide Institute) dissolved in DMSO. The concentrations of all treatments were determined in optimization experiments prior to the experiments. Extraction of total protein and isolation of protein aggregates. Frozen cell samples (100 mg) were disrupted and homogenized with stainless steel beads and a TissueLyser LT (Qiagen) in 1 mL of protein extraction buffer (100 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM DTT, 1% Triton X-100, 0.2% β-mercaptoethanol) under ice-cold conditions. The total protein content in homogenates was determined on an aliquot of the homogenate www.nature.com/scientificreports www.nature.com/scientificreports/ using the RCDC Protein Assay (Bio-Rad Laboratories). Protein aggregates were isolated from the rest of the homogenates as the insoluble protein fraction using low-speed centrifugation as described in . Briefly, the homogenates were centrifuged at 2,200 × g for 5 min, the supernatant was discarded, and precipitates were resuspended twice more in 1 mL of the same extraction buffer followed by centrifugation to wash out the soluble proteins. The last pellets were resuspended with the same extraction buffer and used to calculate the amount of insoluble proteins using the RCDC Protein Assay. Using the amount of total proteins and insoluble proteins, the percentage of insoluble proteins was calculated.
TEM. TEM imaging of cells was performed by Tokai Electron Microscopy Inc., as described in detail in Ueno et al.. TEM images were randomly selected, and more than 10 images were analyzed to measure the total area, number, and size of electron-dense bodies in each image using ImageJ 36 .
## Lc-ms/ms data acquisition of insoluble proteins.
Insoluble proteins were isolated from hybrid cell cultures incubated at 28 or 36 °C for 3 h in medium containing E-64 or DMSO. Insoluble proteins (5 μg) were separated by SDS-PAGE on 15% polyacrylamide gels at 30 mA and stained by Coomassie brilliant blue R-250. The stained gels were divided equally into ten slices, dried in a vacuum centrifuge, and reduced with 10 mM DTT in 100 mM NH 4 HCO 3 for 1 h at 56 °C, and then alkylated with 55 mM iodoacetamide in 100 mM NH 4 HCO 3 at ambient temperature in the dark with occasional vortexing for 45 min. In-gel proteins were digested by 12.5 ng/ μl Trypsin Gold (Promega) in 50 mM NH 4 HCO 3 at 37 °C overnight. The peptides were extracted three times with 50% acetonitrile and 5% formic acid, and dried down by vacuum centrifuge.
LC-MS/MS analysis was conducted with an LTQ Orbitrap XL (Thermo Fisher Scientific) according to Yoshikawa et al.. Peptide separation was carried out at room temperature on a C18 column at a flow rate of 100 nL/min with a 0%-80% gradient of acetonitrile. Mass spectra were recorded under data-dependent mode to automatically switch between Orbitrap-MS and linear ion trap-MS/MS acquisition with a centroid mode. Survey full-scan MS spectra (450 to 1500 m/z) were acquired in the Orbitrap with resolution set to 15,000 after accumulation to a target value of 500,000 in the linear ion trap. The top 10 precursor ions from each MS scan were isolated for fragmentation for every 0.2 s in the linear ion trap using collision induced dissociation at a target value of 30,000.
# Lc-ms/ms data analysis.
Data analysis was performed as described in Yoshikawa et al.with little modification. The raw data acquired by Xcalibur version 2.0.7 (Thermo Fisher Scientific) was converted to an mgf file by Proteome Discoverer version 1.1 (Thermo Fisher Scientific). A search was performed by MASCOT version 2.2.07 against the N. tabacum cv. TN90 protein database in NCBI RefSeq with the parameters of fixed modification and carbamidemethyl (Cys); variable modifications of oxidation (Met) and pyroglutamine; maximum missed cleavages of 2; peptide mass tolerance of 25 ppm; and MS/MS tolerance of 0.8 Da. The candidate peptides were identified as having significant homology (p < 0.05) and are referred to as "hits". Further, we a set more strict criteria for protein assignment: any peptide candidate with an MS/MS signal number of <2 was eliminated from the "hits" candidates. For estimation of the absolute protein amount assigned by the database search, we used the exponentially modified protein abundance index (emPAI). The %emPAI (normalized amount of proteins) was used for further analysis.
Gene ontology (GO) analysis. Blastp program was used to compare protein sequences of N. tabacum cv.
TN90 from NCBI RefSeq and Arabidopsis thaliana from the TAIR10 database (E-value < 1e −5 ) to convert the protein ID of N. tabacum to blastp top-hit A. thaliana AGI code. The converted gene list was analyzed on the DAVID database (https://david.ncifcrf.gov/) 39 to identify gene ontology (GO) classifications and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Using these data, Fisher's exact test was conducted on R 41 and the adjusted p-value by the Benjamini-Hochberg procedure (q-value) was calculated to determine which GO and pathway were significantly enriched in the different groups.
Proteasome activity assay. Frozen cell samples (100 mg) were disrupted and homogenized with stainless steel beads and a TissueLyser LT in proteasome extraction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT, 5 mM EDTA) under ice-cold conditions. After incubation on ice for 30 min, cell debris was removed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatant was immediately used for enzyme assays performed in reaction buffer (25 mM HEPES, pH 7.5, 2 mM DTT, 5 mM EDTA) incubated for 60 min at 37 °C. 50 μM Succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine 4-methylcoumaryl-7-amide (Suc-LLVY-AMC) (Peptide Institute) was used as the substrate of proteasome; the amount of 7-amino-4-methyl-coumarin released was determined with a SpectraMax Paradigm Multimode Microplate Reader (Molecular Devices) based on fluorescence emission at 460 nm upon excitation at 360 nm. Protein content was determined using a Qubit Protein Assay Kit (Life Technologies) with a bovine serum albumin solution as the standard.
Statistics. All the numeric data are reported as mean values with standard error (SE). Statistical analysis was performed with the Student's t-test or Welch's t-test for the comparison of two groups and the Tukey-Kramer test for multiple comparisons. Student's t-test was carried out under the assumption of equal variance and Welch's t-test was conducted for the assumption of unequal variance using the two-sample F-test. In comparisons of percentage data, statistical analysis was done following arcsine transformation. In all cases, differences with P < 0.05 were considered statistically significant.
## Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary Information files. |
Breast implant-associated anaplastic large-cell lymphoma: first case detected in a Japanese breast cancer patient
This paper details the first breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) case detected in Japan. The patient, a 67-year-old Japanese woman, was diagnosed with left unilateral breast cancer 17 years ago. Induration and redness presented in the left breast, which had undergone immediate breast reconstructive surgery using a tissue expander, later replaced by a silicone breast implant (SBI). Breast ultrasound showed fluid collection around the SBI. Surgery was performed to remove the left breast implant and the fragmented capsule surrounding the implant. Postoperative pathological findings did not indicate malignancy. Nine months later, a contralateral axillary lymphadenopathy was observed, and an excisional biopsy of the axillary lymph node was performed. The patient was diagnosed with BIA-ALCL and successfully underwent adjuvant CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy.
# Introduction
Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) was first reported by Keech and Creech in 1997and is classified as T/NK-cell non-Hodgkin's lymphoma. Many cases of BIA-ALCL are found with late-onset seromas or masses. Pathological findings show large neoplastic cells with abundant cytoplasm and pleomorphic nuclei.
BIA-ALCL may be diagnosed if CD30 is positive and anaplastic lymphoma kinase (ALK) protein is negative. In many cases, BIA-ALCL is recognized within 0.8-27 years (9.75 years on average) after breast reconstruction surgery with a silicone breast implant (SBI). The onset risk is said to be latent in one of every 2,207-86,029 patients who underwent textured implant insertion. Detecting BIA-ALCL, removing the SBI, and performing complete capsulectomy in the early stages reportedly lead to a good prognosis. In this paper, the first Japanese breast cancer case of BIA-ALCL presenting with distant metastasis in the contralateral axillary lymph node is reported.
## Case report
A 50-year-old woman diagnosed with left unilateral breast cancer underwent mastectomy, axillary lymph-node dissection, and tissue expander insertion (model unknown) in 2002. Subsequently, the tissue expander was replaced with a textured surface SBI (McGhan Limited/410LM 220g/REF 27-LM115-220/LOT 161276). In the process of caring for her elderly parent for several years postoperatively, the patient used her pectoralis major muscles frequently. As a consequence, "breast stiffness" worsened, and prompted the patient to frequently massage the reconstructed breast. Redness on the reconstructed breast was recognized in December 2017, 15 years postoperative, but was left unattended. Symptoms worsened and she came to the hospital in June 2018. She presented with balloon-like swelling of left breast, upward shifting of the nipple and ipsilateral shoulder, and reddened and indurated areas on the medial and lateral side of the breast. Ultrasound showed fluid collection around the SBI, also presenting with rippled shell, and slightly enlarged bilateral axillary lymph nodes (data not shown). Because fluid collection was small, puncture was not performed before surgery. Blood tests revealed WBC 6100/μl and CRP 0.22 mg/dl. A damaged or infected breast implant was suspected, as well as the possibility of BIA-ALCL, and the SBI was removed along with as much surrounding tissue (capsule) as possible. No damage to the SBI was found, but fragmented capsules were observed during the surgical operation. As shown in, yellow and serous discharge with scrambled egg-like floating matter was observed in the capsular tissue. Cytological examination of the intraoperative fluid showed a small cluster of atypical cells with large, pleomorphic, hyperchromatic, and severely irregular nuclei. Malignant lesions were suspected due to the clear enlargement of the nucleolus and the uneven distribution of the nuclei. The cytological finding was Class IIIb. Moderate nuclear atypia was recognized in large lymphoid cells with degeneration of the capsule and tissues surrounding the SBI. Fragmented capsules showed scattered chronic inflammatory cells in the necrotic area near the capsule. Atypical and hyperchromatic macrophages were seen. Results of immunohistochemistry (IHC) staining revealed CD68 (+), vimentin (+), and CK7 (−), and cells were determined to be histiocytes. Therefore, neither CD30 nor ALK was not performed. Bacterial cultures from fluid collection were negative. The postoperative diagnosis was considered to be sterile inflammation, but the possibility of BIA-ALCL could not be denied. Three months after the removal operation on the left breast, the contralateral axillary lymphadenopathy began to grow larger, and core needle biopsy was performed. In the histopathological diagnosis, most of the needle biopsy samples showed non-neoplastic changes. In a small number of regions, however, focal atypical CD30-positive cells were observed. The results of blood tests at 3 months after the removal operation showed WBC 6500/μl (neutrophil 51.4%, eosinophil 8.3%, and basophil 1.3%), CEA 0.8 ng/ ml, CA15-3 9.2 U/ml, NCC-ST-439 < 1.0 U/ml, and soluble interleukin-2 receptor 477 U/ml. By 5 months postoperatively, the enlargement of the contralateral axillary lymph node had progressed even more. Now, fine needle aspiration cytology resulted in a Class IIIb diagnosis. Excisional biopsy was then performed on the contralateral axillary lymph node. The pathological findings showed proliferation of large atypical lymphoid cells with pleomorphic nuclei.. The result of IHC staining revealed CD30 (+), ALK (−), CD4 (weakly positive), CD8 (−), CD3 (−), CD20 (−), CD56 (−), GranzymeB (+), AE1/3 (−), EMA (−), and CK5/6 (−).The patient was diagnosed with BIA-ALCL due to CD30 positivityand ALK negativity. The patient was in Stage IV and was transferred to a specialized institution to receive adjuvant CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy every 21 days for 6 cycles. Seven months have passed, since the excisional biopsy was performed, and a complete metabolic response was confirmed by PET-CT.
# Discussion
The widespread use of silicone implants in breast reconstruction started in the 1960s. In Japan, SBIs were not covered by insurance until July 2013, when the national health insurance system allowed implants for breast reconstruction. However, the Japan Oncoplastic Breast Surgery Society (JOPBS) allows breast reconstruction surgery to be performed only by physicians and facilities certified by the Society. In Japan, only Allergan breast implants/tissue expander are approved involving "Biocell": macro-texture. According to the 2018 annual report of the JOPBS, 6,582 SBIs were used in that year . In contrast, the 2018 Plastic Surgery Statistics Report issued by the American Society for Plastic Surgeons says that there were 78,814 SBIs, about 12 times more than the figure in Japan, used that year. By ethnicity, Caucasians accounted for 71% of the women who had reconstructive surgery, African-Americans 13%, Asian/Pacific Islanders 4%, Hispanics 11%, and others 1%. Asians use SBIs less frequently. According to Average breast size worldwide on WorldData.info, the most common cup size of women is A (54-304 ml, 179 ml on average) in Japan in marked contrast with F or greater (645-2986 ml, 1668 ml on average) in the United States (Caucasians) and C (147-831 ml, 489 ml on average) in Spain. By race, Caucasians account for 92.1% of BIA-ALCL patients, according to the patient registry and outcomes for breast implants and anaplastic large-cell lymphoma etiology and epidemiology (PROFILE). Furthermore, among major lymphoma subtypes, ALCL-ALK negative was 7.8% in North America and 2.6% in Asia. The findings suggested that certain ethnic backgrounds and also breast size and the number of SBIs used may be associated with the incidence of BIA-ALCL.
Additionally, bacterial biofilm is one possible cause of BIA-ALCL. Reportedly, BIA-ALCL may easily occur in patients undergoing textured implants. These types of implants do not require postoperative massage, creating a great advantage over smooth implants. However, in this case, the patient frequently massaged her breast reconstructed with a macro-textured implant. We hypothesize that the frequent massage may have been the cause of "latent effusion" and "inflammatory rash" along with persistent chronic inflammation. In addition, a differential diagnosis of the symptoms recognized in this case, namely seroma, redness, and induration of the breast, requires consideration of the possibility of local recurrence as well as BIA-ALCL.
The development of BIA-ALCL is also associated with persistent chronic inflammation. Chronic inflammation may induce B-cell-derived lymphomas such as MALT (mucosa-associated lymphoid tissue) lymphoma and pyothorax-associated lymphoma (PAL) induced by Helicobacter pyloriand Epstein-Barr virus, respectively. Comprehensive next-generation sequence analysis of BIA-ALCL cases revealed sequence variants that activate JAK/STAT signaling. High copy-number amplification of TNFRSF11A and PDGFRA was detected, which could be expected as therapeutic targets. Currently, five cases of BIA-ALCL associated with germline TP53 mutation have been reported.
BIA-ALCL may be diagnosed if a neoplasm composed of large lymphoid cells that are CD30 positive and ALK negative is found. However, CD30 can also be positive in Hodgkin's lymphoma, Epstein-Barr virus-associated lymphomas, and non-lymphoid neoplasms. CD30 positivity in ALCL is defined as 75% or more of tumor cells expressing positive CD30. For this reason, it should be noted that being CD30 positive does not always equal to ALCL. The pathological diagnosis was difficult in this case, because the samples collected at the time of SBI removal or axillary lymph-node needle biopsy contained only a few tumor components with degeneration. Primary lesion showed widely necrotic tendency. Histologically, there was no solid collection of the living lymphoma cells near the capsule. Therefore, it would be difficult to diagnose BIA-ALCL even if the capsule could be excised in en bloc at the first operation. Excisional biopsy of the axillary lymph node allowed for the collection of more tissue and made the correct diagnosis possible. In hematoxylin and eosin staining, ALCL may morphologically look like a poorly differentiated carcinoma. As this patient had a history of breast cancer, our first impression of the H&E specimen suspected of axillary lymph-node metastasis from poorly differentiated adenocarcinoma. However, the patient had undergone breast reconstruction with the SBI, and BIA-ALCL needed to be included in the differential diagnosis. Compared to BIA-ALCL after breast augmentation, BIA-ALCL after breast cancer reconstruction appears to be more difficult to diagnose, because it requires differentiation from cancer recurrence.
According to the Food and Drug Administration (FDA) Safety Communication, 573 BIA-ALCL patients were analyzed as of July 6, 2019, and 33 of these patients were reported to have died. Of the 33 died patients, the manufacturer was identified in 13, of which 12 used Allergan breast implants. This led the FDA to request Allergan to voluntarily recall the implants and tissue expanders in question from the global market (as written in an FDA release dated July 24, 2019). We should take into account the fact that the environment surrounding breast surgery and reconstruction in Japan has been changing after the confirmation of the first case of BIA-ALCL in Japan.
# Conclusion
The number of BIA-ALCL cases is expected to increase in the future, and conducting lifetime surveillance postoperatively is important. Since BIA-ALCL must be detected and treated early, it may be imperative to raise awareness about the disease not only among breast surgeons, plastic surgeons, and pathologists but also among patients. Patients with a difficult diagnosis of BIA-ALCL at the time of SBI removal need to undergo strict follow-up. Diagnosing lymphoma is often difficult in needle biopsies performed on an axillary lymphadenopathy. Therefore, an excisional biopsy should be performed to ensure an accurate differential diagnosis of BIA-ALCL from recurrent and poorly differentiated breast adenocarcinoma.
Funding The authors received no financial support for the case report, authorship, and publication of this manuscript.
## Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of interest regarding the publication of this manuscript.
Ethical approval Our hospital does not require ethical approval for reporting individual cases.
## Informed consent
The written informed consent was obtained from the patient for anonymized information to be published in this manuscript.
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Multitargeting Compounds: A Promising Strategy to Overcome Multi-Drug Resistant Tuberculosis
Tuberculosis is still an urgent global health problem, mainly due to the spread of multi-drug resistant M. tuberculosis strains, which lead to the need of new more efficient drugs. A strategy to overcome the problem of the resistance insurgence could be the polypharmacology approach, to develop single molecules that act on different targets. Polypharmacology could have features that make it an approach more effective than the classical polypharmacy, in which different drugs with high affinity for one target are taken together. Firstly, for a compound that has multiple targets, the probability of development of resistance should be considerably reduced. Moreover, such compounds should have higher efficacy, and could show synergic effects. Lastly, the use of a single molecule should be conceivably associated with a lower risk of side effects, and problems of drug-drug interaction. Indeed, the multitargeting approach for the development of novel antitubercular drugs have gained great interest in recent years. This review article aims to provide an overview of the most recent and promising multitargeting antitubercular drug candidates.Generally, LTBI can switch into the active form of TB in case of a strong immune system weakening, such as in case of HIV co-infection, or autoimmune diseases, but it can occur in not-high-risk patients, as well[5].Treatment for active drug-susceptible TB is expensive and long, taking up to six months of daily doses of four first line drugs: Isoniazid, rifampicin, ethambutol, and pyrazinamide. Treatment success rate is generally higher when the patient follows the treatment to its completion. Low treatment success can depend on multiple factors, poor counseling during the treatment phase with consequent drop-out, high-risk populations (refugees, poor), co-morbidity with other pathologies[6]. For these reasons, supporting and informing the patients is pivotal. Furthermore, treatment outcomes depend on the spreading of MTB drug-resistant strains, for which the canonical therapy does not work[7]. Drug susceptibility tests should be performed, not only before, but also during therapy, to allow for an early identification of developing drug-resistant TB (DR-TB)[7].Therefore, DR-TB should be carefully treated based on the resistance phenotype of the MTB drug-resistant strain. As recommended by the WHO consolidated guidelines on drug-resistant tuberculosis treatment of 2019, rifampicin-susceptible and isoniazid-resistant TB should be treated with daily doses of rifampicin, ethambutol, pyrazinamide, and levofloxacin or other fluoroquinolones[8].Poor management of DR-TB can develop in multi-drug resistant TB (MDR-TB) with acquired rifampicin-resistance (RR-TB) with or without resistance to other first line drugs. MDR-TB/RR-TB treatment can be highly challenging, usually needing personalized strategies for each patient[9].Treatment regimen for MDR-TB requires eight months of daily administration of pyrazinamide in association with at least four more second-line drugs. The duration of the treatment can vary from 12 to 20 months, but mostly depends on the patient response and TB evolution[9]. In the treatment of MDR-TB, fluoroquinolones, injectable anti-TB drugs, ethionamide, and cycloserine should be used in association to pyrazinamide for the first intensive phase, while pyrazinamide should be continued for the entirety of the treatment[9]. If cycloserine cannot be used, the para-aminosalicylic acid (PAS) should be used instead[9]. Streptomycin should be used as a second-line drug only for amikacin-resistant MDR-TB, as it correlates with a reduced treatment success rate[8].The spreading of MDR MTB strains has become a major health problem worldwide, especially in high burden countries, where in depth analysis, exhaustive follow-up information retrieval, and organic procedures are challenging [10]. Furthermore, due to poor management of treatment administration and overall inadequate antibiotic distribution, MDR-TB can dramatically evolve into extensively drug resistant TB (XDR-TB) resistant to isoniazid and rifampicin, associated with resistance to at least one fluoroquinolone and one injectable second line drug[11]. The use of multiple drugs in combination over a long period of time, in association with uninformed antibiotic administration, scarce thorough analysis, inadequate patients support and follow-up, paves the way for the onset of drug-resistance. Currently, there is the need to find new and more efficient ways to fight MTB infections, that can help bypass the problems of the current therapy. The tempting idea behind the concept of multitargeting drugs is the reduction of the causalities behind the development of drug resistance, which could help also reduce the time of antibiotics exposure and the number of antibiotics administrated to the patients, further reducing the risk of developing mechanism of resistance and consequently enhancing the possibility of complete recovery.How Polypharmacology Can Help in Fighting MDR-TB?Drug resistance mechanisms are a main problem to face in fighting MDR-and XDR-TB strains. A pathogen, indeed, requires often just a single base mutation to became resilient to antibiotics or chemotherapeutics. The development of a single pharmaceutical molecule that acts on different targets is a possible strategy to bypass this problem. This multitargeting system is known as "polypharmacology". SQ109, for example, is a promising multitarget drug that putatively hits four different targets in the respiratory chain of M. tuberculosis[12].
# Introduction
Tuberculosis (TB) is an airborne infectious disease caused by the bacillus Mycobacterium tuberculosis (MTB). To date, MTB still represents one of the most dangerous pathogens, claiming millions of lives each year worldwide. As reported by the WHO Global report of 2019, in 2018 at least 10 million people fell ill with TB, of which 1.2 million died among HIV-negative subjects, but the number grows with an additional 251,000 deaths among HIV-positive patients. Even if the numbers are still high, there is an overall reduction in recent years, this can be correlated to the improvements in the EndTB Strategy, that started in 2015, whose aim is to reduce by 80% the incidence of TB worldwide and by 90% the number of deaths caused by this single infectious agent before 2030.
Understanding the dynamics of MTB transmission is fundamental to control and prevent TB spreading, especially in high burden countries where this pathogen is still endemic. Genotyping and spoligotyping are established strategies for the molecular characterization and identification of MTB strains. The complex interactions between MTB and the host during first contact, infection, and persistence are yet to be fully understood. The human body response to infectious agents calls for the mobilization of the innate immune system cells, but MTB can evade the immune response and persist in the human body thanks to its high genome plasticity. In addition, upon MTB exposure, only a small percentage of patients will develop an active form of TB, while the majority of them will have a latent TB infection (LTBI). LTBI is a persistent immune response to MTB antigens in the absence of clinical symptoms of TB and can be diagnosed with the interferon-γ release assay (IGRA) and the tuberculin skin tests (TST), but the diagnosis of LTBI is not predictive of developing active TB.
Polypharmacology is in contraposition with the more classical "polypharmacy", the use of different drugs, each with high affinity for one target, that are taken together as cocktails or multicomponent drugs. Even if both strategies are effective, polypharmacology possesses important features to underline. Firstly, the use of only one molecule instead of many is conceivably associated with a possible lower cytotoxicity and side effects. Moreover, polypharmacology is expected to have a higher therapeutic efficacy, compared to the classical approach of hitting only one best target at a time. Moreover, multitarget drugs show synergic or additive effects, this means that they can modulate complex diseases in lower time and with smaller doses thanks to simultaneous targeting. Lastly, they may avoid the problem of drug-drug interaction. All these features allow the consequential improvement of the patient quality of life.
For these reasons, polypharmacology is an emerging strategy in therapeutic development of drugs against synergistic bacterial diseases, neurological diseases, and cancer. Even if the development of multitarget drugs is a relatively novel field, some classifications have been already proposed according to their mechanisms of action, or their structures. Based on mechanisms of action, a molecule that affects different targets within the same metabolic pathway acts in "vertical targeting". We can distinguish a "series inhibition" if the two targets are related, as for example consequential or in the same pathway, while a "parallel inhibition"when they are unrelated, but should have for example a common substrate that can be mimicked. The vertical targeting is a strategy that can fight the insurgence of certain kinds of resistance mechanisms such as mutations. A drug that acts in "network targeting" instead, hits different targets in different pathways, and it is able to prevent compensatory homeostatic responses and the adaptive resistance. By structure, multitarget drugs may be classified according to the optimization and modifications that are introduced by chemical tailoring to the original molecule. Another possibility is to rationally design a multitarget drug by virtual studies. This strategy gives rise to three possibilities: Linked, fused, or merged pharmacophores.
Linked pharmacophores are molecules bound together by a stable or biodegradable linker. The result is a larger molecule that does not need further improvement, but the position of the linker is crucial for its final effect on target. Moreover, this molecule may fail to reach the intracellular compartment of interest because of its size. From another point of view, some linkers can improve solubility and polarity of the molecule and reduce unspecific diffusion into an unwanted cellular compartment. Nonetheless, linked pharmacophores are currently used to produce antibody drug conjugates, that is, a drug (usually a small molecule) conjugated with an antibody that works as a vessel to reach the target. Another example is to conjugate a drug, as for example an antibiotic, to bacterial siderophores. The resulting sideromycin exploits the bacterial uptake mechanisms to be internalized.
A promising example is Cefiderocol. This compound, that binds the penicillin-binding protein 3 (PBP3) inhibiting cell wall biosynthesis, is active against different multidrug-resistant Gram-negative pathogens such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae, and is currently in phase II clinical trials.
Fused pharmacophores are the result of two joint small molecules without a linker. According to the type of final bond (imine, ester, or hydrazine bonds) they may be cleavable or not. Moreover, the position of the bond is crucial for the final effect of both molecules and the resulting pharmacophore may be a large molecule with all the consequent problems for its delivery. According to Sterling et al., for example, the "compound 9" described in their article is active against both acetylcholinesterase (AChE) and monoamine oxidases (MAOs), and it was created by fusion of rivastigmine and rasagiline.
Finally, merged pharmacophores are possible only when a part of two different molecules can overlap, resulting in their integration. Anyway, to be active, the resulting molecule must possess the correct molecular geometry and charge distribution to interact with both targets. For example, Ziprasidone is an antipsychotic drug optimized by the resulting fusion of dopamine and 5-HT 2 R pharmacophore. It targets both type 2 dopamine receptors D2 and type 2 serotonin synergic receptors 5-HT 2 R. . Cefepime derivative is the linker between ceftazidime derivative and catechol moiety in cefiderocol (A). Ladostigil, an analogue of compound 9, is derived from the fusion of rasagiline with rivastigmine (B). Finally, dopamine is merged with 5-HT 2 R pharmacophore and the obtained molecule is optimized to ziprasidone (C).
## Multitargeting compounds against m. tuberculosis
The single-target strategy for the development of novel antimicrobial drugs involves single proteins essential for survival of the pathogen. However, this approach shows a weakness, since a single mutation in the target protein could be sufficient to confer resistance. For this reason, drug combinations in TB treatment are preferred over single-drug therapies; in this context multitarget drug discovery may offer a novel opportunity. In recent years, several multitargeting antitubercular compounds have emerged. Many compounds have been firstly discovered through phenotypic screening, then they were retrospectively found to target multiple proteins simultaneously, such as the case of the MmpL3 inhibitors. Interestingly, in some cases the compound has been designed or selected as an inhibitor of a specific enzyme or pathway, then it was found to have additional targets.
For instance, the recently described 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazoles, have been selected as potential inhibitors of the arylamine N-acetyltransferase enzyme, and were found to be also potent efflux pump inhibitors. Another example is the case of the tetrahydroisoquinoline compounds, that have been selected as inhibitors of the ATP-dependent MurE ligase, but were found to have pleiotropic mechanisms of action, not fully clarified yet. On the other hand, compounds are also emerging from biochemical screens against specific targets, as for instance the GroEL/ES chaperonin and protein tyrosine phosphatase B inhibitors. Finally, multitargeting compounds have been specifically designed, as for instance the fatty acids bypass biosynthetic pathways inhibitors. The most recent and significant examples of the different multitargeting antitubercular compounds are here described.
## Mmpl3 inhibitors
The trehalose monomycolate (TMM) transporter MmpL3 is an essential protein involved in the translocation of TMM and cell wall mycolates across the membrane. This protein has been defined as a promiscuous target since several compounds with different scaffolds have been demonstrated to affect its activity. Among the reported MmpL3 inhibitors, a very promising compound is SQ109 (1), a derivative of ethambutol but with a different mechanism of action with respect to its original structure. Actually (1) is now in phase II clinical trial. As the substrates of MmpL3 are part of the cell wall structure, (1) seems to act as a cell wall inhibitor. However, the direct inhibition of MmpL3 has been debated, since (1), as well as another inhibitor, the 1,5-diarylpyrrole BM212 (2), have been shown to be active also against latent MTB and against other bacterial species lacking this transporter. For this reason, an indirect effect on MmpL3 translocation was suggested, due to the disruption of the proton motive force (PMF) driven by these compounds. Moreover, Li et al.demonstrated that (1) and some derivatives inhibit in vitro two other MTB enzymes, MenA and MenG, both involved in the biosynthetic pathway of quinone structures. MenA catalyzes the formation of demethylmenaquinol by isoprenylation of 1,4-dihydroxy-2-naphthoic acid using one molecule of isoprenoid diphosphate. MenG acts directly on demethylmenaquinol, catalyzing its S-adenosylmethionine-dependent methylation to synthesize the demethylmenaquinone. Since this quinone is involved in electron transport, the final effect is an impairment of ATP biosynthesis.
However, the binding of several inhibitors to MmpL3 has been recently demonstrated, using a fluorescent competition assay and surface plasmon resonance with the purified protein, further confirmed by the crystal structure of the protein in complex with different compounds, (1) included. Nevertheless, among these inhibitors only (1) and (2) were demonstrated to dissipate both ∆pH and ∆Ψ, thus confirming the effects of these two compounds on the PMF.
It is worth noting that all the attempts to directly isolate any spontaneous resistant MTB mutant to these two compounds failed, further demonstrating the usefulness of compounds that have multiple targets in preventing the resistance insurgence.
## Designed dual inhibitors against fatty acids bypass biosynthetic pathways
Mycobacterium tuberculosis possesses a very thick cell wall that allows a certain resilience to molecule diffusion inside the cell and is essential for the survival during the host infection. One main component of the cell wall is a plethora of fatty acids that are synthetized primarily by the fatty acid synthesis (FAS) pathway. MTB possesses two main pathways: FAS-I, a multidomain enzyme that catalyzes the synthesis of short-chain fatty acids, and the FAS-II system involved in long-chain fatty acids production. Some fatty acids, anyway, depend on alternative bypass biosynthetic pathways, such as for example the metabolism of interlinked CoA dependent fatty acid, which could represent an attractive target for novel antitubercular compounds. In this context, Banerjee et al. selected the two enzymes, encoded on a single operon, FabG4 and HtdX for the development of novel multitargeting compounds. The first enzyme is a β-ketoacyl CoA reductase, while the second is a 3(R)-hydroxyacyl CoA dehydratase that acts on the reduced ketoacyl, the product of the reaction catalyzed by FabG4. Both enzymes are reported as essential for MTB survivaland being involved in consecutive steps of a metabolic pathway, a common inhibitor is supposed to have synergistic effects. To this purpose, the authors used a blended structure-based and ligand-based design approach. Firstly, based on the structural information of the catalytic sites they selected as pharmacophores the β-lactam and the isoniazid scaffolds, as well as several aromatic rings. The pharmacophores were then combined to form a small library, that was used for docking studies to select the potential inhibitors. Through this analysis they selected seven scaffolds, three of them showing significant activity against both enzymes. Moreover, the three compounds displayed interesting antimycobacterial activity against M. smegmatis, both planktonic and in biofilm, demonstrating the validity of this approach to achieve novel drug candidates.
## Oxadiazolone derivatives targeting (ser/cys)-enzymes
The oxadiazolone core is an interesting scaffold, which characterizes compounds known to have antimycobacterial activity. Among these compounds, 5-methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (6), was demonstrated to be an inhibitor of the hormone-sensitive lipase family of enzymes, which forms a covalent slowly reversible bond, such as carbamate or thiocarbamate, with the catalytic serine or cysteine residue. Thus, since lipolytic enzymes are important for pathogen and host cross-talk, for pathogenic reactivation or survival during dormancy state, it could represent an interesting scaffold for the development of therapeutic compounds. Recently, starting from these results, Nguyen et al. developed eighteen oxadiazole derivatives, by modifying the R chain and the positioning of the phenoxy group. Four compounds (7-10) ( In order to identify the target(s) of the compounds, the researchers applied an activity-based protein profiling (ABPP) approach, that allowed the identification of 18 different proteins that bind oxadiazolone derivatives. As expected, all the identified proteins were Ser/Cys-based enzymes, including several hydrolases, five of them reported as essentials. Three of these putative targets, the proteins TesA, Cfp21, and Rv0183, have been expressed in recombinant forms, and were confirmed to be directly inhibited by the compounds. This work allowed the identifications of novel unexploited pathways and enzymes as potential targets for novel antitubercular multitargeting compounds.
## Ctp and coa biosynthesis inhibition
One advantage of multitargeting compounds is that the simultaneous inhibition of two different interconnected pathways should lead to synergistic effects. This is the case for the PyrG and PanK inhibitors. The two compounds 5-methyl-N-(4-nitrophenyl) thiophene-2-carboxamide (11) and 3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide (12), have been firstly identified in a high throughput phenotypic screening study as inhibitors of the CTP synthetase PyrG. Both compounds were demonstrated to be indeed prodrugs, which need activation by the monooxygenase EthA to exert their antimycobacterial activity, although only for (11) the active sulfone product (13) has been identified. Moreover, during further investigations, these compounds were found to possess at least another target, being able to inhibit the pantothenate kinase PanK. This enzyme catalyzes the first step of coenzyme A (CoA) biosynthesis, by conversion of the pantothenate (vitamin B5) into 4 -phosphopantothenate. Moreover, two other compounds (14) and (15), identified through in silico and in vitro target based screenings against PyrG inhibitors, respectively, were demonstrated to inhibit also the PanK activity. CoA is an essential cofactor for many key enzymes of different metabolic pathways, such as fatty acids biosynthesis and catabolism, for this reason limiting its bioavailability results in an attractive therapeutic strategy. Nevertheless, target knockdown studies demonstrated that PanK is not useful as a potential drug target, showing poor vulnerability. However, it is worth noting that, although (15) is a relatively moderate inhibitor of the two enzymes, with IC 50 values about 5-fold higher than (13), the compound has a similar MIC value against MTB. Moreover, metabolic labelling studies demonstrated a similar impairment in lipid metabolism, which derives from the combined depletion of activated CDP-derivatives, necessary for the biosynthesis of phospholipids, and decreased CoA levels. This work thus confirmed how polypharmacology should improve the efficacy of compounds, through the synergistic effect of the simultaneous inhibition of two targets.
## Salicylanilide carbamate compounds
Salicylanilide derivative compounds have multiple biological targets described in literature such as d-Ala-d-Ala ligase, transglycosidase, isocitrate lyase and methionine aminopeptidase, L-alanine dehydrogenase, lysine -aminotransferase, chorismate mutase, pantothenate synthetase, and they are also able to act as uncouplers disrupting the proton gradient. Moreover, many compounds were found to be active against MTB, as well as against different nontuberculous mycobacterial strains, with MIC values in order of few micromolar or submicromolar.
Since carbamates have been reported useful to protect phenolic drugs, Férriz et al. masked the phenolic hydroxyl salicylanilide through carbamate formation, in order to increase the hydrophobicity of the structure thus putatively conferring a better permeability through the MTB cell wall, achieving a series of salicylanilide N-n-alkyl carbamate compounds (16)with MIC values ranging from 0.5-2 µM. Subsequently, further N-cycloalkyl/phenyl/phenylalkyl carbamates have been synthetized. This series (compounds (17),showed significant antibacterial and antitubercular activity, with a reduced cytotoxicity, although the selectivity in many cases remained rather low.
Several salicylanilide derivatives are known to target, among others, the isocitrate lyase ICL1, an enzyme involved in the glyoxylate and methylcitrate cycles, important for MTB persistence, thus offering an advantage in fighting TB at different stages. For this reason, the compounds belonging to the (17) series have been assayed against the recombinant enzymes but resulted only in a very weak inhibition of the activity. Thus, the actual targets of these very promising antitubercular compounds are still to be clearly identified.
## Dualtargeting groel/es chaperonin and protein tyrosine phosphatase b
Protein homeostasis pathways, particularly molecular chaperones are emerging targets for antimicrobial drug discovery. M. tuberculosis possesses two different GroEL/ES chaperonins (GroEL1 and GroEL2) that, together with proteases, maintain cellular protein homeostasis, the former helping proteins to fold, while the latter committed to their proper degradation. Among the two chaperonins, that have a sequence identity of only 61%, only GroEL2 is essential for MTB survival, while GroEL1 is important for the granuloma formation. Thus, the simultaneous inhibition of these two proteins should be efficacious against both active and latent TB. To this purpose, Johnson et al. in 2014 performed a biochemical screen of 700,000 small molecules, identifying 235 compounds that inhibit GroEL/GroES-mediated refolding, 22 of them have been then evaluated for the antibacterial activity. Starting from the benzimidazole based compound (18), the authors developed a series of derived benzoxazoles (19), with variable sulfonamide end caps, showing good activity against Gram positive bacteria. Based on these results, these compounds have been evaluated against MTB, but, to determine if simplifying these inhibitors could reduce their cytotoxicity, the "half-molecules" containing only one sulfonamide end-capping on either right or left sides of the molecule, have also been developed. However, the "full-molecules" were always more potent than the "half-molecules", confirming the importance of both aryl-sulfonamide moieties for inhibition.
Furthermore, the authors noticed some similarity within these compounds and the manner in which the two (oxalylamino-methylene)-thiophene sulfonamide (OMTS) molecules bind the active site of their target, the protein tyrosine phosphatase B (PtpB). Indeed, the analysis of the crystal structure of PtpB in complex with the inhibitor, revealed that the sulfonamides moiety of each OMTS molecule were ∼ 11-12 Å apart, similarly to the (19) compounds, suggesting that they could bind the protein, bridging the distal and proximal parts of the active site. PtpB is an MTB phospho-tyrosine phosphatase secreted into the macrophages cytoplasm that interacts and blocks Erk1/2, p38 mediated IL-6 production, and the Akt signaling, subsequently causing the interference of macrophages immune response, and promoting intracellular survival. In light of this evidence a dual targeting compound able to inhibit both GroEL/ES chaperonin systems and PtpB, should be an effective strategy to treat all stages of tuberculosis. With this aim, two compounds were identified (21) and (22), characterized by a 5-chlorothiophene and a primary amine either on the right or left-hand sides of the structure. These compounds showed moderate antitubercular activity, but a good selectivity over the human counterpart of the target enzymes, being a promising starting point for the development of more potent multitargeting inhibitors.
## Multitargeting of the folate pathway
Folate metabolism has been recently considered an interesting target for potential antitubercular compounds, as this cofactor is essential for several pathways, such as the synthesis of methionine, purines, and of the deoxythymidine monophosphate.
Folate antimetabolites, or antifolates, should block the production of reduced folate through the inhibition of the key enzymes in these metabolic pathways. Among these enzymes, dihydrofolate reductase (DHFR), that catalyzes the reduction of dihydrofolate to tetrahydrofolate has gained interest, as a target for anticancer and antimicrobial therapies. Nevertheless, the currently used antifolates, are not particularly active against MTB, for several reasons. For instance, trimethoprim has a low affinity for the mycobacterial DHFR, while other compounds, such as methotrexate or pyrimethamine show good potency against the target in vitro but are characterized by a low cell membrane permeability. However, promising antifolates are the propargyl-linked antifolates (PLAs) designed as inhibitor of the Staphylococcus Aureus DHFR. As these compounds are supposed to enter cells through passive diffusion, they were supposed to have good permeability also over the MTB cell wall. Indeed, a screening of a PLAs library identified a series of active compounds, that were named ionized nonclassical antifolates (INCAs) (23)-(26).
Interestingly, these compounds have been shown to inhibit also an alternate folate pathway, which relies on two recently discovered enzymes: Rv2671 the second DHFR found in MTB, and the flavin-dependent thymidylate synthase (FDTS). In particular, INCAs showed good activity against both DHFR and Rv2671, with an inhibitory constant in the low nM range, and good selectivity over the human dihydrofolate reductase. Thus, inhibiting two enzymes belonging to an alternate pathway for the same metabolite, INCAs frustrate the functional redundancy that should ensure high levels of reduced folates, leading to considerable potency against MTB, with MIC values in the nM-low µM range. Moreover, a special mention is mandatory for the para-aminosalicylic acid (PAS). Indeed, PAS targets DHFR as a prodrug, and its metabolite PAS-M has shown to act as a competitive substrate for two other enzymes: The dihydropteroate synthase DHPS along the same biosynthetic route and the flavin-dependent thymidylate synthase FDTS in the alternative one. Nowadays, PAS is the only successfully used inhibitor of folate pathway in TB treatments.
## Ethionamide and ethionamide booster co-administration
Several antitubercular compounds, including drugs in clinical practice, are indeed prodrugs, that need enzymatic activation to fulfill their inhibitory effects. Ethionamide (ETH) (27), one of the most widely used second-line drugs for MDR-TB treatment, is converted into the nicotinamide adenine dinucleotide adduct by the Bayer-Villiger monooxygenase EthA. Then, the active adduct acts inhibiting the enoyl-acyl-carrier-protein reductase InhA, impairing the mycolic acids biosynthesis. Since EthA is regulated by the transcriptional repressor EthR, the limited expression of the activator can limit the bioactivation of (27). Thus, to overcome this issue several EthR inhibitors have been developed, that allowed to greatly increase the in vivo potency of (27).
However, the co-administration of ETH and booster have been found to be hampered by several factors, particularly the low water solubility of these compounds and the propensity of (27) to crystallize. To address the issue of the solubility of the compounds, β-cyclodextrin based nanoparticles for the simultaneous co-administration of (27) and the BDM43266 (28) boosterhave been developed. However, these nanoparticles displayed drug loading only of 5%. A great improvement was achieved by the synthesis of a co-drug (29), able to self-associate into nanoparticles. The new compound was obtained by tethering N-hydroxymethyl derivatives of both (27) and (28) through a glutaric linker, potentially cleavable intracellularly by esterase. Interestingly, (29) was found able to self-assemble into nanoparticles in a very stable way, which displayed a drug loading of about 80%. Moreover, a suspension of these nanoparticles was found very active in a mouse model, upon direct intranasal administration into the lung. This compound represents a further example of the flexibility and effectiveness of the multitargeting approach.
## In silico approaches for multitargeting compounds development
The development of effective multitargeting compounds could benefit also from in silico approaches. For instance, Janardhan et al. proposed and implemented an in silico guided polypharmacological approach, based on a combination of pharmacophore and QSAR based virtual screening strategy. Starting from three well recognized antitubercular drug targets, such as InhA, the N-acetyl-glucosamine-1-phosphate uridyltransferase GlmU, and the dihydrodipicolinate reductase DapB, they selected 784 hits from a large database, by structure based and ligand based virtual screening protocols. These structures were then further subjected to docking studies against 33 potential targets, and the 110 potential multitargeting hits identified were subjected to different screening protocols to evaluate different parameters, including cell permeability, drug-likeness, and structural alerts. Finally, they achieved nine structures potentially active against more than 10 different targets, as scaffolds for future designs of selective inhibitors, although to date none of them have been assayed yet. However, considering this approach, Volynets et al.identified a hit compound that efficiently inhibits in vitro the two enzymes leucyl-tRNA synthetase LeuRS and methionyl-tRNA synthetase MetRS, thus confirming the usefulness of in silico studies to develop multitargeting antitubercular agents.
## Conclusions and future perspectives
The global problem of the spread of MDR and XDR MTB strains leads to the necessity of novel approaches for the development of the so called "resistance resistant" drugs. In this context polypharmacology appears very promising, as demonstrated by the numbers of compounds that have been designed or discovered in recent years, here described. Moreover, it is worth noting that the two compounds PAS and SQ109, that are already in clinical practice or trials for TB treatment, have been demonstrated to be multitargeting compounds, thus demonstrating the great potential of this approach for the development of new drugs to eradicate TB, and contrast the spreading of resistant MTB strains.
## Conflicts of interest:
The authors declare no conflict of interest. |
Transcript levels of spindle and kinetochore-associated complex 1/3 as prognostic biomarkers correlated with immune infiltrates in hepatocellular carcinoma
The spindle and kinetochore-associated protein complex (Ska) is an essential component in chromosome segregation. It comprises three proteins (Ska1, Ska2, and Ska3) with theorized roles in chromosomal instability and tumor development, and its overexpression has been widely reported in a variety of tumors. However, the prognostic significance and immune infiltration of Ska proteins in hepatocellular carcinoma (HCC) are not completely understood. The bioinformatics tools Oncomine, UALCAN, gene expression profiling interactive analysis 2 (GEPIA2), cBioPortal, GeneMANIA, Metascape, and TIMER were used to analyze differential expression, prognostic value, genetic alteration, and immune cell infiltration of the Ska protein complex in HCC patients. We found that the mRNA expression of the Ska complex was markedly upregulated in HCC. High expression of the Ska complex is closely correlated with tumor stage, patient race, tumor grade, and TP53 mutation status. In addition, high expression of the Ska complex was significantly correlated with poor diseasefree survival, while the high expression levels of Ska1 and Ska3 were associated with shorter overall survival. The biological functions of the Ska complex in HCC primarily involve the amplification of signals from kinetochores, the mitotic spindle, and (via a MAD2 invasive signal) unattached kinetochores. Furthermore, the expression of the complex was positively correlated with tumorinfiltrating cells. These results may provide new insights into the development of immunotherapeutic targets and prognostic biomarkers for HCC.Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is one of the most common cancers and ranks second among causes of cancer-related deaths 1 . Hepatitis virus infection, alcohol consumption, obesity, and aflatoxin are considered risk factors for HCC 2,3 . In recent years, the treatment of liver cancer has been greatly developed, including arterial chemoembolization, hepatectomy, radiotherapy, and targeted therapy. However, due to frequent late-stage diagnosis, recurrence, and metastasis, the overall 5-year survival rate (7%) remains poor 4 . Many studies have explored the role of immune infiltration-related mechanisms in HCC, in search of specific targets for immunotherapy 5 . Thymocyte selection-associated high mobility group box protein (TOX) and P-selectin glycoprotein ligand-1 (PSGL-1) modulate the tumor microenvironment by depleting CD8 + T cells, and they hold promise as targets for tumor immunotherapy 6,7 . Due to the poor treatment results and low survival rate of HCC, it is particularly important to identify reliable predictive biological targets for early diagnosis and to improve the prognosis of patients through immunotherapy.
Mitotic abnormalities are a common feature of most tumors, and the separation of chromosomes during mitosis is mainly driven by kinetochores attached to specific regions of spindle microtubules [bib_ref] A Kinesin-5, Cin8, recruits protein phosphatase 1 to kinetochores and regulates chromosome..., Suzuki [/bib_ref]. The spindle and kinetochore-associated (Ska) complex is composed of three protein subunits: Ska1, Ska2, and Ska3, which are necessary for the stabilization of kinetochore-spindle microtubule attachment during mitosis [bib_ref] Kinetochore recruitment of the spindle and kinetochore-associated (Ska) complex is regulated by..., Lange [/bib_ref] [bib_ref] Multitasking Ska in chromosome segregation: its distinct pools might specify various functions, Zhang [/bib_ref]. Many studies have shown that dysregulation of the SKA family of genes is associated with a variety of cancers. For example, upregulation of SKA1 expression in esophageal squamous cell carcinoma tissues is associated with tumor differentiation and pathological tumor node metastasis (TNM) stage. Esophageal cancer patients with high expression of SKA1 have a poorer prognosis than patients with low expression [bib_ref] SKA1 overexpression is associated with the prognosis of esophageal squamous cell carcinoma..., Hu [/bib_ref]. SKA2 is significantly upregulated in breast cancer tissues and is associated with TNM stage and lymph node metastasis. High expression of SKA2 promotes invasion and metastasis of breast cancer cells via epithelial-mesenchymal transition (EMT) [bib_ref] SKA2 mediates invasion and metastasis in human breast cancer via EMT, Ren [/bib_ref]. SKA3 expression is also increased in cervical cancer tissues, and cervical cancer patients with high SKA3 expression have a poor prognosis. SKA3 overexpression promotes cervical cancer cell proliferation and migration and accelerates tumor growth [bib_ref] SKA3 promotes cell proliferation and migration in cervical cancer by activating the..., Hu [/bib_ref]. In pancreatic cancer, high expression of SKA1 and SKA3 is associated with poor prognosis and immune cell infiltration. Furthermore, SKA1 has emerged as a prognostic indicator associated with tumor cell infiltration and holds promise as a therapeutic target in adrenocortical carcinoma [bib_ref] Identification of tumor-infiltrating immune cells and prognostic validation of tumor-infiltrating mast cells..., Tian [/bib_ref].
In recent years, the SKA gene family has been increasingly studied in HCC, and previous studies have shown that these genes are highly expressed in HCC [bib_ref] SKA1 overexpression is associated with poor prognosis in hepatocellular carcinoma, Chen [/bib_ref] [bib_ref] SKA2 promotes proliferation and invasion of hepatocellular carcinoma cells via activating the..., Wang [/bib_ref] [bib_ref] SKA3 Promotes tumor growth by regulating CDK2/P53 phosphorylation in hepatocellular carcinoma, Hou [/bib_ref]. However, the potential importance of SKA genes in this disease, especially in prognostic development and immune infiltration, has not been comprehensively elucidated.
The role of the SKA gene family in HCC has been explored with gene sequencing and the use of various bioinformatics databases. In this study, several databases were used for data mining of HCC patients, aiming to systematically and comprehensively explore the gene expression, prognostic value, immune correlation, and potential function of SKA genes in HCC patients. Our study may reveal the molecular mechanisms involved in the expression and regulation of the Ska complex and the development of HCC and could provide reliable targets for HCC diagnosis and treatment.
# Materials and methods
Oncomine database. The Oncomine database (www. oncom ine. org) is a publicly accessible online database that provides an analysis of genome-wide expression with a range of cancer microarray information [bib_ref] ONCOMINE: a cancer microarray database and integrated data-mining platform, Rhodes [/bib_ref]. The expression data of SKA genes in diverse cancer types were obtained from Oncomine. In this study, a Student's t-test was performed on this data with the significance threshold set as follows: P value = 0.05; fold change = 2; gene rank: 10%; data type: mRNA.
UALCAN. UALCAN (http:// ualcan. path. uab. edu/ analy sis. html) is an interactive web resource for in-depth analysis of cancer data from The Cancer Genome Atlas (TCGA) database [bib_ref] UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses, Chandrashekar [/bib_ref]. It was used to analyze the expression of SKA1-3 in both normal and cancerous tissues. Student's t-test was used to generate P values. The P value cutoff was set at 0.05. GEPIA2. GEPIA2 (http:// gepia2. cancer-pku. cn/) is a website for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and Genotype-Tissue Expression (GTEx) projects, using a standard processing pipeline [bib_ref] GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis, Tang [/bib_ref]. In this study, we explored the expression differences of SKA genes in HCC tissues and normal tissues, the analysis of pathological stages, and the related prognostic analysis using the "Single Gene Analysis" module of GEPIA. Student's t-test was used with a critical value for the P value of 0.05. cBioPortal. cBioPortal (www. cbiop ortal. org), an open online tool, can visualize and analyze multidimensional cancer genomics [bib_ref] The cBio cancer genomics portal: an open platform for exploring multidimensional cancer..., Cerami [/bib_ref] [bib_ref] Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal, Gao [/bib_ref]. Based on the TCGA database, genetic alterations and the summary of gene types were analyzed using cBioPortal, as well as the relationship between gene mutations and the prognosis of HCC patients. Statistical significance was set at P < 0.05.
GeneMANIA. GeneMANIA (http:// www. genem ania. org) is a flexible web interface that can generate hypotheses about gene function, analyze gene lists, and prioritize genes for functional assays [bib_ref] The GeneMANIA prediction server: biological network integration for gene prioritization and predicting..., Warde-Farley [/bib_ref]. Using GeneMA-NIA, it was possible to identify the relationships between the Ska proteins and their interactive genes.
Metascape. Metascape (http:// metas cape. org) is a web-based portal that provides gene annotation, enrichment analysis, and a protein-protein interaction (PPI) network based on over 40 independent knowledgebases [bib_ref] Metascape provides a biologist-oriented resource for the analysis of systems-level datasets, Zhou [/bib_ref]. To verify the enrichment of the Ska proteins and the SKA genes, the "Express Analysis" module of Metascape was used.
TIMER. TIMER (http:// timer. cistr ome. org/) is a freely available web server for investigating the infiltration of diverse immune cells and their clinical impact [bib_ref] TIMER2.0 for analysis of tumor-infiltrating immune cells, Li [/bib_ref]. The Ska proteins were submitted to the "Gene module" of TIMER and their correlation with immune cells (B cells, CD4 + T cells, CD8 + T cells, macrophages, neutrophils, and dendritic cells) was explored.
# Results
Abnormal expression of SKA genes in HCC patients. To explore the expression of SKA genes in HCC patients, we first quantified the mRNA expression level using the Oncomine database, which showed that SKA1 expression was dramatically elevated in HCC tissues compared with normal tissues [fig_ref] Figure 1: mRNA expression levels of SKAs in different types of human cancer [/fig_ref]. Specifically, using Chen's dataset, the analysis showed that SKA1 was overexpressed 2.534-fold in liver hepatocellular carcinoma www.nature.com/scientificreports/ (LIHC) specimens. Using the Wurmbach liver dataset [fig_ref] Table 1: Transcription expression of SKAs family members between LIHC and normal liver tissues [/fig_ref] , SKA1 was overexpressed 1.701-fold. UALCAN was also used to analyze the expression of SKA genes in HCC and normal tissues, and the results showed that the expression of SKA1 (P = 1.62e−12), SKA2 (P = 1.62e−12), and SKA3 (P < 1e−12) increased significantly in HCC tissues . We also used the GEPIA2 database to verify the expression of SKA genes in HCC tissues. Consistent with the previous results, the protein expression levels of SKA1, SKA2, and SKA3 were significantly higher in HCC patients . In summary, all three genes in the SKA family were significantly upregulated in HCC patients.
Clinicopathological parameters of SKA genes in HCC patients. We explored the relationship between the SKA gene expression level and the clinical characteristics of patients with HCC, including tumor stage, patient race, tumor grade, and TP53 mutation status. As shown in [fig_ref] Figure 3: Correlation between mRNA expression levels of SKAs and clinicopathological parameters of HCC... [/fig_ref] , SKA gene expression was significantly associated with the stage of HCC, with increased expression correlating with a higher stage. The expression of SKA family genes was significantly upregulated in stages 1, 2, 3, and 4 compared with normal liver tissue, while there was no significant difference between normal tissue and stage 4 HCC, possibly due to the small number of stage 4 cases (n = 6). Among these, there were also significant differences in the expression of SKA genes between stages 1 and 2 and between stages 2 and 3. We also examined the relationship between SKA expression levels and the race of the patients, which showed that Caucasian, African American, and Asian patients all presented with significantly higher expression levels compared with normal tissues, and that Asian patients showed higher expression differences than Caucasian patients [fig_ref] Figure 3: Correlation between mRNA expression levels of SKAs and clinicopathological parameters of HCC... [/fig_ref]. In terms of tumor grade, an increasing grade correlated with increased SKA gene expression [fig_ref] Figure 3: Correlation between mRNA expression levels of SKAs and clinicopathological parameters of HCC... [/fig_ref]. Furthermore, there was significant variability in the expression of SKA genes between levels. Finally, we also examined the relationship between the expression of SKA genes and the mutation status of TP53, which showed that expression of the Ska protein complex was significantly upregulated in patients with TP53 mutations compared with patients without mutations [fig_ref] Figure 3: Correlation between mRNA expression levels of SKAs and clinicopathological parameters of HCC... [/fig_ref]. In general, SKA gene expression levels were significantly correlated with tumor stage, tumor grade, and TP53 mutation status, and Asian patients presented a higher relative increase in the expression of SKA genes than Caucasian and African American patients.
## Prognostic value of ska genes in hcc patients.
To determine the relationship between differential expression of SKA genes and prognosis of HCC patients, we first analyzed the correlation between differential expression and overall survival using GEPIA2. This showed that patients with high expression of SKA1 (P = 0.0023) and SKA2 (P = 0.00042) were primarily associated with shorter overall survival [fig_ref] Figure 4: The prognostic value of mRNA expression level of SKAs in HCC patients [/fig_ref]. The relationship between the differential expression of SKA genes and disease-free survival was also evaluated. We found that patients with high expression of SKA1 (P = 0.00037), SKA2 (P = 0.024), and SKA3 (P = 0.0027) were significantly associated with shorter disease-free survival [fig_ref] Figure 4: The prognostic value of mRNA expression level of SKAs in HCC patients [/fig_ref]. These results suggest that SKA gene expression plays a crucial role in the prognosis of patients with HCC and may become a reliable predictor of survival in these patients.
Frequency changes of SKA genes in HCC patients. We used the cBioPortal database to analyze frequency changes in SKA genes in HCC patients. Fifty-nine (17%) patients had significant alterations in the SKA genes, including missense mutations, amplifications, deep deletions, and transcriptional upregulation. Specifically, the percentages of gene alterations in SKA1, SKA2, and SKA3 were 4%, 10%, and 8%, respectively [fig_ref] Figure 5: Alteration frequency of SKAs and their prognostic value in HCC patients [/fig_ref]. We further explored the impact of gene alterations in the SKA family on the prognosis of patients with HCC, which showed that patients with genetically altered HCC had shorter overall survival than patients with unchanged SKA genes (P = 0.0294). However, there was no relationship between the SKA family gene alterations and disease-free survival (P = 0.0963) in HCC patients [fig_ref] Figure 5: Alteration frequency of SKAs and their prognostic value in HCC patients [/fig_ref].
Co-expression and enrichment of SKA genes in HCC patients. To further explore the role of the SKA genes in HCC patients, we analyzed their regulatory network and their functionally similar genes using the GeneMANIA database. The results showed 20 genes with the strongest correlation, which were nudixhydrolase 5 (NUDT5), kinetochore protein SPC24 (SPC24), kinetochore-associated protein DSN1 homolog (DSN1), kinetochore protein NDC80 homolog (NDC80), SS18-like protein 1 (SS18L1), centromere protein E (CENPE), mitotic checkpoint serine/threonine-protein kinase BUB1 (BUB1), aurora kinase B (AURKB), kinetochore protein SPC25 (SPC25), centromere protein U (CENPU), centromere protein K (CENPK), centromere protein M (CENPM), protein MIS12 homolog (MIS12), DNA excision repair protein ERCC-6-like (ERCC6L), baculoviral IAP repeat containing protein 5 (BIRC5), kinetochore protein Nuf2 (NUF2), shugoshin 1 (SGO1), centromere protein A (CENPA), kinesin family member 18A (KIF18A), ZWILCH kinetochore protein (ZWILCH) [fig_ref] Figure 6: Gene-gene network of SKAs in HCC patients [/fig_ref]. Subsequently, we used Metascape to explore the biological functions of SKA genes and the aforementioned coexpressed genes. The top nine most abundant terms are shown in [fig_ref] Figure 7: The enrichment analysis of SKAs and the 20 co-expressed genes in HCC... [/fig_ref] : amplification of signals from the kinetochores, cell division, chromosome segregation, PID PLK1 pathway, kinetochore organization, microtubule cytoskeleton organization, NDC80 kinetochore complex, microtubule polymerization or depolymerization, and meiotic nuclear division. In addition, we constructed a network map of the enriched terms [fig_ref] Figure 7: The enrichment analysis of SKAs and the 20 co-expressed genes in HCC... [/fig_ref]. To further analyze the relationship between SKA genes and HCC, PPI network maps were constructed, and MCODE component analysis was also performed [fig_ref] Figure 7: The enrichment analysis of SKAs and the 20 co-expressed genes in HCC... [/fig_ref]. The most significantly different MCODE components were extracted from the PPI network graph, and the results showed that there were two significantly different components. MCODE1 was associated with amplification of signals from unattached kinetochores via a MAD2 inhibitory signal, amplification of signals from the kinetochores, and the mitotic spindle checkpoint. MCODE2 was associated with the CENP-H-I complex, CENP-A NAC-CAD complex, and CEN complex. [fig_ref] Figure 8: The relationship between SKAs and tumor immunological features of HCC patients [/fig_ref].
# Discussion
Ska1, Ska2 and Ska3, which constitute the major components of the Ska complex, play a key role in the normal segregation of chromosomes during mitosis. Chromosomal malformation, a peculiar phenomenon of tumors, leads to genomic instability, thereby promoting tumor occurrence and development [bib_ref] Using telomeric chromosomal aberrations to evaluate clastogen-induced genomic instability in mammalian cells, Bolzan [/bib_ref]. An increasing number of studies have demonstrated that Ska proteins play an important role in tumorigenesis, cancer cell proliferation, and apoptosis [bib_ref] SKA3 promotes cell growth in breast cancer by inhibiting PLK-1 protein degradation, Ruan [/bib_ref] [bib_ref] High expression of spindle and kinetochore-associated protein 1 predicts early recurrence and..., Jiang [/bib_ref]. In recent years, immunotherapy has drawn increasing attention in the treatment of cancer; however, the prognostic value and immune infiltration of the Ska proteins in HCC have not been comprehensively explored. This study demonstrated that the Ska proteins and the SKA genes were abnormally highly expressed in HCC, suggesting a link between the dysregulation of these genes and HCC. We further examined the relationship between the expression of SKA genes and the clinical characteristics of patients with HCC. The results showed www.nature.com/scientificreports/ that a higher expression of SKA genes was significantly correlated with tumor stage and pathological grade and that this expression also differed with the ethnicity of the patients, with a greater increase in expression levels in Asian than in Caucasian patients. In addition, there was a correlation between Ska protein complex expression and TP53 mutations. TP53 mutations are the most common mutations in HCC, and they lead to the downregulation of the immune response and differential expression of immune-related genes in HCC [bib_ref] Development and validation of a TP53-associated immune prognostic model for hepatocellular carcinoma, Long [/bib_ref]. It has been shown that receptor activity-modifying protein 3 (RAMP3) in HCC patients may reduce the detrimental effect of TP53 mutations on survival [bib_ref] RAMP3 is a prognostic indicator of liver cancer and might reduce the..., Fang [/bib_ref]. Furthermore, in HCC patients, high expression of SKA1 and SKA2 was notably associated with shorter overall survival, while high expression of the Ska protein complex was markedly associated with shorter disease-free survival. To date, many studies have confirmed that SKA genes play an important role in HCC. One study examined 166 HCC and paired adjacent normal tissues and found that SKA1 was highly expressed in HCC and correlated with tumor size and TNM stage [bib_ref] SKA1 overexpression is associated with poor prognosis in hepatocellular carcinoma, Chen [/bib_ref]. Another study found that LINC00339 could [bib_ref] LINC00339 promotes growth and invasiveness of hepatocellular carcinoma by the miR-1182/SKA1 pathway, Xiao [/bib_ref]. It has also been confirmed that SKA2 can accelerate HCC progression by upregulating Wnt/β-catenin signaling [bib_ref] Spindle and kinetochore-associated protein 2 facilitates the proliferation and invasion of hepatocellular..., Jiang [/bib_ref]. Our findings regarding SKA gene expression in HCC are in agreement with those of a previous study. Therefore, we speculated that individual SKA genes or SKA family genes could serve as potential prognostic biomarkers for patients with HCC. However, the effects of SKA genes on the development, metastasis, cell proliferation, and apoptosis of HCC have not been comprehensively studied. The occurrence and development of HCC is complex and multifaceted, and genetic changes play a role in this process [bib_ref] Integrative analysis reveals novel driver genes and molecular subclasses of hepatocellular carcinoma, Yang [/bib_ref]. Therefore, we explored the molecular characteristics of SKA genes in HCC. In HCC, the differential expression of SKA genes often undergo genetic changes, which are relevant to the overall survival rate. The most significant genetic change was the increase in mRNA expression.
Previous studies have revealed that Ska proteins function in other diseases, mainly by regulating chromosome segregation [bib_ref] Integrating pathology, chromosomal instability and mutations for risk stratification in early-stage endometrioid..., Li [/bib_ref] [bib_ref] Ensemble-level organization of human kinetochores and evidence for distinct tension and attachment..., Roscioli [/bib_ref]. In this study, we explored the core genes underlying the function of Ska proteins, some of which have been identified as regulators of these proteins. Redli et al. [bib_ref] The Ska complex promotes Aurora B activity to ensure chromosome biorientation, Redli [/bib_ref] reported that Ska proteins promote AURKB activity to limit their own microtubule and mitochondria association and ensure that kinetochore-microtubule (KT-MT) dynamics and stability fall within an optimal bi-directional equilibrium range. Sivakuma et al. [bib_ref] Phosphatase-regulated recruitment of the spindle-and kinetochore-associated (Ska) complex to kinetochores, Sivakumar [/bib_ref] demonstrated that NUF2 binding to Ska1 promotes the recruitment of the Ska complex to kinetochores, reducing metaphase arrest upon chromosome segregation. This implicates NUF2 as a potential regulator of Ska1. NDC80 has also been shown to influence the recruitment of the Ska complex [bib_ref] Mechanism of Ska recruitment by Ndc80 complexes to kinetochores, Janczyk [/bib_ref]. In our study, we performed functional enrichment analysis to understand the biological functions of the SKA genes. The results suggest that these genes are mainly involved in the amplification of signals from the kinetochores and mitochondrial spindle checkpoint, and further amplification of signals from unattached kinetochores occurs via a MAD2 inhibitory signal. Our results concur with those of a previous report 42 that high expression of the mitotic checkpoint protein MAD2 in the mammary gland of mice resulted in mitotic checkpoint hyperactivation, mitotic arrest, and retarded tumor growth. This suggests that SKA genes play a crucial role in the development and progression of tumors.
Accumulating evidence suggests that immune cell infiltration can influence tumorigenesis and recurrence and serve as an important determinant of immunotherapy response and clinical outcome [bib_ref] The prognostic landscape of tumor-infiltrating immune cell and immunomodulators in lung cancer, Liu [/bib_ref]. Similarly, the immune microenvironment of tumors plays an important role in HCC [bib_ref] Deviations of the immune cell landscape between healthy liver and hepatocellular carcinoma, Rohr-Udilova [/bib_ref]. CD8 + cytotoxic T lymphocytes can specifically identify major histocompatibility complex (MHC) antigens, which are widely used in tumor-targeted therapies [bib_ref] RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize..., Duewell [/bib_ref]. One study showed that an elevated ratio of CD4+/CD8+ T cells was associated with a favorable prognosis in HCC [bib_ref] High-intensity focused ultrasound ablation combined with transcatheter arterial chemoembolization improves long-term efficacy..., Sun [/bib_ref]. It has also been shown that the co-expression of PD-1 and T-cell immunoglobulin and tyrosine inhibitory motif domain (TIGIT) in CD4+ and CD8+ T cells of HCC patients was significantly increased and negatively correlated with the overall survival and disease-free survival of patients [bib_ref] PD-1(+) TIGIT(+) CD8(+) T cells are associated with pathogenesis and progression of..., Liu [/bib_ref]. Our study suggests that the expression levels of SKA genes may be significantly associated with the infiltration of immune cells (B cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells). This suggests that SKA genes not only respond to the prognosis of HCC but also reflect the immune status of the disease and can provide new insights into HCC immunotherapy. www.nature.com/scientificreports/ In addition, it must be acknowledged that our study has some limitations. First, we obtained data from several different databases, and it was difficult to guarantee that the data were consistent. Moreover, experimental validation of these data has not been performed at the time of writing, but this will be performed in our future work.
# Conclusions
We found that SKA gene expression levels were highly elevated in HCC. Ska1 and Ska3 could be considered potential prognostic markers. SKA gene expression was also significantly associated with the infiltration of immune cells (B cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells), which indicated that Ska proteins may regulate the development of HCC by influencing the immune microenvironment. Inhibition of Ska protein expression and function, potentially in combination with immunotherapies, could represent a promising treatment strategy for patients with HCC.
[fig] Figure 1: mRNA expression levels of SKAs in different types of human cancer (Oncomine). Red indicates high expression. Blue indicates low expression. P < 0.05, fold-change > 2 and gene rank = 10% were considered statistically significant. Numbers in each cell represent the data set meeting the threshold. SKA spindle and kinetochore-associated complex.Immune cell infiltration of Ska proteins in HCC patients.Immune infiltration is associated with the development of cancer. In the present study, the TIMER database was used to analyze the correlation between SKA gene expression (and therefore Ska protein expression) and immune infiltration.All three SKA genes showed positive correlation between expression and immune cell infiltration. The expression of SKA1 was positively correlated with B cells (Rho = 0.38, P = 2.90e−13), CD4 + T cells (Rho = 0.206, P = 1.16e−4), macrophages (Rho = 0.304, P = 8.41e−9), neutrophils (Rho = 0.412, P = 8.32e−3), and dendritic cells (Rho = 0.477, P = 4.93e−21) (Fig. 8a). SKA2 was also positively correlated with B cells (Rho = 0.241, P = 5.97e−6), CD8 + T cells (Rho = 0.135, P = 1.23e−2), CD4 + T cells (Rho = 0.205, P = 1.3e−4), macrophages (Rho = 0.31, P = 34.29e−9), neutrophils (Rho = 0.237, P = 8.47e−6), and dendritic cells (Rho = 0.336, P = 1.47e−10) (Fig. 8b). Similarly, there was a positive correlation between the expression of SKA3 and the infiltration of B cells (Rho = 0.42, P = 3.66e−12), CD4 + T cells (Rho = 0.237, P = 8.34e−6), macrophages (Rho = 0.298, P = 1.67e−8), neutrophils (Rho = 0.146, P = 6.43e−3), and dendritic cells (Rho = 0.498, P = 4.73e−23) [/fig]
[fig] Figure 3: Correlation between mRNA expression levels of SKAs and clinicopathological parameters of HCC patients. (UALCAN). (a) Correlation between mRNA expression levels of SKAs and tumor stages of HCC patients. (b) Correlation between mRNA expression levels of SKAs and race of HCC patients. (c) Correlation between mRNA expression levels of SKAs and tumor grade of HCC patients. (d) Correlation between mRNA expression levels of SKAs and TP53 mutation status of HCC patients. [/fig]
[fig] Figure 4: The prognostic value of mRNA expression level of SKAs in HCC patients (GEPIA2). (a) The relationship between SKAs expression and OS in HCC patients. (b) The relationship between SKAs expression and DFS in HCC patients. OS overall survival; DFS disease-free survival. [/fig]
[fig] Figure 5: Alteration frequency of SKAs and their prognostic value in HCC patients (cBioPortal). (a,b) Summary of alterations in the SKAs in HCC patients. (c,d) K-M plots curve of OS and DFS in HCC patients with/without the SKAs alterations. [/fig]
[fig] Figure 6: Gene-gene network of SKAs in HCC patients (GeneMANIA). GeneMANIA database identified 20 genes most associated with SKAs. [/fig]
[fig] Figure 7: The enrichment analysis of SKAs and the 20 co-expressed genes in HCC patients (Metascape). (a) Bar chart of the first nine enriched terms for SKAs and the 20 co-expressed genes. (b) Net graph of enriched terms, Different colors represent different cluster ID. (c,e) PPI network and MCODE components identified. Scientific Reports | (2021) 11:11165 | https://doi.org/10.1038/s41598-021-89628-z [/fig]
[fig] Figure 8: The relationship between SKAs and tumor immunological features of HCC patients. (a) The relationship between SKA1 and immune infiltrating cells. (b) The relationship between SKA2 and immune infiltrating cells. (c) The relationship between SKA3 and immune infiltrating cells. [/fig]
[table] Table 1: Transcription expression of SKAs family members between LIHC and normal liver tissues (Oncomine). [/table]
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The glutamate transport inhibitor DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells
Background: Colorectal cancer (CRC) is a leading cause of cancer death globally and new biomarkers and treatments are severely needed.Methods: Here, we employed HCT116 and LoVo human CRC cells made resistant to either SN38 or oxaliplatin, to investigate whether altered expression of the high affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3 (EAAT3, EAAT1) is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability and glutathione content. Results were evaluated using one-and two-way ANOVA and Students two-tailed t-test, as relevant.Results: In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated~60 % and up-regulated~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [ 3 H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu 2+ -transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction.Conclusions: SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point to novel treatment options in treatment-resistant CRC.
# Background
Colorectal cancer (CRC) is the fourth most common cause of cancer death worldwide [bib_ref] Cancer incidence and mortality patterns in Europe: estimates for 40 countries in..., Ferlay [/bib_ref] [bib_ref] Estimates of worldwide burden of cancer in 2008: GLOBOCAN, Ferlay [/bib_ref]. Currently, treatment of CRC is based on combination of 5-fluorouracil (5-FU) and leucovorin [bib_ref] Clinical, laboratory and molecular factors predicting chemotherapy efficacy and toxicity in colorectal..., Chua [/bib_ref] [bib_ref] Molecular predictors of response to chemotherapy in colorectal cancer, Dienstmann [/bib_ref] [bib_ref] In vitro drug responses in primary and metastatic colorectal cancers, Mechetner [/bib_ref] with other chemotherapeutic drugs. In addition, despite frequent resistance development, targeted treatment with the epidermal growth factor receptor (EGFR) inhibitor cetuximab or the angiogenesisinhibitory antibody bevacizumab is successful in some patients [bib_ref] Molecular predictors of response to chemotherapy in colorectal cancer, Dienstmann [/bib_ref]. The combination treatments FOLFOX (5-FU + leucovorin + oxaliplatin) [bib_ref] Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer, Andre [/bib_ref] and FOLFIRI (5-FU + leucovorin + irinotecan) [bib_ref] CPT-11 (irinotecan) addition to bimonthly, high-dose leucovorin and bolus and continuous-infusion 5-fluorouracil..., Andre [/bib_ref] significantly prolong progression-free survival in advanced CRC, the choice between irinotecan and oxaliplatin being largely dictated by toxicity issues [bib_ref] Differential toxicity biomarkers for irinotecan-and oxaliplatin-containing chemotherapy in colorectal cancer, Cortejoso [/bib_ref]. Oxaliplatin is a diaminocyclohexane platinum derivative which induces formation of DNA adducts, and irinotecan is the precursor of the topoisomerase-I inhibitor 7-ethyl-10-hydroxycamptothecin (SN38). Both compounds induce DNA damage, upregulation of p53 and p21 WAF1/Cip1 , cell cycle arrest, and cell death [bib_ref] Cellular pharmacology of the combination of the DNA topoisomerase I inhibitor SN-38..., Zeghari-Squalli [/bib_ref] [bib_ref] p53 dependent and independent sensitivity to oxaliplatin of colon cancer cells, Toscano [/bib_ref] [bib_ref] Antisense Bcl-xl down-regulation switches the response to topoisomerase I inhibition from senescence..., Hayward [/bib_ref]. The majority of patients with metastatic CRC, whether on FOL-FOX or FOLFIRI, will experience treatment resistance and disease progression upon treatment, leaving only limited additional treatment options. Possible remedies to this include the development of drugs that do not exhibit cross-resistance with those currently used, and of predictive biomarkers ensuring that patients receive the treatment with the highest likelihood of effect [bib_ref] In vitro drug responses in primary and metastatic colorectal cancers, Mechetner [/bib_ref]. Although progress has been made in recent years, strong biomarkers predicting response to oxaliplatin or irinotecan are lacking and urgently needed [bib_ref] Clinical, laboratory and molecular factors predicting chemotherapy efficacy and toxicity in colorectal..., Chua [/bib_ref] [bib_ref] Molecular predictors of response to chemotherapy in colorectal cancer, Dienstmann [/bib_ref] [bib_ref] A colorectal cancer classification system that associates cellular phenotype and responses to..., Sadanandam [/bib_ref].
To gain insight into the molecular mechanisms underlying chemotherapy resistance, we developed drug-resistant human CRC cell lines based on the well-characterized HCT116 and LoVo cell lines. Sublines resistant to SN38 and oxaliplatin, respectively, were established by long-term exposure to increasing doses of these drugs. The cell lines developed exhibit little cross-resistance between SN38 and oxaliplatin [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref]. Microarray analyses demonstrated marked changes in mRNA profiles of resistant cells compared to their parental counterparts. Among these, we noted major changes in mRNA levels of the high affinity excitatory amino acid transporters (or glutamate transporters) Solute Carrier (SLC) 1A1 and -1A3 (EAAT3 and EAAT1, respectively), in the resistant cell lines [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref]. Studies of plasma membrane transport proteins in chemotherapy-resistant tumor cells have generally focused on ABC-transporters [bib_ref] Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant..., Januchowski [/bib_ref] [bib_ref] Membrane transporters and channels: role of the transportome in cancer chemosensitivity and..., Huang [/bib_ref]. However, a number of properties make the SLC1A family (SLC1A1-A7) very interesting in this context. Although some isoforms, including SLC1A1 and SLC1A3, are also found in peripheral tissues, the SLC1A family is by far most widely expressed in the brain [bib_ref] The SLC1 high-affinity glutamate and neutral amino acid transporter family, Kanai [/bib_ref] [bib_ref] Primary structure and functional characterization of a high-affinity glutamate transporter, Kanai [/bib_ref] [bib_ref] Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma..., Varini [/bib_ref]. SLC1A family transporters mediate cellular uptake of glutamate, driven by 3Na + ,1H + cotransport, 1 K + counter-transport. In addition, SLC1A1 has high capacity for transporting L-cysteine, a precursor in glutathione synthesis [bib_ref] The SLC1 high-affinity glutamate and neutral amino acid transporter family, Kanai [/bib_ref]. Slc1a1 and Slc1a3 knockout mice show retinal ganglion cell degeneration, altered brain glutamate homeostasis, and increased oxidative stress sensitivity [bib_ref] The potential role of glutamate transporters in the pathogenesis of normal tension..., Harada [/bib_ref] , and Slc1a1 knockout mice exhibit brain atrophy and reduced neuronal levels of the antioxidant tripeptide (glutamate, cysteine, glycine) glutathione [bib_ref] Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse, Aoyama [/bib_ref] , consistent with a role for these transporters in glutathione synthesis. A few studies reported altered expression and localization of glutamate transporters in CNS [bib_ref] Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters..., Ye [/bib_ref] and non-CNS [bib_ref] Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma..., Varini [/bib_ref] cancers. Gliomas down-regulate SLC1A family transporters and switch from net uptake to net efflux of glutamate. This stimulates their growth and motility in an autocrine fashion, while exerting toxic effects on surrounding neurons [bib_ref] Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters..., Ye [/bib_ref] [bib_ref] Glioma cells release excitotoxic concentrations of glutamate, Ye [/bib_ref] [bib_ref] Autocrine glutamate signaling promotes glioma cell invasion, Lyons [/bib_ref]. Furthermore, increased levels of reduced glutathione (GSH) have been associated with chemotherapy resistance in several cancer types [bib_ref] Glutathione in cancer biology and therapy, Estrela [/bib_ref]. However, the possible role of glutamate transporters in CRC chemotherapy resistance has, to our knowledge, never been addressed.
The aim of this study was to investigate the regulation and possible roles of glutamate transporters SLC1A1 and SLC1A3 in SN38-and oxaliplatin-resistance in CRC. We show that SLC1A1 expression and glutamate transporter activity are altered in a parallel manner in SN38-resistant CRC cells. The glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while strongly attenuating that induced by oxaliplatin. Collectively, our findings indicate that changes in glutamate transporter expression and activity may be relevant to the prediction and treatment of CRC chemotherapy resistance, and that cotreatment with DL-TBOA may be beneficial in combination with irinotecan, but detrimental in combination with oxaliplatin treatment.
Part of this work has previously been reported in abstract form.
# Results
## Expression and activity of glutamate transporters are altered in resistant crc cells
Our recent microarray analysis pointed to robust changes in the expression of glutamate transporters SLC1A1 and SLC1A3 upon resistance development in both HCT116 cells and LoVo cells (Additional file 1: [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref]. Strikingly, analysis of publically available CRC patient tissue data (www.oncomine.org; [bib_ref] Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer..., Rhodes [/bib_ref] showed a significant down-regulation of SLC1A1 mRNA levels in CRC compared to normal tissue in 11 out of 15 datasets, while SLC1A3 expression was generally unaltered (Additional file 1: .
We therefore asked whether changes in SLC1A1 and SLC1A3 expression were involved in resistance development in HCT116 and LoVo cells. Consistent with the microarray data, qPCR analysis showed that the SLC1A1 mRNA level was down-regulated in HCT116-SN38 cells compared to that in parental cells . The SLC1A3 mRNA level was increased in oxaliplatin-resistant HCT116 cells and unaffected in SN38-resistant HCT116 cells. In LoVo cells, both SLC1A1 and SLC1A3 mRNA levels were increased in SN38-resistant cells and unaffected in oxaliplatin-resistant cells, compared to the levels in parental cells .
Protein levels of SLC1A1 followed the same pattern as the mRNA levels, i.e. SLC1A1 protein expression was down-regulated in SN38-resistant HCT116 cells, and increased in oxaliplatin-resistant HCT116 cells and SN38resistant LoVo cells, compared to parental levels . For SLC1A3, no protein band of the expected size was detectable for either of the reported splice variants (~60 and 55 kDa) [bib_ref] A novel alternative splicing form of excitatory amino acid transporter 1 is..., Vallejo-Illarramendi [/bib_ref] , using 3 different antibodies which all gave clear bands of correct size in positive control mouse brain tissue (not shown). Although other scenarios are possible, this suggests that the SLC1A3 protein level is very low in CRC cells.
As glutamate transporter activity and membrane localization are heavily posttranslationally regulated [bib_ref] Mechanisms of glutamate transport, Vandenberg [/bib_ref] , expression levels alone do not reveal whether transport activity is altered. We therefore next determined glutamate transporter activity (as uptake of the substrate [ 3 H]-D-Asp following a 6-min incubation in buffer supplemented with a tracer concentration of 100 nM [ 3 H]-D-Asp). Data are shown in [bib_ref] The SLC1 high-affinity glutamate and neutral amino acid transporter family, Kanai [/bib_ref] [bib_ref] Mechanisms of glutamate transport, Vandenberg [/bib_ref] [bib_ref] Discovery of the first selective inhibitor of excitatory amino acid transporter subtype..., Jensen [/bib_ref]. IC 50 values of DL-TBOA for SLC1A1 and SLC1A3 in uptake assays are in the low micromolar range, depending on the system and experimental setup [bib_ref] Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3..., Jensen [/bib_ref] [bib_ref] Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers, Shimamoto [/bib_ref] , whereas UCPH-101 exhibits high-nanomolar IC 50 values for SLC1A3 and is inactive at SLC1A1 at concentration up to > 400 fold higher [bib_ref] Discovery of the first selective inhibitor of excitatory amino acid transporter subtype..., Jensen [/bib_ref]. In all cell lines, basal [ 3 H]-D-Asp uptake was inhibited by DL-TBOA with IC 50 values around 2 μM, whereas it was essentially unaffected by UCPH-101 at concentrations up to 100 μM. Basal [ 3 H]-D-Asp uptake was decreased by about 60 % in SN38-resistant compared to parental HCT116 cells, whereas that in SN38-resistant LoVo cells was nearly tripled compared to parental LoVo cells. In the oxaliplatin-resistant cell lines, [ 3 H]-D-Asp uptake was slightly decreased in the HCT116 model, and unaltered in the LoVo model.
Collectively, these data show that SLC1A1 mRNA and protein expression and DL-TBOA-sensitive, UCPH-101insensitive [ 3 H]-D-Asp uptake are decreased in SN38resistant HCT116 cells and increased in SN38-resistant LoVo cells, compared to their parental controls, while neither SLC1A3 protein or activity could be detected in any of the cell lines.
Viability of SN38-and oxaliplatin-resistant CRC cells is differentially affected by DL-TBOA
To determine whether glutamate transporter activity contributed to the SN38-and oxaliplatin-resistant phenotypes, we next assessed viability, first by MTT assay . Viability of parental HCT116 and LoVo , f ) cell lines was reduced after 48 h exposure to SN38 or oxaliplatin, with about 20 % viable cells remaining after 48 h at the highest dose tested (0.8 μM SN38 or 20 μM oxaliplatin, respectively). Addition of DL-TBOA (70 or 350 μM) concomitantly with the chemotherapeutic drugs if anything slightly exacerbated the SN38-induced loss of viability in parental cell lines . In contrast, DL-TBOA counteracted the effect of oxaliplatin on viability in both parental cell lines . This was particularly evident in LoVo cells, in which 350 μM DL-TBOA essentially abolished the loss of viability induced by 0.8 μM oxaliplatin . Notably, the DL-TBOA-induced increase in viability was specific to oxaliplatin-treated cells, as untreated cells consistently showed a small decrease in viability upon DL-TBOA treatment .
We next determined whether SN38-and oxaliplatinresistance was associated with changes in the impact of DL-TBOA on viability. Indeed, in SN38-resistant HCT116 ( The MTT assay measures mitochondrial conversion of tetrazolium salt to formazan [bib_ref] Use of the tetrazolium assay in measuring the response of human tumor..., Price [/bib_ref]. Although this is generally a good measure of cell viability, artifacts can arise if mitochondrial activity changes without parallel changes in viability. To determine viability by an independent method we therefore DAPI-labeled nuclei and quantified the surviving, still adherent cells by high-throughput confocal microscopy. The opposite effects of DL-TBOA on SN38and oxaliplatin-induced loss of viability are also evident in this assay, strongly indicating that the effects of DL-TBOA primarily reflect changes in cell viability (Additional file 2: .
Taken together, this data shows that DL-TBOA enhances SN38-induced, and counteracts oxaliplatin-induced, cell death.
## Expression of the cu 2+ transporter ctr1 is unaffected by dl-tboa
The marked and specific reversal of oxaliplatin-induced cell death by DL-TBOA suggested that an oxaliplatin import mechanism might be inhibited by DL-TBOA. The high-affinity Cu 2+ transporter CTR1 is a major such pathway [bib_ref] The role of the mammalian copper transporter 1 in the cellular accumulation..., Larson [/bib_ref]. We therefore hypothesized that DL-TBOA-induced rescue of CRC cells from oxaliplatininduced death might reflect CTR1 down-regulation. To avoid confounding effects of the substantial death induction seen at 48 h, CTR1 levels were assessed after 24 h of chemotherapy +/− DL-TBOA. Oxaliplatin treatment tended to reduce CTR1 protein expression in all cell lines except parental HCT116, HCT116-Oxa, and LoVo-Oxa cells, yet without detectable effects of DL-TBOA on the CTR1 protein level .
## Cellular gsh is increased in resistant hct116 cells, but only marginally affected by dl-tboa
In light of the importance of SLC1A1 in regulation of L-cysteine transport and cellular GSH homeostasis [bib_ref] The SLC1 high-affinity glutamate and neutral amino acid transporter family, Kanai [/bib_ref] [bib_ref] The potential role of glutamate transporters in the pathogenesis of normal tension..., Harada [/bib_ref] [bib_ref] Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse, Aoyama [/bib_ref] and the role of increased GSH levels in chemotherapy resistance in several cancer types [bib_ref] Glutathione in cancer biology and therapy, Estrela [/bib_ref] , we next asked whether resistance development and DL-TBOA treatment were associated with changes in cellular GSH level. Notably, the steady state intracellular GSH level was increased in both SN38-and oxaliplatin-resistant HCT116 cells, yet unaltered in the resistant LoVo strains [fig_ref] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels [/fig_ref]. After a 24 h treatment with SN38 or oxaliplatin, parental HCT116 cells showed slightly increased GSH levels, and a trend towards decreased GSH levels was seen in SN38 resistant cells [fig_ref] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels [/fig_ref]. In contrast, oxaliplatin-resistant HCT116 cells [fig_ref] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels [/fig_ref] and all LoVo cell lines [fig_ref] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels [/fig_ref] showed no detectable changes in cellular GSH levels upon treatment. There was no detectable effect of DL-TBOA on GSH levels.
## P53 induction by sn38 and oxaliplatin is decreased by dl-tboa
We next explored the impact of SN38, oxaliplatin and DL-TBOA on protein levels of p53 and p21 WAF1/Cip (p21), major cell survival-and proliferation regulators induced by DNA damage after SN38 and oxaliplatin treatment [bib_ref] Cellular pharmacology of the combination of the DNA topoisomerase I inhibitor SN-38..., Zeghari-Squalli [/bib_ref] [bib_ref] p53 dependent and independent sensitivity to oxaliplatin of colon cancer cells, Toscano [/bib_ref] [bib_ref] Antisense Bcl-xl down-regulation switches the response to topoisomerase I inhibition from senescence..., Hayward [/bib_ref] , and on PARP-1 cleavage, a wellcharacterized indicator of apoptosis induction. In parental HCT116 cells, p53 and p21 were markedly induced by 24 h treatment with SN38 or oxaliplatin [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref] and Additional file 3: , consistent with the known DNA damage induction by both drugs [bib_ref] Cellular pharmacology of the combination of the DNA topoisomerase I inhibitor SN-38..., Zeghari-Squalli [/bib_ref] [bib_ref] p53 dependent and independent sensitivity to oxaliplatin of colon cancer cells, Toscano [/bib_ref] [bib_ref] Antisense Bcl-xl down-regulation switches the response to topoisomerase I inhibition from senescence..., Hayward [/bib_ref]. In SN38resistant HCT116 cells, this response to oxaliplatin was retained, while, as expected, SN38 had essentially no effect on p53 expression, yet modestly increased p21 expression. Conversely, in oxaliplatin-resistant cells, only SN38 induced p53 and p21 expression [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref] and Additional file 3: . PARP-1 cleavage was induced by SN38 in parental and oxaliplatin-resistant, yet not in SN38-resistant, cells (Additional file 3: . A comparable pattern was seen for the LoVo cell lines [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref] and Additional file 4: [fig_ref] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels [/fig_ref]. Notably, treatment with DL-TBOA concomitant to the chemotherapeutic compounds induced an apparent decrease in p53 induction compared to chemotherapy alone, in both parental and drug-resistant cell lines [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref]. As p53 affects both proliferation and death pathways, we next (See figure on previous page.) Expression and activity of SLC1A1 and SLC1A3 is altered in SN38-and oxaliplatin-resistant CRC lines. a Relative mRNA levels of SLC1A1 and SLC1A3 in parental (PAR), SN38-and oxaliplatin-resistant HCT116 and LoVo cells, determined by qPCR analysis. b Protein levels of SLC1A1 in parental, SN38-and oxaliplatin-resistant HCT116 and LoVo cells relative to that in their parental counterparts. Representative Western blots (p150 serves as a loading control) and densitometric quantification of the Western blot data are shown. The qPCR and Western blot data represent 3 independent experiments per condition. *) p < 0.05, **) p < 0.01, and ***) p < 0.001, compared to parental cells by one-way ANOVA and Dunnett post-test. c-d asked whether DL-TBOA affected proliferation, using retinoblastoma protein phosphorylation on Ser 807/811 (pRb) as a well-established marker of active cell cycling (Additional file 5: [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref]. In the resistant (but, unexpectedly not in the parental), cell lines, the pRb level was decreased by the chemotherapy treatment to which the cell lines were sensitive, confirming that the treatments impact on proliferation. While these effects did not reach statistical significance, DL-TBOA also tended to increase pRb levels under control conditions in both Effects of SLC1A1 knockdown and -overexpression on SN38-and oxaliplatin-induced cell death DL-TBOA is a non-selective inhibitor of all SLC1A isoforms, thus the observed effects of DL-TBOA in the cells could potentially arise from its activity at SLC1A1, −A2, and/or -A6-7, whereas the lack of effect of UCPH-101 rules out the involvement of SLC1A3. We therefore asked whether p53 levels were similarly affected by siRNAmediated SLC1A1 knockdown. About 50 % and 30 % SLC1A1 knockdown was obtained in resistant HCT116 and LoVo cell lines, respectively [fig_ref] Figure 6: Effects of SLC1A1 siRNA on cell death and survival parameters after chemotherapy... [/fig_ref]. In LoVo, but not in HCT116 cells, SLC1A1 knockdown tended to reduce the oxaliplatin-induced increase in p53 protein level seen after DL-TBOA treatment, however, this effect was less marked than that seen after DL-TBOA treatment (compare with [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref]. Overexpression of SLC1A1 had no detectable effect on p53, p21, or PARP cleavage in any of the cell lines (Additional file 6: [fig_ref] Figure 6: Effects of SLC1A1 siRNA on cell death and survival parameters after chemotherapy... [/fig_ref].
## Effects of sn38-, oxaliplatin and dl-tboa on subcellular localization of slc1a1 in hct116 cells
To address the question of whether altered SLC1A1 localization was involved in the effects of SN38, oxaliplatin and DL-TBOA, we performed immunofluorescence analysis of the parental and resistant cell lines, in absence and presence of chemotherapeutics and DL-TBOA. SLC1A1 localization is shown in [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref]. In Additional file 7: [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref] the same images are shown merged with DAPI and F-actin staining. SLC1A1 is predominantly localized in intracellular vesicles, from where it undergoes regulated trafficking to the plasma membrane upon specific stimuli [bib_ref] Changes in the expression of the glutamate transporter EAAT3/EAAC1 in health and..., Bianchi [/bib_ref]. In agreement with this, SLC1A1 localized partially to the membrane and partially in a cytosolic compartment in both parental and SN38-resistant cells under control conditions [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref]. In contrast, in oxaliplatin-resistant cells, SLC1A1 staining was predominantly seen in the perinuclear/nuclear region under control conditions [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref].
In parental cells, treatment with SN38 or oxaliplatin induced a marked shift in SLC1A1 localization towards the perinuclear/nuclear region [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref]. In these cells, DL-TBOA had no detectable effect on SLC1A1 localization, either alone or in combination with the chemotherapeutic agents [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref]. In SN38-resistant cells, addition of DL-TBOA to the chemotherapeutic treatment increased the fraction of SLC1A1 fluorescence localized to the perinuclear/nuclear compartment, and a similar trend was seen with DL-TBOA alone [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref]. Notably, in oxaliplatinresistant cells, a greater fraction of SLC1A1 was intracellular under control-and oxaliplatin-treated conditions, and this was partially reversed by DL-TBOA [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref].
# Discussion
Resistance to irinotecan (of which SN38 is the active metabolite) and oxaliplatin is a major problem in CRC treatment, and the mechanisms of resistance remain incompletely understood. A major finding of this study is the striking difference between the effects of the glutamate transport inhibitor DL-TBOA on viability after SN38-and oxaliplatin treatment: DL-TBOA modestly exacerbated the loss of viability in untreated or SN38-treated cells, whereas it markedly counteracted oxaliplatin-induced cell loss. This suggests that glutamate transporter activity has a specific, negative impact on oxaliplatin-induced death, The SLC1A1 protein levels paralleled its mRNA levels, whereas SLC1A3 protein expression was not detectable. Basal glutamate transporter activity largely, but not completely, paralleled SLC1A1 expression and was inhibited by L-glutamate and by the broad glutamate transporter inhibitor DL-TBOA, but not by UCPH-101, a specific SLC1A3 inhibitor. Collectively, this suggests that SLC1A1 is at least partially responsible for the observed glutamate transporter activity. Although this is, to our knowledge, the first study to demonstrate SLC1A1 protein and activity changes in drug-resistant cancer cells, altered SLC1A1 mRNA expression was also reported in ovarian cancer cells and in the NCI-60 cancer cell line panel [bib_ref] Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant..., Januchowski [/bib_ref] [bib_ref] Membrane transporters and channels: role of the transportome in cancer chemosensitivity and..., Huang [/bib_ref] , suggesting a more widespread relevance than CRC. In both HCT116 and LoVo cells, changes in [ 3 H]-D-Asp uptake [fig_ref] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy... [/fig_ref] shows the same images, merged with staining for DAPI and Rhodamine-conjugated phalloidin to visualize localization of nuclei and F-actin, respectively upon SN38 resistance development were quantitatively more pronounced than the changes in SLC1A1 protein level, and in oxaliplatin-resistant HCT116 cells, SLC1A1 expression was modestly increased, yet [ 3 H]-D-Asp uptake modestly decreased. While not further addressed here, this suggests an additional role for posttranslational regulation of SLC1A1 activity, and/or, contributions from other SLC1A isoforms.
## Possible mechanisms involved in the effect of glutamate transporter inhibition on viability
We first hypothesized that the rescue of oxaliplatintreated cells by DL-TBOA might reflect a dependence of oxaliplatin influx pathway(s) on glutamate transporter activity. The protein level of the major such pathway, CTR1, was modestly decreased by oxaliplatin treatment, yet was not regulated by DL-TBOA. Another possibility was that SLC1A1 might modulate cellular GSH levels, which also regulate oxaliplatin uptake via CTR1 [bib_ref] Elevated glutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of..., Chen [/bib_ref]. SLC1A1 can directly transport L-cysteine and plays a major role in supporting GSH production [bib_ref] The SLC1 high-affinity glutamate and neutral amino acid transporter family, Kanai [/bib_ref] [bib_ref] The potential role of glutamate transporters in the pathogenesis of normal tension..., Harada [/bib_ref] [bib_ref] Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse, Aoyama [/bib_ref] , and also SLC1A1mediated changes in extracellular glutamate signaling could contribute, under conditions where extracellular glutamate availability is a limiting factor. Multiple components of the glutamate signaling machinery are expressed in the colon epithelium, including, in addition to glutamate transporters [bib_ref] Primary structure and functional characterization of a high-affinity glutamate transporter, Kanai [/bib_ref] , NMDA receptors (NMDARs) [bib_ref] The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma..., Kim [/bib_ref] and metabotropic glutamate receptors [bib_ref] Glutamate antagonists limit tumor growth, Rzeski [/bib_ref]. Indeed, NMDAR-and mGluR-antagonists inhibit the proliferation of HT29 cells [bib_ref] Glutamate antagonists limit tumor growth, Rzeski [/bib_ref]. On the other hand, excessive NMDAR activation induces increased [Ca 2+ ] i and consequent cell death in CRC cells [bib_ref] The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma..., Kim [/bib_ref]. Similarly, glutamate release autocrinely stimulates gliomas, yet is toxic to surrounding neurons [bib_ref] Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters..., Ye [/bib_ref] [bib_ref] Glioma cells release excitotoxic concentrations of glutamate, Ye [/bib_ref] [bib_ref] Autocrine glutamate signaling promotes glioma cell invasion, Lyons [/bib_ref]. Our microarray data [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref] support the notion that glutamate-and glutathione homeostasis are broadly altered in the SN38-and oxaliplatin resistant CRC cells, in agreement with previous studies showing increased GSH levels and γ-GCS up-regulation in drugresistant cancer cells [bib_ref] Glutathione synthesis, Lu [/bib_ref]. Of note, the SN38-resistant LoVo cells, which exhibited a 4-fold increase in SLC1A1 expression, also show up-regulation of both mGluR and iGluR [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref]. Also glutamate decarboxylase, which is ratelimiting for GABA production and was recently assigned important roles in small-cell lung cancer [bib_ref] Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by beta-catenin..., Kimura [/bib_ref] , shows increased expression in several of the resistant cell lines, as does the GSH-dependent ABCC2/MRP2 GS-X drug efflux pump [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref]. Further supporting the notion that a change in basal GSH metabolism contributes to the resistant phenotype, cellular GSH was increased in SN38-and oxaliplatin-resistant HCT116, yet this did not correlate with the effects of DL-TBOA treatment on viability. p53 induction by chemotherapeutic treatment was reduced by DL-TBOA in both HCT116 and LoVo cells. This differs from the opposite effects of DL-TBOA on viability in HCT116 and LoVo cells, thus, the specific mechanisms involved in the latter must be cell typedependent and/or upstream of p53. Effects related to the cotransport of Na + and H + by the glutamate transporters may also be envisaged. Thus, in mouse astrocytes, glutamate uptake reduced cytosolic and mitochondrial pH and inhibited oxidative metabolism in a manner inhibited by DL-TBOA and only in cells expressing the glutamate transporters [bib_ref] Glutamate transport decreases mitochondrial pH and modulates oxidative metabolism in astrocytes, Azarias [/bib_ref]. Other mechanisms previously implicated in oxaliplatin resistance include upregulation of Breast Cancer Resistance Protein (BCRP, ATPG2) and increased DNA-damage repair via up-regulation of Excision Repair Cross Complementing Protein 1 (ERCC1) [bib_ref] Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant..., Boyer [/bib_ref] , whereas SN38 resistance was proposed to involve down-regulation of topoisomerase-I [bib_ref] Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant..., Boyer [/bib_ref]. Future studies should establish the possible link of these mechanisms to altered glutamate transporter activity.
Finally, the subcellular localization of SLC1A1 was altered in chemotherapy-resistant cells as well as by treatment with chemotherapy or DL-TBOA. Chemotherapy treatment (a reduction in viability), was associated with a shift of SLC1A1 towards a perinuclear/nuclear localization, except in HCT116-Oxa cells, in which SLC1A1 was perinuclear/ nuclear already prior to treatment. Notably, the effects of DL-TBOA on SLC1A1 localization and cell viability correlated: In SN38-resistant cells, DL-TBOA augmented both SN38-induced loss of viability and chemotherapy-induced nuclear/perinuclear shift of SLC1A1, and in oxaliplatinresistant cells, DL-TBOA counteracted both oxaliplatininduced loss of viability and nuclear/perinuclear SLC1A1 localization. The mechanism involved cannot be deduced from the present studies, yet it is notable that regulated nuclear localization of SLC1A3 (GLAST-1) in cancer cells has been reported independently by two groups [bib_ref] Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma..., Varini [/bib_ref] [bib_ref] Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters..., Ye [/bib_ref]. Ye et al. [bib_ref] Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters..., Ye [/bib_ref] showed that SLC1A3 localized to the nucleus in glioma cells and glioblastoma patient brain tissue, but to the plasma membrane in normal astrocytes and normal brain tissue. Varini et al. [bib_ref] Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma..., Varini [/bib_ref] showed that nuclear localization of SLC1A3 was associated with reduced cell density/loss of cell-cell contacts. Neither study provided direct evidence to the mechanisms involved in this phenomenon, but if SLC1A1 localization is similarly regulated by cell-cell contacts, this might suggest that the translocation is downstream of reduced cell numbers in response to chemotherapy treatment, and also this interpretation is consistent with the precise correlation between the effect of DL-TBOA on viability and on SLC1A1 localization.
## Possible involvement of other excitatory amino acid transporters in the effects of dl-tboa
The effect of DL-TBOA was concentration-dependent in the range congruent with known IC 50 -values for inhibition of SLC1A1, yet only a partially similar pattern was seen after siRNA knockdown of SLC1A1. It remains possible, therefore, that the effect of DL-TBOA involved other excitatory amino acid transporters than SLC1A1, and nonspecific effects can obviously not be excluded. However, the fact that DL-TBOA was always protective in oxaliplatin-treated, and always detrimental in SN38treated, cells suggests that glutamate transporter inhibition impacts on a drug-specific, upstream mechanism, either at the level of drug influx/efflux, or, less likely, at the level of upstream interactions with the chromatin and associated DNA damage. Notably, public database information indicates that SLC1A1 is frequently downregulated in CRC tumors (Additional file 1: ; www.oncomine.org; [bib_ref] ONCOMINE: a cancer microarray database and integrated data-mining platform, Rhodes [/bib_ref]. It is therefore tempting to speculate that in some CRC patients, this may confer a growth advantage similar to that exerted by DL-TBOA after oxaliplatin treatment. Future studies should assess excitatory amino acid transporter levels in CRC tumors from SN38-and oxaliplatin resistant and non-resistant patients.
# Conclusions
In conclusion, SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells, and the glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while strongly attenuating that induced by oxaliplatin. Our findings indicate that changes in glutamate transporter expression and activity may be relevant in CRC, diagnostically and in the context of choice of treatment regimen.
# Methods
## Reagents
SN38 was from Sigma-Aldrich, oxaliplatin from Sanofi-Aventis, and DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) from Tocris. UCPH101 was synthesized as described [bib_ref] Discovery of the first selective inhibitor of excitatory amino acid transporter subtype..., Jensen [/bib_ref]. Primary antibodies were from Santa Cruz Biotechnology (SLC1A1, p21 WAF1/Cip1 (p21) and CTR1), BD Transduction (p150), Cell Signaling Technology (poly-(ADP-ribose) polymerase-1 (PARP-1), phospho-Ser807/ 811-retinoblastoma protein, and p53). Rhodamine-phalloidin was from Invitrogen, and AlexaFluor488-conjugated secondary antibody from Life Technologies. 4',6-diamidino-2-phenylindole (DAPI) was from Invitrogen. Alkaline phosphatase-coupled secondary antibodies were from Sigma-Aldrich.
## Cell lines and treatments
HCT116 human CRC cells originate from a primary colon carcinoma [bib_ref] Heterogeneity of malignant cells from a human colonic carcinoma, Brattain [/bib_ref] , and LoVo cells from a metastatic nodule from a CRC patient [bib_ref] Establishment of a human carcinoembryonic antigen-producing colon adenocarcinoma cell line, Drewinko [/bib_ref]. Both cell lines are metastatic in xenograft models [bib_ref] Liver metastasis models of colon cancer for evaluation of drug efficacy using..., Hamada [/bib_ref]. HCT116 cells were obtained from the NCI/Development Therapeutics Program, and LoVo cells from the American Tissue Culture Collection. The cell line identities of parental and resistant cell lines were confirmed using short tandem repeat DNA analysis (IdentiCell Cell Line Authentication Service, Aarhus University Hospital, Aarhus, Denmark). In addition, all cell lines were regularly assessed to be mycoplasma-free. HCT116 and LoVo sub-lines resistant to SN38 and oxaliplatin (hereafter denoted HCT116-SN38, HCT116-Oxa, LoVo-SN38 and LoVo-Oxa), respectively, were established by exposing parental HCT116 and LoVo cell lines to increasing doses of the respective chemotherapeutics for at least 45 passages [bib_ref] Establishment and characterization of models of chemotherapy resistance in colorectal cancer: towards..., Jensen [/bib_ref]. Cells were grown in RPMI 1640 + Glutamax™, 10 % Foetal Bovine Serum (FBS), and 1 % Penicilin/Streptomyocin (Life Technologies) at 5 % CO 2 , 37°C, and propagated by gentle trypsination every 3-4 days. Unless otherwise specified, experiments were carried out at 37°C.
## Real-time quantitative pcr (qpcr) analysis
Total RNA was purified using the Machery-Nagel NucleoSpin® RNA II kit. cDNA was synthesized using SuperScript II RT (Invitrogen) and random primers. qPCR was carried out in triplicate in an ABI7900 Real Time PCR machine, using FastStart universal SYBR Green master mix (Roche Applied Bioscience), and 0.2 μM forward and reverse primers. Thermal profile was: 96°C 10 min (96°C 1 min, 60°C 30 s, 72°C 1 min) × 40. Normalization was done to GAPDH, PBGD and β-actin for LoVo, and PBGD and β-actin for HCT116. Primers were: SLC1A1:fw:5'-GGATGTCACCCTGATCATTGC-3', rv:5'-CCAAGGACGTTGACCATGGT-3; SLC1A3: fw:5'-CG AAGCCATCATGAGACTGGTA-3',rv:5'-TCCCAGCAAT CAGGAAGAGAA-3'; β-actin: fw:5'-AGCGAGCATCCCC CAAAGT-3', rv:5'-GGGCACGAAGGCTCATCAT-3'; PBGD: fw:5'-TCCAAGCGGGAGCCATGTCTG-3', rv:5'-AGAAT CTTGTCCCCTGTGGTGGA-3'; GAPDH: fw:5'-GAAGG TGAAGGTCGGAGTC-3, rv:5'-GAAGATGGTGATGGG ATTTC-3'. Melting curves confirmed the presence of only one amplicon. Relative expression ratios were calculated as in [bib_ref] Induction of group VIA phospholipase A2 activity during in vitro ischemia in..., Poulsen [/bib_ref].
## Immunoblotting
Cells were grown to a confluence of 60-80 % and treated with chemotherapy, siRNA-or plasmid transfection as indicated. Cells were washed once in PBS, lysed in lysis buffer (1 % SDS, 0.1 M Tris pH 7.5, and 1 mM Na 3 VO 4 ), sonicated, and protein content determined (DC assay, BioRad). Lysates were mixed 2:1with NuPage LDS sample buffer (Invitrogen). SDS-PAGE was carried out in NOVEX chambers with NuPAGE 10 % Bis-Tris gels under reducing and denaturing conditions, using Bench-Mark protein ladder (Invitrogen), and 15 μg protein per lane. Proteins were transferred to PVDF membranes, which were Ponceau S stained, blocked for 1 h at 37°C in 5 % nonfat dry milk in TBST (0.01 M Tris/HCl, 0.15 M NaCl, 0.1 % Tween 20, pH 7.4), incubated overnight at 4°C with primary antibodies diluted in blocking buffer, washed in TBST, incubated with secondary antibodies for 1 h, washed in TBST, and developed using BCIP/NBT (KPL). Bands were scanned and quantified using UN-SCAN-IT (Silk Scientific).
## Cell viability assays
Cells were seeded at appropriate density (LoVo 10,000, and HCT116 5000 cells /100 μl) in growth medium in 96-well plates. Next day, cells were treated with inhibitors and/or chemotherapy in a total volume of 200 μl in growth medium and incubated for 48 h at 37°C, 5 % CO 2 . The medium was replaced with 100 μl of 0.5 mg/ ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in growth medium. The reaction was stopped 1.5-2.5 h later by addition of 100 μl 20 % SDS in 0.02 M HCl, and incubation overnight to allow complete lysis of cells and formazan crystals. Absorbance was measured in an ELISA plate reader (Thermo Scientific MultiScan FC) at 550 nm. Data were background subtracted and % viability relative to controls calculated.
Cell counting using the OPERA high throughput confocal Cells were seeded in 96-well plates (Greiner Bio) one day prior to experiments. Cells were treated with chemotherapy and/or inhibitors as indicated and incubated for 48 h. Medium was aspirated, and cells were washed gently in ice cold PBS, fixed in 2 % paraformaldehyde, washed in PBS, and nuclei stained by 5 min incubation with DAPI. Plates were scanned using an OPERA high-throughput microscope (PerkinElmer). Nucleus counting was performed using proprietary OPERA software, based on 100 points per well, and cell counts from non-treated cells as controls.
Constructs, siRNA and transfection 50 nM of non-specific siRNA (Eurofins MWG Operon, #10-5394-1/1) or SLC1A1 siRNA (GTGTTATATGCC ACTAGGT; Mission siRNA, Sigma-Aldrich) was transfected using Lipofectamine 2000, into 40-50 % confluent LoVo and HCT116 cell lines. For overexpression, cells were transfected with full-length SLC1A1 in pcDNA3.1 [bib_ref] Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3..., Jensen [/bib_ref] using 1 μg of DNA per well of a 6-well plate, and Lipofectamine 2000. 48 h after transfection, cells were treated with oxaliplatin or SN38 as indicated, and 24 h later, lysed for immunoblotting.
## [ 3 h]-d-asp uptake assay
The [ 3 H]-D-Asp uptake assay was performed essentially as in [bib_ref] Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3..., Jensen [/bib_ref]. Briefly, cells were split into poly-D-lysinecoated white 96-well plates (PerkinElmer). A similar number of cells were seeded for HCT116 parental, HCT116-SN38 and HCT116-Oxa cell lines (7 × 10 4 cells/well) and LoVo parental, Lovo-SN38 and Lovo-Oxa cell lines (6 × 10 4 cells/well). 16-24 h later, culture medium was aspirated, and cells were washed once with 100 μl assay buffer (Hank's Buffered Saline Solution supplemented with 20 mM HEPES, 1 mM CaCl 2 and 1 mM MgCl 2 , pH 7.4). 50 μl assay buffer supplemented with 100 nM [ 3 H]-D-Asp and test compounds as indicated was added, and the plate was incubated at 37°C for 6 min. Non-specific [ 3 H]-D-Asp uptake was determined in wells with 3 mM L-glutamate. The assay mixture was quickly removed, and wells were washed with 2 × 100 μl ice-cold assay buffer, followed by 150 μl Microscint™20 scintillation fluid (PerkinElmer). The plate was shaken for 1 h and counted in a Wallac 1450 MicroBeta Trilux scintillation counter (GMI, Ramsey, MN).
## Measurement of cellular glutathione levels
Cells were seeded in 24-well plates (10 4 cells per well), treated the next day with chemotherapy and/or DL-TBOA and incubated for 24 h. Medium was removed and cells were washed twice with ice-cold PBS, which was removed and 500 μl ice-cold 1 % Sulfosalicylic acid was added per well. Cells were incubated on ice for at least 10 min. After centrifugation (1 min, 15,000 g), 10 μl lysate was used to determine total GSx content. To measure GSSG content, 130 μl sample was mixed with 55 μl 0.2 M Tris (pH 9) and 5 μl 2-Vinylpyridine. Tubes were vortexed carefully and incubated for at least 1 h at room temperature. 10 μl of this mix was mixed first with 90 μl of water in a 96-well plate and then with a reaction mix (0.1 M sodium phosphate buffer pH 7.5 containing 1 mM EDTA, 10 mM NADPH, 10 mM DTNB and 0.05 μl Glutathione reductase, 2U/μl). Measurements were taken every 30 s for 10 min in an ELISA plate reader (Thermo Scientific MultiScan FC) at 405 nm absorbance. GSH values were obtained by subtraction of GSSG values from GSx values.
## Immunofluorescence analysis of slc1a1
Immunofluorescence analysis was carried out essentially as in [bib_ref] The Na + /H + exchanger NHE1, but not the Na +..., Lauritzen [/bib_ref]. Cells grown on glass coverslips were fixed in 2 % paraformaldehyde, washed in TBST, permeabilized for 5 min (0.5 % Triton X-100 in TBST), blocked for 30 min in 5 % BSA in TBST, incubated with SLC1A1 primary antibody in TBST + 1 % BSA overnight at 4°C, washed in TBST, and with Rhodamine-phalloidin and AlexaFluor488 conjugated secondary antibody (1:600 in TBS + 1 % BSA) for 1 h, followed by washing in TBST, and mounting in N-propyl-galleate mounting medium (2 % w/v in PBS/glycerol). DAPI was added for 3 min following incubation with the secondary antibody. Cells were visualized using the 60X/1.35 NA objective of an Olympus Bx63 epifluorescence microscope. No/negligible labeling was seen in the absence of primary antibody. Overlays were carried out using Adobe Photoshop software. No other image adjustment was performed.
# Statistical analysis
Statistical analysis was carried out in Graphpad Prism-6, using one-way ANOVA with Dunnett post-test, two-way ANOVA with Tukey post-test, or Students two-tailed t-test, as indicated.
# Ethics statement
No human material or human data except established cell lines and publically available information from the Oncomine database were used in the present study.
## Additional files
Below is the link to the electronic supplementary material. and SLC1A3 in CRC tissue relative to that in normal tissue. As seen, SLC1A1 was nearly ubiquitously downregulated, while the SLC1A3 level was generally unaltered. Data from Oncomine (www.oncomine.org; [bib_ref] Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer..., Rhodes [/bib_ref].
Additional file 2: . Nucleus counting after treatment of parental and drug-resistant HCT116 and LoVo cells with DL-TBOA. Parental and drug-resistant HCT116 and LoVo cell lines seeded in 96-well dishes were exposed to SN38 (0.1 or 0. Additional file 3: . Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment of HCT116 cells. Parental and drug-resistant HCT116 cell lines seeded in 6-well dishes were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM), alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein levels of p21, and PARP-1 (full-length and cleaved, the latter indicated by arrowheads) were determined by Western blotting. Top: Representative Western blots, with p150 as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. *) p < 0.05, **) p < 0.01, ***) p < 0.001,****) p < 0.0001 compared to the control group without drug or TBOA treatment; #) p < 0.05 compared to controls without TBOA treatment. Two-way ANOVA with Tukey post-test.
Additional file 4: [fig_ref] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels [/fig_ref]. Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment of LoVo cells. Parental and drug-resistant LoVo cell lines seeded in 6-well dishes were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM), alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein levels of p21, and PARP-1 (full-length and cleaved, the latter indicated by arrowheads) were determined by Western blotting. Top: Representative Western blots, with p150 as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. *) p < 0.05, **) p < 0.01, ***) p < 0.001,****) p < 0.0001 compared to the control group without drug or TBOA treatment; Two-way ANOVA with Tukey post-test.
Additional file 5: [fig_ref] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment [/fig_ref]. Effects of DL-TBOA on proliferation after chemotherapy treatment. Parental and drug-resistant HCT116 (A) and LoVo (B) cell lines seeded in 6-well dishes were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM) alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein levels of phosphorylation of retinoblastoma protein on Ser 807/811 (pRb) were determined by Western blotting. Top: Representative Western blots, with β-actin as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. Two-way ANOVA with Tukey post-test.
Additional file 6: [fig_ref] Figure 6: Effects of SLC1A1 siRNA on cell death and survival parameters after chemotherapy... [/fig_ref]. SLC1A1 overexpression has no detectable effect on cell death-and survival parameters tested. Representative blot data of 3 independent experiments of HCT116 (A) or LoVo (B) cell lines, in absence or presence of transient overexpression of wild type SLC1A1 (pSLC1A1) or corresponding empty vector (pcDNA3.1), followed by 24 h of chemotherapeutic treatment (0.8 μM SN38 or 20 μM Oxa). The protein levels of SLC1A1, p53, p21, and PARP-1 (full-length and cleaved, the latter indicated by arrowheads) were determined by Western blotting. p150 is shown as a loading control.
[fig] Figure 4: Effect of glutamate transporter inhibition on cellular GSH levels. a Basal intracellular GSH levels were measured as described in Materials and Methods, and normalized to total protein in the samples. b-c Normalized basal GSH levels under control conditions, in HCT116 and LoVo parental and resistant cells after 24 h of treatment with 0.8 μM SN38 or 20 μM oxaliplatin, in absence or presence of DL-TBOA (350 μM) as shown. * p < 0.05 vs untreated control group (n = 5). One-way ANOVA followed by Dunnett post-test [/fig]
[fig] Figure 5: Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment. Parental and drug-resistant HCT116 (a) and LoVo (b) cell lines were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM), alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein level of p53 was determined by Western blotting. Top: Representative Western blots, with p150 as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. *) p < 0.05, **) p < 0.01, ***) p < 0.001,****) p < 0.0001 compared to the control group without drug or TBOA treatment; Two-way ANOVA with Tukey post-test yet a modest positive effect on survival/growth in untreated and SN38-treated cells (which may also be present in oxaliplatin-treated cells but be masked by the strong, opposite effect).SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells [/fig]
[fig] Figure 6: Effects of SLC1A1 siRNA on cell death and survival parameters after chemotherapy treatment. Drug-resistant HCT116 and LoVo cell lines were transfected with siRNA against SLC1A1 or corresponding mock siRNA (siCtrl.). 24 h later, cells were exposed to SN38 (0.8 μM), oxaliplatin (20 μM) as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein level of SLC1A1 and p53 was determined by Western blotting. a Representative Western blots, with p150 as loading control. b Densitometric quantifications of relative p53 protein level, based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. *) p < 0.05, **) p < 0.01, ***) p < 0.001,****) p < 0.0001 compared to the control group without drug or TBOA treatment; Two-way ANOVA with Tukey post-test [/fig]
[fig] Figure 7: Subcellular localization of SLC1A1 in parental and resistant CRC cellseffects of chemotherapy and DL-TBOA. a Immunofluorescence images of parental (PAR), SN38 resistant and oxaliplatin resistant HCT116 cells treated or not for 48 h with 350 μM DL-TBOA, and stained with antibody against SLC1A1. b Parental and SN38-resistant HCT116 cells treated for 48 h with 0.8 μM SN38 in the absence or presence of 350 μM DL-TBOA, and stained as in A. c Parental and oxaliplatin-resistant HCT116 cells treated for 48 h with 20 μM oxaliplatin in the absence or presence of 350 μM DL-TBOA, and stained as in A. All conditions are representative of 2 or 3 independent biological replicates in duplicate. Scale bar: 10 μm. Additional file 7: [/fig]
[fig] Additional file 1, Figure S1: Microarray and Oncomine data showing the expression pattern of SLC1A1 and SLC1A3. (A) Microarray data showing the fold change in expression of SLC1A1 and SLC1A3 in SN38-and oxaliplatin (Oxa)-resistant HCT116 and LoVo cell lines compared to respective parental cell line. Data are from [13]. (B) The figure summarizes data from 15 different studies, showing the mRNA expression of SLC1A1 [/fig]
[fig] 8: μM) or oxaliplatin (0.8 or 20 μM), alone or in combination with 70 or 350 μM DL-TBOA as indicated, for 48 h. Cells were washed in PBS, fixed in 2 % paraformaldehyde and nuclei were stained with DAPI. The number of adherent cells was determined by automated counting using an OPERA confocal microscope. (A-B) Parental HCT116 cells. (C) SN38 resistant HCT116 cells. (D) Oxaliplatin-resistant HCT116 cells. (E-F) Parental LoVo cells. (G) SN38 resistant LoVo cells. (H) Oxaliplatin-resistant LoVo cells. Data are means with S.E.M. error bars of 3 independent experiments. Values are normalized to those of untreated cells. [/fig]
[fig] Additional file 7, Figure S7: Subcellular localization of SLC1A1, nuclei, and F-actin in parental and resistant CRC cells-effects of chemotherapy and DL-TBOA. (A) Immunofluorescence images of parental (PAR), SN38 resistant and oxaliplatin resistant HCT116 cells treated or not for 48 h with 350 μM DL-TBOA, and stained with antibody against SLC1A1 (green) and with DAPI (blue) and Rhodamine-conjugated phalloidin (red) to visualize localization of nuclei and F-actin, respectively. (B) Parental and SN38-resistant HCT116 cells treated for 48 h with 0.8 μM SN38 in the absence or presence of 350 μM DL-TBOA, and stained as in A. (C) Parental and oxaliplatin-resistant HCT116 cells treated for 48 h with 20 μM oxaliplatin in the absence or presence of 350 μM DL-TBOA, and stained as in A. All conditions are representative of 2 or 3 independent biological replicates in duplicate. Scale bar: 10 μm. [/fig]
[table] Table 1: Summary of pharmacological properties and basal level [ 3 H]-D-Asp uptake IC 50 values for the three compounds are in μM, with pIC 50 values in brakets. The basal [ 3 H]-D-Asp uptake data are based on the measured uptake in the chemotherapeutic-cells normalized to that in the relevant parental cell line on the experiment performed in duplicate. *) p <0.05,**) p <0.01,and ***) p <0.001, Compared to parental cell by two-tailed Student's t-test [/table]
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Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression
We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAPdependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.Deletion of chromosome 9p21 encompasses the tumour suppressors CDKN2A and methylthioadenosine phosphorylase (MTAP) 1,2 , and is a frequent somatic event in several cancers 1,2 . MTAP is a critical enzyme in the methionine salvage pathway that metabolizes the substrate methylthioadenosine (MTA), leading to regeneration of methionine and adenosine 3 . Deletion of MTAP has been reported to confer a vulnerability to inhibition of the epigenetic regulator protein arginine methyltransferase 5 (PRMT5), which symmetrically methylates arginine on histone H4, leading to chromatin remodelling 2,4,5 .Malignant pleural mesothelioma (MPM) is an incurable cancer caused by asbestos that lacks effective targeted therapies, and harbours high frequency deletion of 9p21 6,7 . Here we report a negative prognostic impact of MTAP on survival of patients with MPM, and demonstrate potential to selectively target somatic deletion of MTAP using a small-molecule based strategy, involving PRMT5 transcriptional suppression.
# Results
MTAP is negatively prognostic in MPM. MTAP copy number loss was assessed in 79 mesotheliomas acquired at radical surgery involving extended pleurectomy decortication (EPD) (Supplementary . Copy number loss of 9p21.3 encompassing CDKN2A/MTAP was observed in 47% (37 samples) of patients and 42% had MTAP deletion (33 samples); co-deletion of MTAP and CDKN2A was frequent (95%). MTAP heterozygous or homozygous deletion was associated with shorter median overall survival (OS) compared to wild-type MTAP, 12.5 (95%CI 6.8-18.2) versus 17.6 (95%CI 6.5-28.7) months respectively, p = 0.042, HR 0.609 (95%CI 0.376-0.987), . In an independent validation cohort of 100 samples (Supplementary , 63% of patients harboured MTAP deletion (63 samples); co-deletion of MTAP and CDKN2A was similarly at high frequency (83%). MTAP deletion was associated with shorter overall survival (OS) compared to wild-type MTAP, 8.7 (95%CI 4.6-12.8) versus 22.7 months (95% CI 11.4-33.9) months respectively, p = 0.021, HR 0.599 (95%CI 0.386-0.930) .
To explore MTAP as a covariate, we performed a univariate analysis showing a statistically significant effect between overall survival with: age (p = 0.02); iMig stage (p = 0.009) and MTAP status (p = 0.008). These variables were taken forward into a Cox multivariate analysis showing that iMig stage (HR 1.36 (95%CI 1.11-1.68), p = 0.003) and MTAP status (HR 1.41 (95%CI 1.03-1.93), p = 0.032) retained statistical significance whilst age (HR 1.32 (95%CI 0.97-1.79), p = 0.08) did not.
PRMT5 silencing mediates growth arrest in MTAP negative mesothelioma. To determine whether MTAP negative MPM cells were dependent on PRMT5, we silenced PRMT5 expression by RNA interference in both MTAP wild-type and negative MPM cell lines . Reduced clonogenic growth was selective for MTAP negative cell lines, with concurrent reduction in symmetrical di-methylation of Histone H4 arginine 3 (H4R3me2S). Silencing of the PRMT5 interactor WDR77 phenocopied PRMT5 silencing in MTAP negative cells, leading to a reduced clonogenic activity and a reduced H4R3me2S . This effect was not phenocopied by siRNA targeting the PRMT5 interactor RIO1 kinase .
To investigate the kinetics of growth arrest, we conducted real time analysis following PRMT5 RNAi which showed a growth arrest after 120 h . Neither apoptosis nor cell cycle perturbation were observed by flow cytometry after PRMT5 silencing .
To determine whether or not PRMT5 silencing-induced chromatin remodelling would upregulate tumour suppressor pathways, we conducted gene expression, examining canonical pathways linked to upregulated genes. Overexpression of tumour suppressors, such as EIF3F, FOXP4, ZBTB4, GANAB, TMEM141 was observed. Gene set enrichment analysis revealed a significant enrichment of the EZH2 target gene ,C, Supplementary .
In common with PRMT5 siRNA, the small molecule EPZ015666 reduced clonogenic growth (Supplementary . Neither apoptosis nor cell cycle perturbation were observed by flow cytometry after inhibition by EPZ015666 .
Quinacrine hydrochloride is a PRMT5 perturbagen. To fully harness the potential for MTAP selective activity via PRMT5 inhibition, we utilised the connectivity map 8 to identify novel transcriptional suppressors of PRMT5 , [fig_ref] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM... [/fig_ref]. Among the top 5 predicted molecules, Quinacrine Hydrochloride led to significant suppression of both PRMT5 mRNA levels [fig_ref] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM... [/fig_ref] and protein with consequent reduction of H4R3me2S and growth arrest in MTAP negative but not MTAP positive cells [fig_ref] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM... [/fig_ref]. Quinacrine Hydrochloride directly inhibited PRMT5 promoter activity as confirmed by luciferase reporter assay [fig_ref] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM... [/fig_ref] , but did not have any direct effect on PRMT5 enzymatic activity [fig_ref] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM... [/fig_ref].
Neither apoptosis nor cell cycle perturbation were observed after treatment with Quinacrine by flow cytometry, in common with both PRMT RNA interference or small molecule inhibitor (Supplementary . We used a functional genetic approach to establish the extent to which the phenotypic effects of Quinacrine in MTAP negative cells were reliant upon PRMT5. Overexpression of wild-type PRMT5 rescued cells from treatment with Quinacrine, however this was not observed with transfection of the methyltransferase dead mutant PRMT5 E444Q compared to empty vector control. This is consistent with modulation of endogenous PRMT5 transcription as an essential mechanism underpinning the effect of Quinacrine in MTAP negative context [fig_ref] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM... [/fig_ref].
## Quinacrine transcriptionally regulates prmt5 via c-jun.
To further explore the possible mechanism of PRMT5 transcriptional perturbation by Quinacrine, we used RNA interference to screen for putative transcription factors (TFs) implicated by PROMO 9 . These TFs included CEBP1, c-JUN and NF-YA were predicted to bind to the PRMT5 promoter. RNAi mediated silencing of c-JUN, but not of CEBP1 or NF-YA, resulted in a significant reduction of PRMT5 mRNA levels in MTAP negative cells and this was comparable to that achieved with Quinacrine [fig_ref] Figure 4: Quinacrine h [/fig_ref]. c-JUN silencing led to reduced clonogenic growth with concurrent downregulation of both PRMT5 and H4R3me2S in MTAP negative cells [fig_ref] Figure 4: Quinacrine h [/fig_ref] , but no apoptosis or cell cycle perturbation . Quinacrine suppressed c-JUN mRNA suggesting that it targets PRMT5 transcription indirectly via this transcription factor [fig_ref] Figure 4: Quinacrine h [/fig_ref]. The effects of c-JUN silencing were MTAP-selective, as MTAP positive cells failed to reduce PRMT5 mRNA [fig_ref] Figure 4: Quinacrine h [/fig_ref] , without evidence of impaired clonogenic growth or reduced H4R3me2S [fig_ref] Figure 4: Quinacrine h [/fig_ref]. Interestingly, Quinacrine did not suppress c-JUN mRNA in MTAP positive cells [fig_ref] Figure 4: Quinacrine h [/fig_ref]. www.nature.com/scientificreports/
# Discussion
Copy number loss of MTAP is one of the most frequent events in MPM. Our data confirmed a marked negative prognostic effect, which warrants novel targeted therapy. It should be noted that given the co-deletion of MTAP with CDKN2A, which has been also shown to be negatively prognostic, it was not possible to deconvolute the impact of MTAP in isolation from our data involving chromosome 9p21.3 deleted cel lines. PRMT5 plays a key role in the regulation of several pathways including DNA damage response, apoptosis, inhibition of tumour suppressors, and activation of survival pathways [bib_ref] The regulation, functions and clinical relevance of arginine methylation, Guccione [/bib_ref] and has been reported to be a dependency in MTAP negative cells [bib_ref] PRMT5 silencing selectively affects MTAP-deleted mesothelioma: in vitro evidence of a novel..., Barbarino [/bib_ref] , which we have verified in MPM. We confirmed global epigenetic modification associated with reduced H4R3me2S and re-expression of tumour suppressors, such as EIF3F, FOXP4, ZBTB4, GANAB, TMEM141, in association with loss of clonogenicity. SAM competitive PRMT5 inhibitors, such as EPZ015666 and GSK3326595, have shown limitations in recapitulating the vulnerability of MTAP negative cells in response to PRMT5 inhibition [bib_ref] The regulation, functions and clinical relevance of arginine methylation, Guccione [/bib_ref]. To address this limitation, we used the connectivity map approach [bib_ref] The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and..., Lamb [/bib_ref] to screen for small molecules with PRMT5 downregulating activity and identified the off-patent small molecule Quinacrine, which was primarily used as an antimalarial drug as well as an intrapleural sclerosing treatment for malignant pleural effusions with an excellent safety profile 12 . Quinacrinemediated growth arrest was PRMT5 dependent as confirmed by a methyltransferase dead PRMT5 mutant.
Apoptosis was not observed as a mechanism of reduced clonogenicity; the phenotype was cytostatic, but not restricted to any phase of the cell cycle. Recent studies suggest that small molecule PRMT5 inhibition, either directly or indirectly through inhibition of cyclin dependent kinases 4 and 6, alters RNA splicing, leading to an MDM4 dependent activation of p53/p21 [bib_ref] Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors..., Abuhammad [/bib_ref]. Whether this axis is exploited by Quinacrine requires further exploration.
Quinacrine has been reported to regulate c-jun phosphorylation at positions 349 to 340 and 266 to 257 [bib_ref] Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2..., Changchien [/bib_ref]. We showed that c-jun is essential in driving PRMT5 expression and mediating Quinacrine-induced apoptosis. This study therefore implicates c-jun or SAPK pathway regulation as a mechanism underlying PRMT5 transcriptional repression by Quinacrine.
Based on early pharmacokinetic studies conducted with Quinacrine in the 1940′s, plasma concentration was measured as 225 nM at a dose of 100 mg, 3 times a day. However, hepatic and leucocyte distributions are high, with high plasma binding. This agent can however be safely instilled into the pleural cavity at a much higher dose of 5 mM, at which dose it acts as a sclerosant. This implies that lower micromole doses are achievable and could be locally administered, achieving localised exposure to mesothelioma at concentrations capable of suppressing PRMT5 expression.
Our results provide a proof of concept to support the use of small molecule transcriptional perturbation to leverage a somatic-mutation based vulnerability, suggesting a repurposing potential that warrants further study. . Overall survival was calculate from the date of surgery. A separate cohort of 100 patients 16 was used for validation and clinico-pathological characteristics are described in . Informed consent to provide research samples was obtained from all patients. All methods were carried out in accordance with local guidelines and regulations.
# Material and methods
Oncoscan analysis. DNA was extracted with the GeneRead DNA FFPE kit (Qiagen, Manchester, UK). 80 ng of gDNA were analysed using the OncoScan FFPE Assay Kit (Affymetrix, Wooburn Green High Wycombe, UK), which utilizes molecular inversion probe (MIPs) technology [bib_ref] Cross-laboratory validation of the OncoScan(R) FFPE Assay, a multiplex tool for whole..., Foster [/bib_ref]. The BioDiscovery Nexus Express 10.0 for OncoScan software was then used to define copy number alterations and loss of heterozygosity as previously described [bib_ref] Ganetespib in combination with pemetrexed-platinum chemotherapy in patients with pleural mesothelioma (MESO-02):..., Fennell [/bib_ref]. Clonogenic assays. 5000 cells per well were seeded in 12 well plates and left untreated or treated with Quinacrine (500 nM, 1 µM, 2 µM). Cells were fixed between days five and seven (once enough colonies had formed in the control) on ice in methanol for 10 min. Cells were then stained with crystal violet (Sigma, Gillingham, UK) for 20 min. Colonies were dissolved in 30% acetic acid to allow quantification [bib_ref] Resistance to HSP90 inhibition involving loss of MCL1 addiction, Busacca [/bib_ref] Real time proliferation assay. The xCELLigence RTCA DP instrument (Acea Bioscience, San Diego, CA) was used as described in the manufacturer´s instruction manual. Cells (5,000 cells/ well) were seeded in E16-Plates. The cell indices were measured every 15 min for 120 h. Each treatment condition was measured in triplicate.
## Reagents and antibodies.
## Connectivity mapping.
A PRMT5-centred gene signature was created from co-expression analyses of 9 independent Gene Expression Omnibus (GEO) datasets GSE37745, GSE50081, GSE28571, GSE77803, GSE43580, GSE19804, GSE18842, GSE10245, and GSE19188. Within each dataset, PRMT5 was used as the seed gene, with which the gene expression correlation coefficients for other genes (probes) were calculated. All genes were then ranked based on the magnitude and statistical significance of their correlations with the seed, following the ranking method described in [bib_ref] Ganetespib in combination with pemetrexed-platinum chemotherapy in patients with pleural mesothelioma (MESO-02):..., Fennell [/bib_ref]. The genes' ranks were then combined across these datasets to obtain an overall rank for each gene to determine its inclusion to the PRMT5 gene signature for subsequent connectivity mapping analysis. A gene signature progression approach [bib_ref] Resistance to HSP90 inhibition involving loss of MCL1 addiction, Busacca [/bib_ref] determined that an 8-gene signature was the optimal length including PRMT5 and its 7 strongest co-expression correlates PSMB5, HNRNPC, APEX1, HNRNPC, IPO4, TOX4, and TUBB. This 8-gene signature was used as an input to query a collection of 83,939 reference drug gene expression profiles 20 covering 1353 FDA approved drugs (http:// www. lincs cloud. org). This connectivity mapping analysis was conducted in the framework of sscMap [bib_ref] A simple and robust method for connecting small-molecule drugs using gene-expression signatures, Zhang [/bib_ref] [bib_ref] sscMap: an extensible Java application for connecting small-molecule drugs using gene-expression signatures, Zhang [/bib_ref]. In our analysis all the individual reference profiles with the same drug formed a reference set. A set score was then calculated between the gene signature and each reference set, and the associated p-value was estimated by generating a large number of random gene signatures of the same length. Any signature-drug connections with a p-value no greater than a pre-set threshold (1/1353 = 7.4e−4) were declared as statistically significant. Additionally, gene signature perturbation analysis [bib_ref] Identification of candidate small-molecule therapeutics to cancer by gene-signature perturbation in connectivity..., Mcart [/bib_ref] was performed to obtain the robustness (perturbation stability) of the significant signature-drug connections.
Only the significant drugs that had 100% perturbation stability were selected for further consideration. Finally, significant drugs were ranked by the absolute value of their connection z-score to the PRMT5 gene signature.
PRMT5 enzymatic activity. PRMT5 chemoluminescent assay was purchased from AMS Biotechnology (Europe) Ltd (Abingdon, UK). Quinacrine or EPZ015666 was added to the plate pre-coated with histone H4 peptide substrate. PRMT5 enzymatic activity was measured after reaction with the antibody against methylated arginine3 residue of Histone H4, the secondary HRP-labeled antibody, S-adenosylmethionine, methyltransferase assay buffer, and purified PRMT5 enzyme, according to the manufacturer instructions.
Protein extraction and immunoblotting. Seventy-two hours after treatment cells were lysed in RIPA buffer containing protease inhibitors (Roche, Burgess Hill, UK). Lysates were clarified by centrifugation at 4 °C at 13,000 rpm for 10 min. 40 µg of total cell lysates were loaded on SDS-PAGE gels. Signal detection was performed with ECL-plus chemiluminescent system (GE Healthcare, Little Chalfont, UK).
Real time quantitative RT-PCR. www.nature.com/scientificreports/
[fig] Figure 1, Figure 2: MTAP is negatively prognostic in MPM (A) Kaplan-Meier survival curve for OS of MTAP positive and MTAP negative patients (n = 79). (B) Kaplan-Meier survival curve for OS of MTAP positive and MTAP negative patients from validation cohort (n = 100). (C) MTAP negative cells (NCI-H2052, NCI-H2591) and MTAP positive cells (MPP89) were transfected with siNT or siPRMT5 20 nM (Q: Qiagen sequence, D: Dharmacon sequence). Cell proliferation was measured by clonogenic assay 5-7 days after transfection. Data were normalized to siNT controls (NCI-H2591: Q p = 0.0001 D p = 0.0001; NCI-H2052: Q p = 0.0001 D p = 0.0001; NCI-H2452: Q p = 0.0001 D p = 0.0001; MPP89: Q p = n.s. D p = n.s.). The levels of PRMT5 expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot 72 h after transfection. These gels have been cropped and full length gels are presented in Supplementary Fig. 3A-C. (D) Cells were transfected with siNT or siWDR77 20 nM. Cell proliferation was measured by clonogenic assay 5-7 days after transfection. Data were normalized to siNT controls (NCI-H2591: p = 0.0001; NCI-H2052: p = 0.0001; NCI-H2452: p = n.s; MPP89: p = n.s.). The levels of PRMT5 expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot 72 h after transfection. These gels have been cropped and full length gels are presented in Supplementary Fig. 3D-F. PRMT5 silencing mediates growth arrest in MTAP negative mesothelioma. (A) NCI-H2591 cells were transfected with siNT or siPRMT5 (Q: qiagen sequence, D: Dharmacon sequence) 20 nM. Cell proliferation was measured for 168 h with the excelligence real-time cell analyser. Data were normalized to siNT controls (Q p = 0.0015 D p = 0.0019). (B) Venn diagram showing upregulated and downregulated genes comparing the PRMT5 siRNAs versus siNT. (C) Heatmaps and representative GSEA plots showing a significantly enriched up regulated signatures (siPRMT5 vs. siNT). [/fig]
[fig] Figure 3: Identification qRT-PCR on RNA extracted from cells treated with Quinacrine 1 µM for 72 h. Data were normalized to untreated control (NCI-H2052 p = 0.0308; NCI-H2591: p = 0.0063; MPP89 p = 0.005). (C) Cells were left untreated or treated with Quinacrine 0.5 µM and 1 µM for 72 h. Cell proliferation was measured by clonogenic assay 5-7 days after treatment. Data were normalized to untreated controls (NCI-H2591: 0.5 µM p = n.s. 1 µM p = 0.0001; NCI-H2052: .5 µM p = n.s. 1 µM p = 0.0017; MPP89: 0.5 µM p = n.s. 1 µM p = n.s.). The levels of PRMT5 expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot 72 h after transfection. These gels have been cropped and full length gels are presented inSupplementary Fig. 3G-I. (D)The PRMT5 promoter activity was measured by a luciferase reporter assay in NCI-H2591 cells transfected with pGL2 basic (EV) or pGL2-PRMT5 and then treated with Quinacrine 1 µM for 72 h. Data were normalized to pGL2 basic (pGL2 basic vs. pGL2-PRMT5 NT p = 0.0162; pGL2 basic vs. pGL2-PRMT5 1 µM p = n.s.; pGL2-PRMT5 NT vs. pGL2-PRMT5 1 µM p = 0.0335). (E) Cells were left untreated or treated with Quinacrine or EPZ015666 10 nM, 100 nM, 1 µM and 10 µM. PRMT5 enzymatic activity was measured after 2 h. (F) NCI-H2591 cells were transfected with GFP empty vector, PRMT5 WT and PRMT5 E444Q and left untreated or treated with Quinacrine 0.5 µM and 1 µM for 72 h. Cell proliferation was measured by clonogenic assay 5-7 days after treatment. Data were normalized to untreated controls (Empty vector: NT vs. 0.5 µM p = 0.0004, NT vs1 µM p < 0.0001; PRMT5 E444Q: NT vs. 1 µM p = 0.0026; Empty vector 1 vs. PRMT5 WT p < 0.0001). [/fig]
[fig] Figure 4: Quinacrine h. Data were normalized to siNT (siCEBP1 p = n.s.; sic-JUN p = 0.0001; siNF-YA p = n.s). CEBP1, c-JUN and NF-YA mRNA expression was evaluated by qRT-PCR on RNA extracted from cells transfected with siNT, siCEBP1, sic-JUN, siNF-YA 20 nM, for 72 h. Data were normalized to siNT (NCI-H2591: siCEBP1 p = 0.0001; sic-JUN p = 0.0006; siNF-YA p = 0.0033. NCI-H2052: siCEBP1 p = 0.0001; sic-JUN p = 0.0009; siNF-YA p = 0.0001). (B) Cell proliferation was measured by clonogenic assay 5-7 days after treatment. Data were normalized to siNT (NCI-H2591: siCEBP1 p = n.s.; sic-JUN p = 0.0073; siNF-YA p = n.s. NCI-H2052: siCEBP1 p = n.s.; sic-JUN p = 0.0001; siNF-YA p = 0.0001). The levels of PRMT5 and c-JUN expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot. These gels have been cropped and full length gels are presented inSupplementary Fig. 3J-K.(C) C-JUN mRNA expression was evaluated by qRT-PCR on RNA extracted from cells treated for 72 h with Quinacrine 1 µM. Data were normalized to untreated controls (NCI-H2591 p = 0.0001. NCI-H2052 p = 0.0276). (D) PRMT5 and cJUN mRNA expression was evaluated by qRT-PCR on RNA extracted from MPP89 cells transfected with siNT, sic-JUN 20 nM, for 72 h. Data were normalized to siNT (PRMT5 p = n.s. c-JUN p = 0.0205). (E) Cell proliferation was measured by clonogenic assay 5-7 days after treatment. Data were normalized to siNT (sic-JUN p = n.s). The levels of PRMT5 and c-JUN expression and H4 arginine 3 symmetrical di-methylation (H4R3me2S) were measured by western blot. This gel has been cropped and the full length gel is presented inSupplementary Fig. 3L.(F) C-JUN mRNA expression was evaluated by qRT-PCR on RNA extracted from cells treated for 72 h with quinacrine 1 µM. Data were normalized to untreated control p = n.s.Scientific Reports| (2021) 11:7434 | https://doi.org/10.1038/s41598-021-86834-7 [/fig]
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The multimodal nature of communicative efficiency in social interaction
How does communicative efficiency shape language use? We approach this question by studying it at the level of the dyad, and in terms of multimodal utterances. We investigate whether and how people minimize their joint speech and gesture efforts in face-to-face interactions, using linguistic and kinematic analyses. We zoom in on other-initiated repair-a conversational microcosm where people coordinate their utterances to solve problems with perceiving or understanding. We find that efforts in the spoken and gestural modalities are wielded in parallel across repair turns of different types, and that people repair conversational problems in the most cost-efficient way possible, minimizing the joint multimodal effort for the dyad as a whole. These results are in line with the principle of least collaborative effort in speech and with the reduction of joint costs in non-linguistic joint actions. The results extend our understanding of those coefficiency principles by revealing that they pertain to multimodal utterance design.In joint actions, people coordinate their behaviors in order to achieve joint goals 1,2 . Whether people are moving a couch or having a chat, joint action appears to be organized according to a principle of efficiency or effort minimization 3-7 . Empirical work on joint action shows that this effort minimization appears to target overall joint effort (or coefficiency) rather than individual effort 8-10 . Work on spoken language likewise suggests that people work together to minimize the cost for the dyad as a social unit-known as the principle of least collaborative effort[11][12][13]. One consequence for the study of efficiency in language is that language use is not about idealized speakers producing optimal one-off utterances; instead, we need to consider the work that interacting participants jointly undertake to actively construe possible meanings.The notion of coefficiency is in principle agnostic to the type of behavior involved. That is, joint action is recognized to involve a complex interplay of efforts exerted through various types and levels of behavior. However, when it comes to language use, efficiency is usually studied in unimodal ways (by focusing on written representations of speech), where communicative acts are considered to be linear (one word after the other). Complementary or parallel contributions across modalities are overlooked in accounts of efficiency in human languages 4,5 , despite the communicative capacities of composite utterances as revealed by research on multimodal interaction[14][15][16][17][18]. So, work on efficiency in coordinated spoken language use has yet to take into account how simultaneous articulators are concurrently employed to convey information (for work on sign language, see 19 ). Here we take on the challenge to study how people efficiently coordinate multiple types of communicative behavior, by investigating if and how people minimize joint speech and gesture efforts in a task-based conversational setting.We focus on stretches of conversation where people explicitly coordinate their utterances with the goal of jointly solving problems of perceiving or understanding-known as other-initiated repair 20,21 . In a typical sequence of other-initiated repair, one participant temporarily halts the conversation in order to ask for clarification with a repair initiation like "huh?" (open request), "who?" (restricted request), or "like this [gesture]?" (restricted offer) 22-26 , to which their conversational partner responds with a repair solution. After having jointly resolved the trouble, the participants end the repair sequence and the main conversation continues 27,28 . Repair initiations and solutions are defined strictly in terms of sequential positions in conversation, where the turns themselves can recruit any combination of communicative modalities[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45].Sequences of other-initiated repair have played a key role in the development of the notion of least collaborative effort for English task-based and telephone interactions 12,13 and in its generalization to co-present OPEN
## Scientific reports
| (2022) 12:19111 | https://doi.org/10.1038/s41598-022-22883-w www.nature.com/scientificreports/ conversational interaction across diverse languages [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref]. This work revealed that people collaboratively resolve trouble while minimizing their joint efforts in two ways. First, recipients who signal trouble prefer to use restricted formats (e.g., 'Which one?' or 'You mean X?') over open formats (e.g., 'Huh?'), meaning that they initiate repair in the most specific way possible (the specificity principle). Second, the more specific the repair initiation, the longer the repair initiation (involving more speech effort), thereby minimizing the efforts needed for the sender to resolve the trouble in the repair solution (the division of labor principle). However, this prior work focused exclusively on unimodal utterances, either by classifying the referential formats of noun phrases, or by computing the orthographic length of turns. Gesture efforts in spoken language have been overlooked, even though prior research has shown that manual co-speech gestures can play an important role in repair initiations [bib_ref] The body as a resource for other-initiation of repair: Cupping the hand..., Mortensen [/bib_ref] [bib_ref] Holding gestures across turns: Moments to generate shared understanding, Sikveland [/bib_ref] [bib_ref] Depictive hand gestures as candidate understandings, Jokipohja [/bib_ref] and repair solutions [bib_ref] On what happens in gesture when communication is unsuccessful, Hoetjes [/bib_ref] [bib_ref] An experimental investigation of how addressee feedback affects co-speech gestures accompanying speakers'..., Holler [/bib_ref] [bib_ref] Teachers' gestures and speech in mathematics lessons: Forging common ground by resolving..., Alibali [/bib_ref] [bib_ref] Gesturally-enhanced repeats in the repair turn communication strategy or cognitive language-learning tool?, Olsher [/bib_ref] [bib_ref] Repairing person reference in a small Caribbean community, Sidnell [/bib_ref]. Since gestural efforts to convey meaning have not been incorporated in the division of labor equation, we cannot be sure that the speech-centered findings hold water for interactions in their true multimodal form. Adopting a multimodal perspective is also warranted in light of recent studies showing that various interactional strategies (initially discovered based on speech-centered research) extend to the design of multimodal utterances. For example, people modulate both speech and gesture when trying to get a message across in noisy environments (multimodal Lombard effect 51 ); adapt both speech and gestures to the degree of knowledge that is shared with a recipient (multimodal audience design 52 ); and are likely to use cross-speaker repetition of both speech and gesture for establishing joint reference to novel objects (multimodal alignment [bib_ref] The primacy of multimodal alignment in converging on shared symbols for novel..., Rasenberg [/bib_ref]. These findings reinforce the notion that speech and co-speech gestures operate as part of an integrated system [bib_ref] Semiotic diversity in utterance production and the concept of 'language, Kendon [/bib_ref] [bib_ref] What does cross-linguistic variation in semantic coordination of speech and gesture reveal?:..., Kita [/bib_ref] , with people flexibly deploying and coordinating their use of both modalities to engage in joint meaning-making [bib_ref] Holding gestures across turns: Moments to generate shared understanding, Sikveland [/bib_ref] [bib_ref] The primacy of multimodal alignment in converging on shared symbols for novel..., Rasenberg [/bib_ref] [bib_ref] Mimicked gestures and the joint construction of meaning in conversation, Chui [/bib_ref] [bib_ref] Co-speech gesture mimicry in the process of collaborative referring during face-to-face dialogue, Holler [/bib_ref] [bib_ref] Understanding as an embodied, situated and sequential achievement in interaction, Mondada [/bib_ref] [bib_ref] Action and embodiment within situated human interaction, Goodwin [/bib_ref] [bib_ref] Gesture and coparticipation in the activity of searching for a word, Harness Goodwin [/bib_ref] [bib_ref] The return gesture: Some remarks on context, inference, and iconic gesture, De Fornel [/bib_ref].
In work on co-speech gesture, the notion of division of labor is sometimes used in reference to how effort is divided between the modalities of speech and gesture in one speaker's utterances [bib_ref] The interplay between gesture and speech in the production of referring expressions:..., De Ruiter [/bib_ref]. Here instead we focus on the dyad as a social unit, where our primary interest is how effort is distributed across contributions of different speakers, taking into account both speech and gesture.
To investigate the distributions of multimodal effort at the dyad level we focus on sequences of other-initiated repair in task-based interaction. Other-initiated repair has several properties that make it an ideal testing ground for studying efficiency in social interaction. First, it is a miniature coordination problem solved in real-time by two participants, making it a relevant domain for understanding joint actions more broadly [bib_ref] Repair: The interface between interaction and cognition, Albert [/bib_ref]. Second, its sequential structure-an insert sequence composed of an initiation and proposed solution-is cross-linguistically well-attested and highly frequent [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref] [bib_ref] Resources and repair: A cross-linguistic study of syntax and repair, Fox [/bib_ref]. Third, it comes in a small number of formats that we can compare in terms of multimodal effort and frequency of use. By tracking participants' speech and gesture efforts in these conversational enclosures, we test whether multimodal contributions are optimized for least collaborative effort.
We use a director/matcher task in which people describe and find 3D shapes they have not seen before [fig_ref] Figure 3: Panel [/fig_ref]. The shapes, displayed in a randomized array on two screens, are designed to present participants with coordination problems to be solved on the fly using multimodal communication. Standing face-to-face and instructed to communicate in any way they want, participants recruit multimodal utterances in relatively free-form interactions in order to negotiate mutual understanding and jointly solve the task. Speech and gesture behaviors were recorded with head-mounted microphones, cameras and markerless motion tracking devices.
For the spoken modality, we operationalized effort as the number of orthographic characters per repair turn, as this allows us to compare our findings to those of previous work [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref]. Though not a direct measure of talk-ininteraction, orthographic length may be a reasonable proxy because (i) it is not affected by speech rate (while turn duration is) and (ii) it normalizes length across different speakers. In our dataset orthographic length strongly correlates with the duration of the repair turn (r = 0.93, p < 0.001), in line with earlier work [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref]. For the gesture modality, we use the number of submovements of manual co-speech gestures. This has been used as a kinematic measure of complexity and effort before [bib_ref] A Systematic investigation of gesture kinematics in evolving manual languages in the..., Pouw [/bib_ref] [bib_ref] When is ostensive communication used for joint action?, Vesper [/bib_ref] and measures akin to it have been shown to correlate with the number of information units in gestures as interpreted by human coders [bib_ref] A Systematic investigation of gesture kinematics in evolving manual languages in the..., Pouw [/bib_ref]. While perfect equivalence of measures across modalities is impossible, those proposed here are comparable in the sense that (a) both speech and co-speech gesture are used to negotiate meaning in other-initiated repair sequences, and (b) orthographic characters and submovements can both be used as a quantitative proxy for the amount of information that is (verbally or visually) conveyed by a repair turn.
We first investigate speech and gesture efforts separately, where we explore how these efforts are distributed across sequential positions (repair initiation and solution) and repair types (open request, restricted request and restricted offer). To investigate the division of multimodal effort between people, we compute a measure of multimodal effort by summing (standardized) speech and gesture efforts. We hypothesize that the type of repair initiation predicts how the joint amount of multimodal effort is divided between people, similarly to what has been found for the division of speech efforts [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref]. That is, we hypothesize that the more specific the repair initiation (open request < restricted request < restricted offer), the higher the proportion of the multimodal cost paid in the repair initiation relative to the total cost paid in the initiation and solution together. Finally, in line with the principle of least collaborative effort [bib_ref] Grounding in communication, Clark [/bib_ref] [bib_ref] Collaborating on contributions to conversations, Clark [/bib_ref] [bib_ref] Referring as a collaborative process, Clark [/bib_ref] , we predict that people design their utterances so as to minimize the total amount of multimodal effort for the dyad as a whole. Specifically, we hypothesize that the repair type which yields the smallest amount of joint multimodal effort will be used most frequently.
# Results
Overall, 378 repair initiations were found in our dataset of task-based interactions from 20 dyads (comprising about 8 h of audio, video and motion tracking recordings in total). We found a mean of 18.9 repair initiations per dyad (SD = 9.92, range = 6-45), which amounts to a repair initiation occurring once every 1. [bib_ref] Efficiency in human languages: Corpus evidence for universal principles, Levshina [/bib_ref] www.nature.com/scientificreports/ Speech and gesture effort. We start by reporting speech and gesture effort separately to allow for comparisons with prior unimodal work on the division of speech efforts in other-initiated repair [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref]. The effort that people invest through the spoken modality to collaboratively resolve interactional trouble is shown in [fig_ref] Figure 1: joint multimodal effort Division of multimodal effort [/fig_ref]. Division of multimodal effort. In accordance with the inherently multimodal nature of our dataset, we analyze how the total amount of multimodal effort is divided across participants [fig_ref] Figure 1: joint multimodal effort Division of multimodal effort [/fig_ref]. Multimodal effort is the sum of the effort invested through the two available modalities, so it can be speech-only or speech-plusgesture (we found no cases of gesture-only). Adopting a narrow notion of multimodality (focusing on visual information in manual co-speech gestures only), we can consider 63% of the repair sequences as being multimodal in nature, containing one or more gestures by at least one of the participants.
We find that the proportional cost paid by the person initiating repair versus the person resolving the trouble varies as a function of the repair type, and deviates from a case of equal division (where each person would invest 50% of the total effort). The proportion of multimodal effort invested in the repair initiation was higher for restricted requests compared to open requests (β = 0.14, SE = 0.06, t = 2.35, p = 0.02), and higher for restricted offers compared to restricted requests (β = 0.47, SE = 0.04, t = 11.42, p < 0.001), as revealed by mixed effects models (with random intercepts for dyads). Overall, we find a trade-off between the efforts invested by the two members of the dyad; the more multimodal effort is invested by the person initiating repair, the less multimodal effort is used to respond to it (r = − 0.14, p = 0.007).
Minimization of joint multimodal effort. The total amount of multimodal effort that was invested by the dyad to resolve interactional trouble (in the repair initiation and repair solution combined) is shown for each repair type in [fig_ref] Figure 2: 37, SD = 1 [/fig_ref]. On average, we find that the joint multimodal effort is smallest when the repair initiation was a restricted offer. A mixed effect model (with random intercepts and slopes for dyads, and random intercepts for target items) revealed that joint effort is less in sequences involving restricted offers (M = 1. www.nature.com/scientificreports/ that these repair types yield the lowest amounts of joint effort thus means that people appear to do repair in the most cost-efficient way possible, minimizing the joint multimodal effort for the dyad as a whole.
# Discussion
The present study investigated how language use is shaped by communicative efficiency from a multimodal and interactional perspective. We focused on short time windows in turn-by-turn interaction where people work together to achieve a particular joint goal: repairing a problem with perceiving or understanding talk. We analyzed how speech and co-speech gesture efforts are distributed across repair types (open requests, restricted requests and restricted offers) and sequential positions (the repair initiation and the repair solution), with the aim to test whether the division of multimodal effort is optimized for least collaborative effort. There are three main findings. First, we find that speech and gesture efforts rise and fall together across repair types and sequential positions. This corroborates the view that speech and gesture are integral parts of a single multimodal communicative system [bib_ref] Semiotic diversity in utterance production and the concept of 'language, Kendon [/bib_ref] [bib_ref] What does cross-linguistic variation in semantic coordination of speech and gesture reveal?:..., Kita [/bib_ref] [bib_ref] Why we should study multimodal language, Perniss [/bib_ref] , and matches speech-gesture parallelism as reported for other interactional phenomena; for example, people increase both speech and gesture efforts in noisy environments, and people use fewer words and fewer gestures as common ground increases (for a review, see [bib_ref] Multi-modal communication of common ground: A review of social functions, Holler [/bib_ref].
Second, we show in detail how people orchestrate efforts in speech and gesture to achieve rapid coordination. In particular, the type of repair initiation used predicts how people divide their multimodal efforts: the more specific the repair initiation, the more multimodal effort is invested by the person initiating repair, leaving less work for the sender of the original message to resolve the trouble. This replicates prior unimodal work showing systematicity in how verbal effort is distributed across repair initiation and solution [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref] , and shows that the pattern is robust enough to hold in both naturalistic as well as task-based conversations. Our results extend this division of labor principle to composite utterances, providing an unprecedented view of multimodal contributions to the coordination of joint action.
Third, we find that people overwhelmingly converge on the repair format (i.e., restricted offer) that minimizes multimodal effort for the dyad as a social unit. This is a novel, direct attestation of the principle of least collaborative effort 11-13 that is made possible by combining quantifications of speech effort with new, reproducible methods to measure gestural effort in terms of kinematics.
Taken together, these findings provide a novel unifying perspective on studies of language use 11-13 and nonlinguistic joint action [bib_ref] Etiquette and effort: Holding doors for others, Santamaria [/bib_ref] [bib_ref] Rationality in joint action: Maximizing coefficiency in coordination, Török [/bib_ref] [bib_ref] Computing joint action costs: Co-actors minimize the aggregate individual costs in an..., Török [/bib_ref]. The coordination of joint action minimally involves dynamically updated task representations, monitoring processes, and adjustable behaviors [bib_ref] A minimal architecture for joint action, Vesper [/bib_ref]. Although linguistic coordination has sometimes been cast as a qualitatively different form of coordination [bib_ref] A minimal architecture for joint action, Vesper [/bib_ref] , here we have shown that the micro-environment of interactive repair-which occurs at frequencies and timescales more commensurate with joint action-provides a unique window onto the real-time negotiation of distributed agency. In repair, people provide public evidence of representations and monitoring processes, allowing them to rapidly hone in on optimal coordinative solutions 70 . By zooming in on these miniature coordination problems, we reveal systematicity in how people adjust multiple types of communicative behavior to minimize joint efforts, thereby unravelling the multimodal nature of coefficiency in conversational joint action.
Beyond the empirical findings, our study also makes conceptual and methodological contributions. One is to extend and enrich standard notions of efficiency in language use. Prior work has usually considered efficiency in terms of unimodal message length 4 . One limitation of such operationalizations is that they easily lose sight of the fact that in conversation, the interactional work of achieving mutual understanding is often distributed across turns and participants [bib_ref] The distribution of repair in dialogue, Colman [/bib_ref] [bib_ref] A simple repair mechanism can alleviate computational demands of pragmatic reasoning: simulations..., Van Arkel [/bib_ref]. We argue that communicative effort and efficiency are best studied at the level of the dyad as a social unit, and we show how interactive repair provides a microcosm that allows us to study the public negotiation of mutual understanding over multiple turns.
Another challenge of the most common unimodal operationalizations is that, when applied to co-present conversational settings, they are incomplete and reductive, focusing on language as a unimodal discrete symbol system while overlooking multimodal, continuous and dynamic properties of language use [bib_ref] A Systematic investigation of gesture kinematics in evolving manual languages in the..., Pouw [/bib_ref]. Our contribution towards solving this challenge consists of using methods and insights from studies of joint action and behavioral dynamics [bib_ref] Joint-action coordination in transferring objects, Meulenbroek [/bib_ref] [bib_ref] Kinematics fingerprints of leader and follower role-taking during cooperative joint actions, Sacheli [/bib_ref] [bib_ref] Strategic communication and behavioral coupling in asymmetric joint action, Vesper [/bib_ref]. Our measures capture how people use both categorical and gradient semiotic resources in multiple modalities to make meaning together, where we operationalized effort with (a) a linguistically informed quantification of speech in terms of orthographic characters 46,77 and (b) a kinematic measure of submovements derived from continuous manual movement [bib_ref] A Systematic investigation of gesture kinematics in evolving manual languages in the..., Pouw [/bib_ref] [bib_ref] When is ostensive communication used for joint action?, Vesper [/bib_ref] [bib_ref] Measuring conventionalization in the manual modality, Namboodiripad [/bib_ref] [bib_ref] Toward the markerless and automatic analysis of kinematic features: A toolkit for..., Trujillo [/bib_ref]. These measures do not fully capture the multi-semiotic dimension of social interaction, as we disregard dynamic properties in the spoken modality (see e.g., research on the phonetic characteristics of repair solutions 80 ) as well as non-manual embodied resources (further discussed below). However, we believe the combination of measures for the spoken and gestural modality used in the present study are a step in the direction of a truly multimodal linguistics that takes semiotic diversity seriously Limitations. The nature of our task may have invited more representational gestures than some other conversational settings, as the 3D objects lack conventional names and lend themselves well to iconic depiction 81 . We also found relatively high amounts of restricted offers (83,3%), compared to restricted requests and open requests (6,3%; a pattern reported for other task-based datasets as well [bib_ref] Contextualizing conversational strategies: Backchannel, repair and linguistic alignment in spontaneous and task-oriented..., Dideriksen [/bib_ref] [bib_ref] Measures and mechanisms of common ground: Backchannels, conversational repair, and interactive alignment..., Fusaroli [/bib_ref]. This might partially follow from the lab setting, in which people were unlikely to be troubled by noise interference or other low-level perceptual problems (which are associated with open requests [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref]. Perhaps because of the overall low amounts of open and restricted requests, we found no statistical difference in the frequency of these two types. Consequently, our conclusion that people repair trouble in the most cost-efficient way possible is based on the use of restricted offers: repair sequences of this type are the most frequent and require the smallest amount of joint multimodal effort. Future research could test whether frequency and cost-efficiency patterns together across all repair types in more naturalistic settings. Though the lab-and task-based setting can affect the use of gesture and www.nature.com/scientificreports/ repair types, our findings are largely in line with studies on other-initiated repair in more naturalistic settings, where people also prefer to use restricted formats over open requests [bib_ref] Universal principles in the repair of communication problems, Dingemanse [/bib_ref] , and take advantage of iconic properties of gestures for repair initiations and solutions [bib_ref] Holding gestures across turns: Moments to generate shared understanding, Sikveland [/bib_ref] [bib_ref] Depictive hand gestures as candidate understandings, Jokipohja [/bib_ref] [bib_ref] Gesturally-enhanced repeats in the repair turn communication strategy or cognitive language-learning tool?, Olsher [/bib_ref]. We therefore believe that the observed distribution of multimodal effort across repair types is likely to be robust enough to generalize to everyday language use. That is, while we might expect to find fewer manual gestures and more open and restricted requests, we would still predict people's multimodal productions to be more effortful in repair initiations of the type restricted offer compared to open and restricted requests (and vice versa for solutions).
We have investigated the cost-efficient use of words and manual co-speech gestures. By foregrounding efficiency in a task-based setting and focusing on speech and gestures, we have of course captured only a partial view of what it means for people to coordinate their multimodal utterances to resolve conversational problems. Future studies could broaden this view, for example by incorporating eye gaze, eyebrow movements, head movements and forward leans, which are known to play a role in signaling trouble [bib_ref] Gestures as requests for information: Initiating repair operations in German native-speaker conversation, Andrews [/bib_ref] [bib_ref] Timing of visual bodily behavior in repair sequences: Evidence from three languages, Floyd [/bib_ref] [bib_ref] Leaning and recipient intervening questions in Mandarin conversation, Li [/bib_ref] [bib_ref] Inclined to better understanding-The coordination of talk and 'leaning forward' in doing..., Rasmussen [/bib_ref] [bib_ref] A conversation analytic study of gestures that engender repair in ESL conversational..., Seo [/bib_ref] [bib_ref] Context-sensitivity in conversation: Eye gaze and the German repair initiator bitte, Egbert [/bib_ref]. This could be complemented by a consideration of other social or expressive factors which influence communicative behaviors, as people can for example opt to use an open request rather than a restricted offer for face-saving purposes [bib_ref] Other-initiated repair sequences in Korean conversations, Kim [/bib_ref]. More empirical and theoretical work is needed to understand how pressures and constraints in human sociality interact with principles of efficiency in joint action. We take this to be an important avenue for future research, where moving our attention from efforts of the individual to those of the dyad as a cooperative social unit is an important first step.
# Conclusion.
In summary, in this study we investigated communicative efficiency from a multimodal and interactional perspective by zooming in on other-initiated repair sequences. As a conversational environment in which there is a clear goal, a limited set of turns to reach that goal and a limited inventory of communicative resources to use in those turns, other-initiated repair is a natural laboratory for the systematic study of coefficiency in language use. Our findings reveal that people divide the total amount of multimodal effort between them in such a way as to minimize the overall amount of speech and gesture efforts for the dyad as a whole. By investigating how communicative efficiency is realized in multimodal language use at the level of the dyad as social unit, we have shown how minimizing effort in language use is an interactional achievement.
# Methods
Participants. Twenty dyads took part in the study (10 mixed-gender, 6 female-only and 4 male-only dyads, Apparatus and materials. The stimuli were 16 images of novel 3D objects, called 'Fribbles' (adapted from Barry et al. [bib_ref] Meet the Fribbles: Novel stimuli for use within behavioural research, Barry [/bib_ref] , see [fig_ref] Figure 3: Panel [/fig_ref]. During the interaction, participants were standing face-to-face, where each had their own button box and screen (24′ BenQ XL2430T), slightly tilted, and positioned at hip height to ensure mutual visibility of upper torso and gesturing area (see [fig_ref] Figure 3: Panel [/fig_ref]. The Fribbles were presented on these screens on a grey background in rows of 5, 6, and 5 figures respectively, in a size of about 4 × 4 cm per figure, with a corresponding label next to it. The order of the Fribbles was random and varied for the participants (but was constant across dyads). Audio was recorded with head-mounted microphones (Samson QV) and videos were made with three HD cameras (JVC GY-HM100/150). 3D motion tracking data was collected using two Microsoft Kinects V2 (for 25 joints, sampled at 30 Hz).
Procedure. Participants were assigned director/matcher roles. In each trial, a red triangle highlighted a single target Fribble on the director's screen. The participants were instructed to communicate in order for the matcher to find the target item on their screen. To indicate their selection, the matcher said the corresponding label out loud and pressed a button to go to the next trial, where the participants switched director/matcher roles. After matching all 16 Fribbles, a new round would start; in total six rounds were completed, yielding a total of 96 trials. No time constraints were posed and the participants did not receive feedback about accuracy. Participants were told that they were 'free to communicate in any way they want' (an instruction phrased to be agnostic about communicative modality, i.e., speech and/or gesture), and that their performance would be a joint achievement. Dyads spent 24.4 min on the task on average (range = 14.2-34.6 min).
Analysis. The audio-video data were annotated in ELAN (version 5.8); data processing and statistical analyses were performed with the R statistical program (version 4.0.2).
Speech was segmented into Turn Constructional Units (TCUs; i.e., potentially complete, meaningful utterances [bib_ref] Turn-constructional units and the transition-relevance place, Clayman [/bib_ref] and orthographically transcribed based on the standard spelling conventions of Dutch. Other-initiated repair was coded based on a modified version of the coding scheme by Dingemanse et al. [bib_ref] A coding scheme for other-initiated repair across languages, Dingemanse [/bib_ref]. We annotated the trouble source, repair initiation and repair solution, where the boundaries of those annotations corresponded to the speech annotations (where a single repair annotation could correspond to a single TCU or span multiple TCUs). Subsequently, repair initiations were categorized into three types: open request, restricted request and restricted offer. Details on the coding procedure including examples can be found as Supplementary Information (S1.1). Inter-rater reliability for repair identification, segmentation and coding was moderate to high (all yielded minimally 75% agreement; for details and additional reliability measures, see Supplementary Information; S1.2). www.nature.com/scientificreports/ For co-speech gestures, the stroke phase was annotated for gesture units (i.e., the meaningful part of the gestural movement, for the left and right hand separately. Inter-rater reliability was substantial for gesture identification, segmentation and coding (minimally 75% agreement; for details and additional measures, see Supplementary Information; S1.3). We considered gestures to be part of a repair turn when the gesture stroke completely overlapped with the repair annotation (which was the case for 91.5% of the gestures), but used finegrained rules and manual inspection in case of partial or no overlap (see Supplementary Information; S1.4). All types of manual co-speech gestures were included in the analysis, but the majority of the gestures in the dataset are iconic (89.8%).
Submovements were computed for each gesture stroke, of which the onset and offsets were determined by the manual annotations. The calculation was based on the position of the left-and right-hand tips in 3D space. To ignore noise-related jitter, we smoothed the position traces, and their derivatives (3D speed) with a third order Kolmogorov-Zurbenko (KZ) filter with a span of 2. 3D gesture speed was used to determine submovements, which was based on the speed of the left or right hand for one-handed gestures, or the summed speed of both hands in the case of two-handed gestures. The number of submovements was then computed by identifying local maxima peaks in the 3D speed time series [bib_ref] A Systematic investigation of gesture kinematics in evolving manual languages in the..., Pouw [/bib_ref] [bib_ref] Toward the markerless and automatic analysis of kinematic features: A toolkit for..., Trujillo [/bib_ref]. To this end, we used R-package pracma and considered a peak to be a submovement when it exceeded at least 10 cm/s and only if they had at least 100 ms distance between adjacent peaks. The minimum amount of submovements per gesture stroke is 1 (i.e., static strokes are considered to consist of 1 submovement). Two examples of gestures along with their submovement profile are presented in [fig_ref] Figure 4: In panel [/fig_ref] (for more examples, see Supplementary Information; S1.5).
In order to analyze the division of multimodal effort, we combined the speech and gesture efforts to yield a multimodal effort variable. We first standardized the individual speech and gesture measures (as their distributions differed greatly, with gesture submovements being zero-inflated, see [fig_ref] Figure 4: In panel [/fig_ref] , and then summed them. We then calculated the proportion of multimodal effort in the repair initiation as compared to the total multimodal effort in the repair initiation and repair solution. To subsequently inspect how the total amount of joint effort varies across repair types, we summed the multimodal effort in the repair initiation and repair solution for each repair sequence. The resulting measures (i.e., the proportion of effort in the repair initiation and total joint effort) were used as dependent variables in mixed effects models with random intercepts and slopes for dyads and target items (unless reported otherwise, when a maximal model was not possible due to convergence issues) and repair initiator type as predictor. We used backward difference contrast coding to compare restricted requests to open requests, and restricted offers to restricted requests.
## Data availability
All data and analysis scripts used for this study are openly available on the Donders Repository at: https:// doi. org/ 10. 34973/ 12dp-9q56. The gesture is produced by a matcher as part of a restricted offer with the following speech: "ah en is zijn arm uh rond maar ook een beetje met hoeken?" [literal translation: ah and is his arm round but also a bit with corners?]. The right arm is extended to model the 'arm' , while the left hand is moved around it to depict the angular shape (number of submovements: 4). The bottom row shows a gesture which was produced by a director in a repair solution in response to a restricted offer. The gesture depicts the rectangular subpart on the front side of the Fribble, while saying "ja precies" [yes exactly]. The multimodal utterance thereby confirms the preceding restricted offer (which contained an identical gesture). The hands are kept somewhat apart (to depict the width of the rectangle), and moved straight downwards (number of submovements: 1). The density plots in panel (B) show the distributions for the speech and gesture effort measures (the dots are the median and the lines the 95% quantile interval).
[fig] Figure 1: joint multimodal effort Division of multimodal effort (proportion of joint multimodal effort) C Boxplots showing the effort invested in the repair initiation (orange) and repair solution (blue), for repair formats of increasing specificity (open request < restricted request < restricted offer). The boxes represent the interquartile range; the middle line the median; the whiskers the minimum and maximum scores (outliers excluded). Every dot represents a repair initiation or solution. Absolute speech effort (A) and absolute gesture effort (B) both go up in repair initiations and down in repair solutions as repair formats become more specific. Proportional multimodal effort (C) shifts from repair initiation to repair solution as we move towards more specific repair formats. The dashed line represents equal division of effort across participants. For the gestural modality, there is considerable individual variation, with some people gesturing very rarely or not at all. In total, 479 co-speech gestures were produced across all repair initiations and solutions, with 37.2% of the turns containing at least one gesture. But the likelihood of encountering a gesture in a turn differs greatly across repair types and sequential positions; ranging from 4.2% in repair initiations of the type open request, to 79.2% in repair solutions in response to open requests. When quantifying gesture effort in terms of submovements, we find a similar pattern as for speech effort (Fig. 1B). As repair initiations become more specific, more gesture submovements are used in the initiation (open requests: M = 0.04, SD = 0.20; restricted requests: M = 0.15, SD = 0.43; restricted offers M = 1.02, SD = 1.57), and fewer are used in the solution (open requests: M = 3.63, SD = 3.84; restricted requests: M = 2.67, SD = 3.15; restricted offers M = 0.53, SD = 1.74). [/fig]
[fig] Figure 2: 37, SD = 1.21) compared to restricted requests (M = 2.45, SD = 1.74; β = -1.15, SE = 0.30, t = -3.80, p = 0.006), but that joint effort in restricted requests does not differ significantly from open requests (M = 3.51, SD = 2.58; β = -0.86, SE = 0.55, t = -1.58, p = 0.137). In terms of how often the different types of repair initiations are used, we found that restricted offers are by far the most frequent (83,3%). A mixed effect model (with random intercepts for dyads and target items) revealed that restricted offers occur significantly more than restricted requests (10,3%; β = 0.91, SE = 0.06, t = 15.89, p < 0.001), while restricted requests do not differ in frequency from open requests (6,3%; β = 0.05, SE = 0.06, t = 0.82, p = 0.413). The preference for using restricted offers paired with the finding Boxplots showing the joint amount of multimodal effort invested by both participants to resolve the interactional trouble. The boxes represent the interquartile range; the middle line the median; the whiskers the minimum and maximum scores (outliers excluded). Every dot represents a repair sequence, i.e., repair initiation and repair solution together. As the specificity of repair formats goes up, joint multimodal effort invested goes down. Scientific Reports | (2022) 12:19111 | https://doi.org/10.1038/s41598-022-22883-w [/fig]
[fig] M: age = 22.3 years, Range age = 18-32 years). The unacquainted participants were recruited via the Radboud SONA participant pool system. Participants provided informed consent and were paid for participation. The participants who are visible in the figures provided informed consent to publish the images in an online open access publication. The study met the criteria of the blanket ethical approval for standard studies of the Commission for Human Research Arnhem-Nijmegen (DCCN CO 2014/288), and was conducted in accordance with relevant guidelines and regulations. [/fig]
[fig] Figure 3: Panel (A) shows the "Fribbles" that were used as stimuli. Panel (B) shows the set-up by means of screenshots from the three cameras. [/fig]
[fig] Figure 4: In panel (A), the top row shows a gesture which depicts the subpart on the left side of the Fribble. [/fig]
|
Traumatic Pulmonary Pseudocyst Mimicking a Congenital Cystic Lung Disease
Traumatic pulmonary pseudocyst (TPP) is a rare entity that occurs following a trauma to the chest. It usually presents as multiple cystic lesions on thoracic imaging. It is treated conservatively and tends to completely resolve after few months. In the absence of striking signs of trauma such as rib fractures, TPP can be mistaken for other cystic lung diseases. We present a case of TPP in a 17-year-old male who was seen for mild hemoptysis after falling off a cliff. The extent of his right lower lobe cystic lesions along with the lack of major signs of trauma led to an incorrect diagnosis of congenital pulmonary airway malformation. The patient was considered for lobectomy, which he refused. Imaging of the chest repeated one and three years later showed complete resolution of the lesions.
# Introduction
Traumatic pulmonary pseudocysts (TPPs) are cystic and cavitary lesions that can develop in the lung parenchyma after a trauma to the chest [bib_ref] Cystic and cavitary lung lesions in children: Radiologic findings with pathologic correlation, Odev [/bib_ref]. Although TPP remains a rare entity, awareness of this benign condition is imperative as it may mimic other more serious pulmonary cystic diseases. [bib_ref] Traumatic pulmonary pseudocyst, George [/bib_ref] We present a case where TPP was initially misdiagnosed as congenital pulmonary airway malformation (CPAM) and highlight the potential consequence of such misdiagnosis.
## Case report
A previously healthy 17-year-old male presented with the complaint of mild hemoptysis after sustaining a blunt trauma to the chest. He fell off a 3-foot cliff while hiking and landed on the right side of his chest. On presentation, the patient's pain was tolerable and he was breathing comfortably. His vital signs showed a pulse of 98 beats per minute, blood pressure of 110/60 mmHg, and an oxygen saturation of 98% on room air. His exam revealed minimal abrasions, ecchymosis, and tenderness over the right lower chest wall at the anterior axillary line. His lung exam revealed decreased breath sounds over the right lower lung field. A chest Xray obtained within 2 hours of the trauma showed alveolar opacities in the right lower lobe with multiple cystic air spaces containing air-fluid levels [fig_ref] Figure 1: Chest X-ray obtained 2 hours after the trauma showing multiple cystic lesions... [/fig_ref]. There were no associated pleural effusions, pneumothorax, or rib fractures. A Computed Tomography (CT) scan of the chest showed thick-walled multicystic lesions with patchy air space opacities and consolidations in the right lower lobe [fig_ref] Figure 2: CT scan of the chest showing thick-walled multicystic lesions with patchy air... [/fig_ref]. No previous chest imaging was available for comparison. The described CT scan abnormalities, in the absence of extrapulmonary posttraumatic findings, were suggestive of CPAM with superimposed bleeding. The patient was admitted for observation and evaluation and placed on intravenous Amoxicillin/Clavulanate. Spirometry done the next day was normal. His complete blood count, basic metabolic panel and bleeding profile were normal. His C-reactive protein was elevated at 32.0 mg/L. Gram stain, acid fast stain, and sputum cultures for bacteria, fungi, and tuberculosis were all negative. Alpha-1 antitrypsin and immunoglobulin levels were within normal limits.
The patient was evaluated by a cardiothoracic surgeon and a right lower lobectomy was being considered. However, given the indolent course of his disease and his negative history for pulmonary infections thus far, the patient elected to defer further surgical evaluation and, instead, followup with clinical observation. He remained asymptomatic throughout the interval period and a chest X-ray repeated after one year was normal [fig_ref] Figure 3: Chest X-ray of patient repeated 1 year later showing complete resolution of... [/fig_ref]. Finally, a CT scan of the chest obtained two years later showed complete resolution of the previously described abnormalities [fig_ref] Figure 4: A normal chest CT scan of the patient obtained three years after... [/fig_ref]. Due to the fact that his cysts resolved spontaneously with time after his trauma, the patient was finally diagnosed with TPP.
# Discussion
TPP develops as a result of high compressive forces transmitted to the pulmonary parenchyma during chest wall trauma. It is primarily described following a blunt trauma to the chest but is also seen with penetrating injuries [bib_ref] Editorial: Diagnosing traumatic pulmonary pseudocyst, Gulbahar [/bib_ref]. The rapid compression and decompression of the chest result in lacerations and cavitary lesions within the lung tissue, particularly when the lungs are compressed against a closed Clinically, TPP can vary widely in presentation. Patients can be entirely asymptomatic or can have life-threatening respiratory compromise. Commonly reported symptoms include hemoptysis, which is seen in almost half of the patients. Other manifestations include shortness of breath, cough, chest pain, fevers, and leukocytosis. Additionally, TPP is almost always associated with rib fractures or other evidences of trauma such as large contusions and pneumothoraces [bib_ref] Traumatic pulmonary pseudocyst: An underreported entity, Phillips [/bib_ref].
Radiologically, cavitary lesions in TPP appear on chest X-ray within the first 24 hours of trauma in almost half the patients. The size of the pseudocysts ranged from 2 to 14 cm in diameter in previous reports. [bib_ref] Traumatic pulmonary pseudocyst, George [/bib_ref] They can be single or multiple and can be seen either on the same side of trauma or on the opposite side as a result of contrecoup injury [bib_ref] Blunt thoracic trauma in children: Review of 137 cases, Balci [/bib_ref]. Notably, TPP lesions can change quickly in size and shape on serial X-rays done over the course of days. This can help distinguish TPP from other cystic conditions. An even more sensitive imaging modality is CT scan of the chest. The finding of single or multiple cysts with thin walls along with air space consolidation of the surrounding parenchyma has been suggested as diagnostic in the setting of a preceding chest wall trauma [bib_ref] Chest case of the day. Traumatic pneumatocele, Suhocki [/bib_ref].
TPP is usually self-remitting and the overall prognosis of the condition itself is excellent. Complications are rare but may include infection, pneumothorax, or bleeding [bib_ref] Diagnosis and treatment of traumatic pulmonary pseudocysts, Melloni [/bib_ref]. The rate of complications increases when the patient is exposed to unnecessary procedures from failure to accurately diagnose this condition. Our patient, for instance, was being considered for what turned out to be an unnecessary surgical lobectomy. This highlights the importance of differentiating TPP from other resembling yet more serious conditions.
In our patient, the extent of the intraparenchymal lesions along with the absence of extrapulmonary posttraumatic findings led to the erroneous diagnosis of CPAM. Furthermore, antibiotics were started for concern of a superimposed infectious process, in the setting of active hemoptysis and right lower lobe opacities and consolidations on chest imaging. CPAM, formerly known as congenital cystic adenomatoid malformation (CCAM), is a unilateral congenital lung disease in which one or multiple cysts replace a lobe of the lungs. It is the most common developmental malformation of the lungs. Cavities are often thick-walled and can range in size from less than 1 cm to more than 2 cm depending on the type of lesions [bib_ref] Congenital cystic adenomatoid malformation -Dangers of misdiagnosis: A case report, Amraoui [/bib_ref].
The pathophysiology of CPAM is unclear, but the condition is thought to arise from an airway obstruction during fetal development [bib_ref] Congenital cystic adenomatoid malformations of the lung: Diagnosis, treatment, pathophysiological hypothesis, Lezmi [/bib_ref]. It generally leads to respiratory distress and infections in neonates and infants, and less than 10% of patients will present after the age of 1 year. In adults, it may manifest as recurrent or persistent pneumonia in the same location. Other features may include lung abscesses, failure to thrive, or a pneumothorax. The decision for elective surgery in asymptomatic patients with CPAM stems from its potential for complications as well as a possible association with lung malignancy [bib_ref] Congenital cystic lung disease: Contemporary antenatal and postnatal management, Azizkhan [/bib_ref].
CT scan remains the gold standard for diagnosing both CPAM and TPP. Unfortunately, there are no specific radiological findings that help distinguish both entities. Cystic lesions typically affect the lower lobes of the lungs in CPAM and TPP and can be single or multiple and unilateral or, rarely, bilateral. Furthermore, the cysts can be filled with air or fluids in both conditions. Nevertheless, TPP becomes more plausible in the presence of preceding trauma. Furthermore, regression of the lesions on repeat chest imaging is a key diagnostic finding that occurs only in TPP as opposed to CPAM. For this reason, patients suspected to have TPP ought to have repeat images demonstrating regression of the lung lesions before a definite diagnosis of TPP is made. The average time for cyst resolution in TPP is estimated at 4 months [bib_ref] Traumatic pulmonary pseudocysts mimicking a congenital malformation of the lung, Ngoo [/bib_ref].
# Conclusion
Unlike CPAM that often requires surgical resection, TPP is a benign, self-resolving cystic lung condition that develops after thoracic trauma. Physicians should be cognizant of TPP as an improper diagnosis can expose the patient to unnecessary drugs, procedures, or even surgery.
[fig] Figure 1: Chest X-ray obtained 2 hours after the trauma showing multiple cystic lesions containing air-fluid levels. [/fig]
[fig] Figure 2: CT scan of the chest showing thick-walled multicystic lesions with patchy air space opacities. [/fig]
[fig] Figure 3: Chest X-ray of patient repeated 1 year later showing complete resolution of the cystic lesions. [/fig]
[fig] Figure 4: A normal chest CT scan of the patient obtained three years after the trauma. glottis impeding the rapid ejection of air through the upper airways. Subsequently, if no communication exists between the cavities and the pleural space, air and fluid escape from the parenchyma and fill up the cavity[2]. [/fig]
|
Alcohol interventions, alcohol policy and intimate partner violence: a systematic review
Background: Intimate partner violence (IPV) is a significant global public health issue. The consistent evidence that alcohol use by one or both partners contributes to the risk and severity of IPV suggests that interventions that reduce alcohol consumption may also reduce IPV. This study sought to review the evidence for effects on IPV of alcohol interventions at the population, community, relationship and individual levels using the World Health Organization ecological framework for violence. Methods: Eleven databases including Medline, PsycINFO, CINAHL and EMBASE were searched for English-language studies and grey literature published investigating whether alcohol interventions/ policies were associated with IPV reduction within adult (≥18) intimate relationships. Eleven studies meeting design criteria for attributing effects to the intervention and ten studies showing mediation of alcohol consumption were included in the review. The heterogeneity of study designs precluded quantitative meta analysis; therefore, a critical narrative approach was used. Results: Population-level pricing and taxation studies found weak or no evidence for alcohol price changes influencing IPV. Studies of community-level policies or interventions (e.g., hours of sale, alcohol outlet density) showed weak evidence of an association with IPV. Couples-based and individual alcohol treatment studies found a relationship between reductions in alcohol consumption and reductions in IPV but their designs precluded attributing changes to treatment. Randomized controlled trials of combined alcohol and violence treatment programs found some positive effects of brief alcohol intervention as an adjunct to batterer treatment for hazardous drinking IPV perpetrators, and of brief interventions with non-dependent younger populations, but effects were often not sustained. Conclusions: Despite evidence associating problematic alcohol use with IPV, the potential for alcohol interventions to reduce IPV has not been adequately tested, possibly because studies have not focused on those most at risk of alcohol-related IPV. Research using rigorous designs should target young adult populations among whom IPV and drinking is highly prevalent. Combining alcohol and IPV intervention/policy approaches at the population, community, relationship and individual-level may provide the best opportunity for effective intervention.
# Background
The World Health Organization (WHO) defines intimate partner violence (IPV) as 'any behaviour within an intimate relationship that causes physical, psychological or sexual harm'. WHO recently estimated the global prevalence of physical and/or sexual IPV to be 30% among everpartnered women. Thus, IPV is a significant global public health and human rights issue that has damaging effects on the health and well-being of women and children, [bib_ref] Intimate partner violence, abortion, and unintended pregnancy: Results from the WHO Multi-country..., Pallitto [/bib_ref] and significant social and economic costs.
Alcohol use, especially heavy drinking and drinking large amounts per occasion, is linked to male-to-female partner violence [bib_ref] Alcohol and intimate partner violence: a meta-analytic review, Foran [/bib_ref]. Across different cultures, violence is more severe when one or both partners (most often the male partner) has been drinking [bib_ref] Alcohol may not cause partner violence but it seems to make it..., Graham [/bib_ref]. Meta-analyses suggest that alcohol plays a causal contributing role in aggression generally; [bib_ref] Effects of Alcohol on Human Aggression: Validity of Proposed Explanations, Bushman [/bib_ref] however, the extent to which alcohol's role in IPV is causal, is complex and contested. In addition, across the globe, IPV is a gendered issue, reflecting the unequal power relationships between men and women. Although under experimental conditions alcohol increases aggression in both men and women, the effect is stronger for men [bib_ref] Men and women, alcohol and aggression, Giancola [/bib_ref] and drinking by men has been shown to play a more important role in IPV perpetration than has drinking by women, [bib_ref] Problem drinking and intimate partner violence, White [/bib_ref] reflecting the gendered nature of both problem drinking and IPV.
Alcohol is thought to influence aggressive behaviour through detrimental effects on the drinker's cognitive executive functioning, [bib_ref] Executive functioning: a conceptual framework for alcohol-related aggression, Giancola [/bib_ref] and problem-solving abilities, [bib_ref] Alcohol and aggression: a social information processing analysis, Sayette [/bib_ref] narrowing the focus of attention, [bib_ref] Alcohol myopia: its prized and dangerous effects, Steele [/bib_ref] increasing their willingness to take risks, [bib_ref] Effects of alcohol intoxication on the perceived consequences of risk taking, Fromme [/bib_ref] and increasing concern about personal power among male drinkers. In the context of an intimate couple, when one of the partners has been drinking, he or she will be less able to address conflicts constructively because of (a) the effects of alcohol on cognitive functioning and problem-solving; (b) the drinking partner may have a disproportionate response to a perceived slight, insult or other apparent wrong done by the partner and be less likely to see the partner's perspective or the situational and environmental factors that may have affected the partner's behaviour (because of the narrowing of their focus of attention on a specific action of the partner related to their drinking); (c) the drinking partner may engage in highly provocative or aggressive behaviour without thinking about the consequences of his or her actions because of alcohol's effects on risk-taking; and, (d) for male partners in particular, perceived slights or aggression by the partner may be interpreted as a threat to their masculinity or social identity generally and therefore require an aggressive response to reassert this identity (see [bib_ref] Aggression as impression management, Felson [/bib_ref] [bib_ref] Apparent motives for aggression in the social context of the bar, Graham [/bib_ref]. When both partners have been drinking, the role of alcohol may be even greater [bib_ref] Alcohol consumption and escalatory aggression in intoxicated and sober dyads, Leonard [/bib_ref] because of the potential for alcohol to affect the thinking, perceptions and risk-taking of both partners. That is, both partners are more likely to misperceive the other's behaviour, be less able to resolve the situation without aggression, and be more likely to engage in risky aggression. Social and cultural perceptions of alcohol can also play a role where the acceptance and tolerance of alcohol-related misbehaviour (including aggression), can influence drinkers' expectations about their behaviour while drinking [bib_ref] Current directions in research on understanding and preventing intoxicated aggression, Graham [/bib_ref]. This means that, regardless of the effects of alcohol, some people who have been drinking may intentionally engage in aggression or violence toward an intimate partner because they have the expectation that their behaviour will be excused on the basis that they had been drinking at the time.
Although drinking can occur without IPV and IPV without drinking, both are sufficiently linked that the WHO proposed that primary prevention interventions to reduce the harm caused by alcohol could potentially reduce IPV. Further investigation of the effects of alcohol prevention on IPV is important because direct interventions addressing violence against women have been shown to have limited impact [bib_ref] Interventions for violence against women: scientific review, Wathen [/bib_ref]. Recognising the multi-dimensional and complex nature of IPV, the WHO recommends an ecological framework for violence prevention wherein factors influence violent behaviour separately and cumulatively at the individual, relationship, community and societal levels [fig_ref] Figure 1: Ecological model for understanding violence [/fig_ref]. Although previous reviews of alcohol interventions have focused exclusively on the individual or relationship level [bib_ref] Review of the association between treatment for substance misuse and reductions in..., Stuart [/bib_ref] [bib_ref] The effects of treatment for substance use problems on intimate partner violence:..., Murphy [/bib_ref] (e.g., individual or couple treatment for alcohol dependency), as this model suggests, alcohol interventions relevant to alcohol-related IPV can occur at the community level and the population level, as well as at the individual/relationship level. Community-level interventions are distinguished from population-level interventions in that they apply to a specific community or area, are often developed in response to local issues or concerns and typically involve community stakeholders in their development and management [bib_ref] Introduction: community action research and the prevention of alcohol problems at the..., Allamani [/bib_ref]. Population or societal-level interventions, by contrast, are implemented at the population level more broadly (country, state, region) and may be more likely to involve formal mechanisms such as taxation, although similar interventions can occur at both community and population levels.
Because alcohol use is 'one of the factors most open to intervention and change, '(p.viii) and broad evidence exists of effective interventions that reduce alcohol consumption and related harms,this review asks the question: Do interventions to reduce alcohol use at the individual, relationship, community and/or population level, reduce intimate partner violence? In this systematic review, we aimed to explore the evidence for the effects of alcohol interventions on IPV, and the extent to which the effects are mediated by changes in alcohol consumption. For this review, we adopted a broad definition of "intervention" to include interventions specifically implemented to reduce alcohol consumption within a target population or community (e.g., alcohol restrictions or addiction treatment) or alcohol policy levers that may affect alcohol consumption indirectly (e.g., alcohol taxes and planning regulations regarding alcohol outlets).
# Methods
Eleven bibliographic databases were searched systematically for English language peer-reviewed and grey literature studies (such as non peer-reviewed government reports) published between 1 January 1992 and 1 March 2013 including: Medline, CINAHL, EMBASE, PsycINFO, Proquest Central, Cochrane Library, Campbell Collaboration Library, ATSI Health, Drug and Rural Health, and Women's Studies International. The search strategy combined three concepts of interest: (i) alcohol use, (ii) IPV, and (iii) interventions, using medical subject headings (MeSH), database-specific thesauri search terms, and text-based keywords. Specific terms for alcohol prevention policies were added. Searches were conducted in two stages -the first searches were conducted between 30 October to 1 November 2012 with a second search to update the review conducted on 1 March 2013. A sample search strategy is at (Additional file 1).
A study was included in the review if it investigated whether an intervention or policy to reduce alcohol consumption was directly or indirectly associated with a change in any form of IPV as a primary or secondary outcome. The review included studies of persons 18 years and older and IPV perpetration by either sex within a current heterosexual or homosexual dating, co-habiting or marital relationship, or from a former partner.
The search retrieved 1,810 citations as outlined in the flowchart [fig_ref] Figure 2: Selection of articles for review of alcohol and policy interventions to reduce... [/fig_ref]. IW conducted the initial review of study titles and abstracts with 93 (5%) full text papers retrieved. A further 24 papers were identified from hand searching reference lists and contacting key experts. A total of 117 papers were examined against the initial eligibility criteria. Commentaries, reviews or articles that reported no original data were excluded. Due to questions regarding the integrity of research by Dr. William Fals-Stewart (State of New York v. William Fals-Stewart, 2010), studies in which he was first author or using his data were excluded.
Forty studies (44 papers) met the initial selection criteria. Data for each study was extracted using an agreed standardised template recording details of the study. This included the PICOS criteria -population/ sample, intervention, controls or comparisons, outcomes (IPV and alcohol consumption and other measures pertaining to IPV) and study design. We also noted year of study, aims and strengths and limitations. Two additional criteria were applied prior to final study selection. First, we assessed whether the study design and sample size allowed outcomes to be attributed, at least partly, to the intervention or policy being evaluated. Eleven studies met these design criteria (Additional file 2: . These included: randomized controlled trials, longitudinal studies that measured IPV over multiple time points before and after the intervention, included multiple replications or used an interrupted time series design. Population and community-level ecological studies that used designs able to rule out explanations other than the intervention to account for changes at the community or population level were included, even when they did not include individual measures necessary for making causal attributions regarding the mechanism of change. Excluded studies used cross-sectional and pre-post designs, small pilot samples and had methodological weaknesses that limited interpretation because of potential bias from regression to the mean and other uncontrolled factors.
Because only a small number of studies met the design criteria and many did not test the assumption that the effects of the intervention on IPV were mediated by the intervention's effect on alcohol consumption, we included in our review a second set of ten studies that support the assumption of mediation (Additional file 3: . That is, while these studies do not meet the design criterion of being able to rule out other explanations for the apparent effects of the intervention, they do provide some evidence that variations in IPV associated with variations in alcohol consumption and that both are associated with the intervention. Selected studies were categorised by level in the ecological frameworkpopulation, community, relationship and individual-level interventions. IW and AT independently reviewed the individual and couple treatment studies for study strengths and limitations (Additional file 2: , and IW and KG independently reviewed the population and community-level interventions, with agreement reached by consensus.
In the Results section below, we refer to the 40 studies (44 papers) that met the initial selection criteria but discuss in detail only the findings from the 21 studies that met either the design criteria or provided evidence that the effects of interventions on IPV were mediated through alcohol consumption.
The breadth of the review and the heterogeneity in design and quality precluded formal meta-analysis; therefore, findings were synthesized using a critical narrative approach. This involved considering the theoretical basis for how the intervention might work within each level, critically appraising studies for methodological quality and juxtaposing findings within the ecological framework to draw conclusions across the body of evidence.
# Results
## Population-level interventions alcohol pricing/taxation and ipv
Alcohol consumption is affected by the price of alcohol, which is largely determined by government policy on taxation. Thus, increasing the price of alcohol, either through market forces or taxation, would be expected to reduce the amount of alcohol consumed by those who perpetrate alcohol-related IPV, and by extension the frequency and severity of IPV. Four studies [bib_ref] The price of alcohol, wife abuse, and husband abuse, Markowitz [/bib_ref] [bib_ref] Taxing sin and saving lives: Can alcohol taxation reduce female homicides?, Durrance [/bib_ref] [bib_ref] Effects of domestic violence policies, alcohol taxes and police staffing levels on..., Zeoli [/bib_ref] [bib_ref] The impact of a large reduction in the price of alcohol on..., Herttua [/bib_ref] evaluated the relationship between alcohol pricing/taxation and IPV. Three met the design criteria [bib_ref] The price of alcohol, wife abuse, and husband abuse, Markowitz [/bib_ref] [bib_ref] Taxing sin and saving lives: Can alcohol taxation reduce female homicides?, Durrance [/bib_ref] [bib_ref] Effects of domestic violence policies, alcohol taxes and police staffing levels on..., Zeoli [/bib_ref] (Additional file 2: . The fourth study [bib_ref] The impact of a large reduction in the price of alcohol on..., Herttua [/bib_ref] , a pre-post comparison in a single country, was excluded on the basis of design.
Only one study, conducted in the USA, [bib_ref] The price of alcohol, wife abuse, and husband abuse, Markowitz [/bib_ref] found a significant relationship between the price of alcohol and IPV. Using a composite price of 1 ounce of pure alcohol based on weighted average annual prices of beer, wine and liquor, the study modelled the effects of changes in the price of alcohol on the probability of self-reported 'husband' and 'wife abuse' from a 1985 national family violence survey and two annual follow ups. The study found that a 1% increase in the price of alcohol was associated with a reduction of 3.1 -3.5% in wife abuse. No association was found for husband abuse. The study used data from two time points over a three-year period; however, the findings are consistent with cross-sectional comparisons and changes in price were small over that period. The study did not include measures of consumption; thus, it was not possible to assess the mediating effect of consumption.
Of the other two studies of pricing/taxation, one longitudinal study [bib_ref] Taxing sin and saving lives: Can alcohol taxation reduce female homicides?, Durrance [/bib_ref] examined the relationship between changes in alcohol taxes, alcohol consumption and female homicide rates across 46 states in the USA (with most women killed by an intimate partner). Their modelling of data from 1990 to 2004 found a significant association between (a) alcohol tax increases and reduced per capita consumption, and (b) reduced consumption and reduced IPV. However, the relationship between alcohol taxes and IPV was not statistically significant, although their results point in this direction. In explaining their results, the authors questioned the extent to which those who consume alcohol and commit homicide are sensitive to price.
The third study [bib_ref] Effects of domestic violence policies, alcohol taxes and police staffing levels on..., Zeoli [/bib_ref] used a multiple time series design to assess the impact of a range of interventions (including changes to State and Federal beer taxes) on intimate partner homicide (IPH) and IPH involving firearms. Their analysis covered 46 of the largest U.S. cities over 24 years. No relationship was found between increased beer taxes and reduced intimate partner homicide. While the study did not include alcohol consumption measures, the authors suggest that tax increases may have been too small to affect drinking behaviour to the extent needed to influence IPH. Further, their outcome measure included all victims of intimate partner homicide regardless of gender, though evidence shows that women are the overwhelming majority of victims of homicide by an intimate partnerand alcohol is more likely to be involved in male-to-female IPV.
In summary, only weak or indirect evidence was found that increasing the price of alcohol reduces IPV.
## Community-level interventions
Alcohol consumption is affected by the physical availability of alcohol as well as other local interventions such as policing and enforcement policies relating to alcohol sales and service. These interventions, such as restricting retail hours or the numbers and density of alcohol outlets within a geographical area, decrease consumption and related harms by increasing the effort to obtain alcohol [bib_ref] The effectiveness of limiting alcohol outlet density as a means of reducing..., Campbell [/bib_ref]. Such community-level interventions would be expected to reduce IPV by decreasing drinking opportunities and overall consumption among those who perpetrate alcohol-related IPV.
## Alcohol sales restrictions and ipv
Only one [bib_ref] The effect of restricting opening hours on alcohol-related violence, Duailibi [/bib_ref] of eight studies (10 papers) [bib_ref] The effect of restricting opening hours on alcohol-related violence, Duailibi [/bib_ref] [bib_ref] Restriction of the hours of sale of alcohol in a small community:..., Douglas [/bib_ref] [bib_ref] Beating the grog: an evaluation of the tennant creek liquor licensing restrictions, Gray [/bib_ref] that evaluated the impact of community-level restrictions on the hours and days of sale of alcohol on IPV met design criteria for inclusion. The remaining seven studies (9 papers) [bib_ref] The price of alcohol, wife abuse, and husband abuse, Markowitz [/bib_ref] [bib_ref] Taxing sin and saving lives: Can alcohol taxation reduce female homicides?, Durrance [/bib_ref] [bib_ref] Effects of domestic violence policies, alcohol taxes and police staffing levels on..., Zeoli [/bib_ref] [bib_ref] The impact of a large reduction in the price of alcohol on..., Herttua [/bib_ref] [bib_ref] The effectiveness of limiting alcohol outlet density as a means of reducing..., Campbell [/bib_ref] [bib_ref] The effect of restricting opening hours on alcohol-related violence, Duailibi [/bib_ref] evaluated alcohol restrictions in remote Australian Indigenous communities, with IPV as one of several outcome measures. All were pre-post designs with no comparison group for IPV outcomes. Although some of these studies found decreases in alcohol consumption following the intervention, overall there was no clear pattern of effects on IPV.
The one study with multiple time points [bib_ref] The effect of restricting opening hours on alcohol-related violence, Duailibi [/bib_ref] examined the effect of a city-wide bar closing time of 11 pm in a mid-sized Brazilian city with high rates of alcohol and violence (Additional file 2: . Analysing homicide rates over a 10-year period and assaults against women over a 5-year period, this study found that earlier bar closing was associated with a significant reduction in homicides in the first three years post-restriction, and a non-significant reduction in assaults against women. Interpretation is limited by the lesser time period for assaults and the inclusion of all assaults against women, not just IPV. The impact of the intervention on alcohol consumption was not assessed.
## Alcohol outlet density and ipv
Eleven studies [bib_ref] A longitudinal analysis of alcohol outlet density and domestic violence, Livingston [/bib_ref] [bib_ref] Alcohol outlets, neighborhood characteristics, and intimate partner violence: ecological analysis of a..., Cunradi [/bib_ref] [bib_ref] Alcohol outlet density and intimate partner violence-related emergency department visits, Cunradi [/bib_ref] [bib_ref] Revealing the link between licensed outlets and violence: counting venues versus measuring..., Liang [/bib_ref] [bib_ref] Alcohol availability and intimate partner violence among US couples, Mckinney [/bib_ref] [bib_ref] Assessing the relationship between alcohol outlets and domestic violence: Routine activities and..., Roman [/bib_ref] [bib_ref] Alcohol outlet density is related to police events and motor vehicle accidents..., Cameron [/bib_ref] [bib_ref] Alcohol availability and domestic violence, Gorman [/bib_ref] [bib_ref] The ecology of domestic violence: the role of alcohol outlet density, Livingston [/bib_ref] [bib_ref] Relationships among alcohol outlet density, alcohol use, and intimate partner violence victimization..., Waller [/bib_ref] conducted in the USA, New Zealand and Australia examined the relationship between alcohol outlet density and IPV. Three studies [bib_ref] A longitudinal analysis of alcohol outlet density and domestic violence, Livingston [/bib_ref] [bib_ref] Alcohol outlets, neighborhood characteristics, and intimate partner violence: ecological analysis of a..., Cunradi [/bib_ref] [bib_ref] Alcohol outlet density and intimate partner violence-related emergency department visits, Cunradi [/bib_ref] used longitudinal designs (Additional file 2: . Three cross-sectional studies [bib_ref] Revealing the link between licensed outlets and violence: counting venues versus measuring..., Liang [/bib_ref] [bib_ref] Alcohol availability and intimate partner violence among US couples, Mckinney [/bib_ref] [bib_ref] Assessing the relationship between alcohol outlets and domestic violence: Routine activities and..., Roman [/bib_ref] provided additional insight into the possible mediating role of alcohol consumption in the relationship between outlet density and IPV (Additional file 3: . The remaining five studies [bib_ref] Alcohol outlet density is related to police events and motor vehicle accidents..., Cameron [/bib_ref] [bib_ref] Alcohol availability and domestic violence, Gorman [/bib_ref] [bib_ref] The ecology of domestic violence: the role of alcohol outlet density, Livingston [/bib_ref] [bib_ref] Relationships among alcohol outlet density, alcohol use, and intimate partner violence victimization..., Waller [/bib_ref] were cross-sectional designs which revealed inconsistent findings regarding the association between outlet density, type of outlet and IPV.
Among the longitudinal studies, Livingston [bib_ref] A longitudinal analysis of alcohol outlet density and domestic violence, Livingston [/bib_ref] examined licensing data and police-recorded IPV incidents in Melbourne, Australia, over ten years and found a positive association between IPV and outlet density, with a large and significant effect found for packaged liquor ("off-premises") outlets. An increase in one packaged liquor outlet per 1,000 residents was associated with a 28.6% increase in the mean domestic violence rate.
A longitudinal study [bib_ref] Alcohol outlets, neighborhood characteristics, and intimate partner violence: ecological analysis of a..., Cunradi [/bib_ref] from California using two police-recorded measures of IPV (IPV-related calls to police and crime reports) also found associations with off-premises outlets but no clear association with onpremises outlet density. However, a second study by the same authors [bib_ref] Alcohol outlet density and intimate partner violence-related emergency department visits, Cunradi [/bib_ref] using data over a shorter time period found an increased risk of an IPV-related emergency department visit associated with higher on-premises outlet density, while off-premises outlet density was associated with a weaker reduced risk.
In terms of support for the mediating role of alcohol consumption in the relationship between outlet density and IPV, a Western Australian study [bib_ref] Revealing the link between licensed outlets and violence: counting venues versus measuring..., Liang [/bib_ref] found a significant association between off-premises sales volume and assaults in private residences, suggesting a potential mediating link between the amount of alcohol sold/consumed (not just number of outlets) and IPV. Similarly, another study using self-reported IPV from a national U.S. survey [bib_ref] Alcohol availability and intimate partner violence among US couples, Mckinney [/bib_ref] found a stronger relationship between outlet density and male-to-female physical IPV for couples who had alcohol problems than for couples without. A further study in the U.S. District of Columbia [bib_ref] Assessing the relationship between alcohol outlets and domestic violence: Routine activities and..., Roman [/bib_ref] found the association between domestic violence police call-outs and off-premises outlet density was greater for calls on weekends, suggesting links between outlet density and IPV during times when heavier drinking was more likely to occur (i.e., weekends).
Overall, evidence from community studies provides weak support for the association between alcohol availability restrictions and IPV.
## Relationship-level interventions couples-based treatment
Couple-based alcohol treatment interventions have been shown to be effective for reducing alcohol consumption and improving relationships among treatment-seeking individuals with alcohol and drug problems who are in a married or cohabiting relationship [bib_ref] Behavioral couples therapy (BCT) for alcohol and drug use disorders: a meta-analysis, Powers [/bib_ref]. To the extent that couple-level interventions reduce problem drinking and improve relationship functioning in relationships where there is violence, they may also reduce IPV.
Five studies (seven papers) [bib_ref] Marital violence before and after alcoholism treatment, O'farrell [/bib_ref] [bib_ref] Domestic violence before and after alcoholism treatment: a two-year longitudinal study, O'farrell [/bib_ref] [bib_ref] Van Hutton V: Verbal aggression among male alcoholic patients and their wives..., O'farrell [/bib_ref] [bib_ref] Partner violence before and after couples-based alcoholism treatment for male alcoholic patients:..., O'farrell [/bib_ref] [bib_ref] Behavioral couples therapy for comorbid substance use disorders and combat-related posttraumatic stress..., Rotunda [/bib_ref] [bib_ref] Fals-Stewart W: Partner violence before and after couples-based alcoholism treatment for female..., Schumm [/bib_ref] [bib_ref] A brief motivational intervention for physically aggressive dating couples, Woodin [/bib_ref] evaluated alcohol interventions involving couples. Of these, only one met design criteria, a trial of a brief intervention [bib_ref] A brief motivational intervention for physically aggressive dating couples, Woodin [/bib_ref] (Additional file 2: that addressed both IPV and alcohol use. This study assessed an individual motivational feedback session on aggression and IPV risk factors (including alcohol use) among a sample of 49 dating university couples. The study found a greater decrease in harmful alcohol consumption and physical aggression in the intervention group compared with those in the control condition who received minimal non-motivational feedback; however, the reductions in alcohol use and physical aggression were not related.
The remaining four studies did not meet design criteria but did provide some evidence of mediation. These involved pre-post evaluations of behavioral couples-based treatment to address alcohol problems in one partner. These clinical samples were predominantly white, middleaged and in long term relationships. Three studies reported significant reductions in male-perpetrated violence and verbal aggression between male alcoholics and their female partners, [bib_ref] Marital violence before and after alcoholism treatment, O'farrell [/bib_ref] [bib_ref] Domestic violence before and after alcoholism treatment: a two-year longitudinal study, O'farrell [/bib_ref] [bib_ref] Van Hutton V: Verbal aggression among male alcoholic patients and their wives..., O'farrell [/bib_ref] [bib_ref] Partner violence before and after couples-based alcoholism treatment for male alcoholic patients:..., O'farrell [/bib_ref] [bib_ref] Behavioral couples therapy for comorbid substance use disorders and combat-related posttraumatic stress..., Rotunda [/bib_ref] and the fourth found decreases in male and female-perpetrated violence in a female alcoholic sample [bib_ref] Fals-Stewart W: Partner violence before and after couples-based alcoholism treatment for female..., Schumm [/bib_ref]. Although conclusions regarding the effectiveness of the treatment are limited by the single group pre-post design, they did find evidence of a possible mediating role of alcohol consumption. Specifically, increases in violence were found more frequently for relapsed compared with remitted patients, though other explanations for the relationship cannot be ruled out. These studies are shown in (Additional file 3: .
Overall, other than weak evidence from uncontrolled studies, the only support for the effectiveness of couple interventions focused on alcohol comes from a single well-designed study of a couples-based brief intervention focused on relationship and lifestyle factors, including alcohol use; however, post intervention reductions in alcohol consumption and IPV were not linked. There was evidence of mediation, however, from the uncontrolled studies of persons in treatment for alcohol problems.
## Individual-level interventions treatment
Individual treatment interventions aim to reduce or eliminate problem drinking in individuals with an alcohol disorder diagnosis or who drink in hazardous or harmful ways. To the extent that their drinking is linked to IPV perpetration, reducing or eliminating alcohol use would be expected to also reduce or eliminate IPV.
Twelve studies [bib_ref] Fluoxetine treatment of alcoholic perpetrators of domestic violence: A 12-week, double-blind, randomized,..., George [/bib_ref] [bib_ref] Development of a brief motivational enhancement intervention for intimate partner violence in..., Schumacher [/bib_ref] [bib_ref] Randomized clinical trial examining the incremental efficacy of a 90-minute motivational alcohol..., Stuart [/bib_ref] [bib_ref] A: A telephone intervention for substance-using adult male perpetrators of intimate partner..., Mbilinyi [/bib_ref] [bib_ref] A cognitive behavioral therapy for alcohol-dependent domestic violence offenders: An integrated substance..., Easton [/bib_ref] [bib_ref] Reduction of intimate partner violence in a gay couple following alcohol treatment, Hellmuth [/bib_ref] [bib_ref] The relationship between relapse to alcohol and relapse to violence, Mignone [/bib_ref] [bib_ref] Partner violence before and after individually based alcoholism treatment for male alcoholic..., O'farrell [/bib_ref] [bib_ref] Longitudinal risk factors for intimate partner violence among men in treatment for..., Taft [/bib_ref] [bib_ref] Marital violence victimization and perpetration among women substance abusers: a descriptive study, Stuart [/bib_ref] [bib_ref] Reductions in marital violence following treatment for alcohol dependence, Stuart [/bib_ref] involved alcohol treatment interventions delivered to individuals. Seven studies of individual alcohol treatment alone on IPV [bib_ref] Reduction of intimate partner violence in a gay couple following alcohol treatment, Hellmuth [/bib_ref] [bib_ref] The relationship between relapse to alcohol and relapse to violence, Mignone [/bib_ref] [bib_ref] Partner violence before and after individually based alcoholism treatment for male alcoholic..., O'farrell [/bib_ref] [bib_ref] Longitudinal risk factors for intimate partner violence among men in treatment for..., Taft [/bib_ref] [bib_ref] Marital violence victimization and perpetration among women substance abusers: a descriptive study, Stuart [/bib_ref] [bib_ref] Reductions in marital violence following treatment for alcohol dependence, Stuart [/bib_ref] did not meet the design criteria. The remaining five studies combined alcohol and batterer treatment using a randomized controlled design [bib_ref] Fluoxetine treatment of alcoholic perpetrators of domestic violence: A 12-week, double-blind, randomized,..., George [/bib_ref] [bib_ref] Development of a brief motivational enhancement intervention for intimate partner violence in..., Schumacher [/bib_ref] [bib_ref] Randomized clinical trial examining the incremental efficacy of a 90-minute motivational alcohol..., Stuart [/bib_ref] [bib_ref] A: A telephone intervention for substance-using adult male perpetrators of intimate partner..., Mbilinyi [/bib_ref] [bib_ref] A cognitive behavioral therapy for alcohol-dependent domestic violence offenders: An integrated substance..., Easton [/bib_ref] ; however, we excluded two [bib_ref] Fluoxetine treatment of alcoholic perpetrators of domestic violence: A 12-week, double-blind, randomized,..., George [/bib_ref] [bib_ref] Development of a brief motivational enhancement intervention for intimate partner violence in..., Schumacher [/bib_ref] because of small sample size, high attrition rates and lack of power. The three included studies are discussed in more detail below and described in (Additional file 2: .
Two studies trialled brief interventions. The first, a well-designed randomized controlled trial of a batterer program with a personalised alcohol component, [bib_ref] Randomized clinical trial examining the incremental efficacy of a 90-minute motivational alcohol..., Stuart [/bib_ref] recruited 252 hazardous drinking males enrolled in batterer programs (98% court mandated). The study compared a standard batterer program (SBP) combined with a personalised brief alcohol intervention to an SBP that included one group substance abuse session. The alcohol intervention was a 90 minute therapist-led motivational interview with personalised feedback provided on the participant's current drinking. The study found significant reductions in the experimental group on drinking outcomes, though these were not sustained. No significant difference was found in the frequency of physical IPV however, the experimental group showed reductions in severe psychological aggression and injuries to partners in secondary analyses. However, improvements dissipated over time.
The second trial [bib_ref] A: A telephone intervention for substance-using adult male perpetrators of intimate partner..., Mbilinyi [/bib_ref] assessed a motivational intervention delivered by telephone with substance using IPV perpetrators recruited from the community (i.e., not receiving counselling or legal sanction). The intervention was based on a personalised assessment of IPV and substance use behaviours compared to the control group who received generalised education materials by mail. Less than half the sample (43%) had a diagnosed substance use disorder. At the 30 day follow-up, men in the treatment condition reported engaging in less violence and consumed fewer drinks per week. The authors did not report whether reductions in alcohol consumption were associated with reductions in IPV.
The third study, [bib_ref] A cognitive behavioral therapy for alcohol-dependent domestic violence offenders: An integrated substance..., Easton [/bib_ref] an integrated substance abusedomestic violence treatment approach using cognitive behavioral treatment with alcohol dependent men, showed a trend towards greater reduction in IPV and significantly more days abstinent among those in the experimental group compared to a comparison group who received substanceonly therapy. However, there were no significant differences at 6 months for either alcohol use or physical IPV.
Of the seven studies of alcohol treatment that did not meet design criteria, three pre-post studies with samples of treatment-seeking male alcoholics [bib_ref] The relationship between relapse to alcohol and relapse to violence, Mignone [/bib_ref] [bib_ref] Partner violence before and after individually based alcoholism treatment for male alcoholic..., O'farrell [/bib_ref] [bib_ref] Longitudinal risk factors for intimate partner violence among men in treatment for..., Taft [/bib_ref] (Additional file 3: found evidence linking alcohol and IPV outcomes, suggesting possible mediation of alcohol consumption in treatment effects on IPV, though design limitations preclude conclusively attributing either outcomes or their relationship to the treatment.
Overall, the evidence for individual-based treatment interventions reducing IPV is limited. Controlled studies of combined alcohol and IPV interventions found significant effects on both behaviors but these effects were not sustained over time and no evidence was provided of mediation. A possible mediating role of alcohol consumption in reductions in IPV was found in three pre-post studies of alcohol treatment alone but the design of these studies precluded the conclusion that these effects were due to the intervention.
# Discussion
For several decades there has been clear and consistent evidence of an association between alcohol consumption and IPV. There is also evidence that alcohol consumption by one or both partners is associated with increased severity of IPV. Thus, interventions that reduce alcohol consumption may also reduce IPV. These interventions can occur at different levels, from individual clinical treatment to state and federal level taxation. An important contribution of the present review is to bring together the disparate literatures relating to alcohol and IPV to examine the effects of alcohol interventions on IPV from the perspective of all levels of the WHO ecological framework -population, community, relationship and individual levels.
Despite the significance of both alcohol misuse and IPV as public health issues, we found the evidence base for assessing the effectiveness of alcohol interventions on IPV from the last 20 years to be disappointingly small. Our review found few studies that had examined the effect of population-level alcohol measures on IPV despite consistent evidence within the alcohol policy science literature that interventions such as alcohol taxation that reduce demand by increasing the cost of alcohol are effective strategies for reducing alcohol consumption and related harms generally [bib_ref] Effects of beverage alcohol price and tax levels on drinking: A meta-analysis..., Wagenaar [/bib_ref]. The small literature available suggested little or weak evidence of an effect of alcohol pricing on IPV, possibly hampered by most studies evaluating very small changes in taxation over time and using a measure of IPV that included both alcohol-related IPV and IPV that was not related to alcohol. While all the reviewed studies were based on the theoretical assumption that changes in price influence consumption, only one study [bib_ref] Taxing sin and saving lives: Can alcohol taxation reduce female homicides?, Durrance [/bib_ref] included alcohol consumption measures in their design, and it failed to find a strong enough link to demonstrate that alcohol tax changes can reduce violence against women, with this effect mediated through a reduction in alcohol consumption.
To address the effectiveness of alcohol policy more conclusively, stronger designs are needed that evaluate meaningful pricing changes using appropriate comparison conditions, and that include measures that can distinguish effects on alcohol-related IPV and measures of alcohol consumption for testing mediation. In addition, given that meta-analyses have shown stronger associations of IPV with heavy and binge-drinking patterns of consumption than with other patterns of drinking [bib_ref] Alcohol and intimate partner violence: a meta-analytic review, Foran [/bib_ref] , it is important to assess the extent that those with heavy or binge drinking patterns are sensitive to changes in alcohol price. To enhance the effectiveness of pricing strategies to reduce alcohol-related harm, such policy approaches should be tailored [bib_ref] Policy options for alcohol price regulation: the importance of modelling population heterogeneity, Meier [/bib_ref] to suit the patterns of consumption of populations highly likely to engage in IPV, for example, younger couples who are most at risk of IPV, and binge drinkers who are most prevalent among adolescents and young adults in many countries. From a public health perspective, population approaches have the opportunity to have the greatest impact; thus, further investigation is warranted regarding how such pricing mechanisms can influence IPV where alcohol use is implicated.
At the community-level, we found policy interventions that restricted the availability of alcohol through reduced trading hours were introduced into communities or areas as a response to significant problems with alcohol and violence. The evidence of an impact on IPV was inconclusive based on the one study in Brazil [bib_ref] The effect of restricting opening hours on alcohol-related violence, Duailibi [/bib_ref] that met design criteria but which measured violence against women generally, not alcohol-related IPV. The remaining studies of alcohol restrictions were implemented in remote Indigenous Australian communities. Although these studies did not meet design criteria, the comprehensive community approaches used in these studies, with multiple interventions directed toward restricting access to alcohol, provide a model for undertaking better controlled evaluation studies in the future to address alcohol-related IPV, which remains a significant problem in many Indigenous communities worldwide [bib_ref] Alcohol policy issues for indigenous people in the United States, Canada, Australia..., Brady [/bib_ref].
Although a relatively strong body of research has linked alcohol outlet density to violence,research relating specifically to IPV is inconsistent with regard to outlet type. This finding may reflect a complex and variable relationship between outlet density and IPV. In particular, given that IPV is much more likely to occur in the home than in other locations [bib_ref] Situational determinants in intimate partner violence, Wilkinson [/bib_ref] , one might expect a stronger link with drinking in the home and therefore IPV would be more strongly associated with off-premises sales. On the other hand, to the extent that IPV is associated with heavier consumption [bib_ref] What factors are associated with recent intimate partner violence? Findings from the..., Abramsky [/bib_ref] and heavier consumption is more likely to occur in licensed premises, [bib_ref] A comparative multi-level analysis of contextual drinking in American and Canadian adults, Kairouz [/bib_ref] [bib_ref] Drinking context and intimate partner violence: evidence from the California community health..., Cunradi [/bib_ref] a stronger association might be expected for on-premise drinking. Further, whether the link is with on or offpremise drinking may vary by culture. For example, in some cultures, it may be common for couples to drink together at home with the associated increased risk of aggression, while in other cultures it may be common for the male partner to drink large amounts in licensed premises and become violent after coming home or for conflict to arise over his drinking on his return. Thus, despite this inconsistency in findings related to type of outlet, there is sufficient evidence to suggest that the association between alcohol outlet density and IPV is worth further investigation. Mixed methodology research is needed to better understand the relationship between alcohol-related IPV and drinking location and how this might be linked to outlet density, including the extent to which this association is affected by cultural, social and individual factors not just availability [bib_ref] Alcohol outlets and community levels of interpersonal violence spatial density, outlet type,..., Pridemore [/bib_ref].
Over half of the studies we reviewed were treatment studies at the relationship and individual levels of the WHO ecological framework. These studies were all conducted in the USA, the majority of these in clinical settings with older, treatment-seeking alcohol dependent men amongst whom IPV is significantly more prevalent than in the general population [bib_ref] Marital violence before and after alcoholism treatment, O'farrell [/bib_ref]. None of the studies of alcohol treatment only -using couple or individual approaches -met design criteria. Thus, although these pre-post studies reported some evidence of reduced IPV after individual or couples-based treatment associated with reduced drinking, excessive drinking and related behaviours such as alcohol-related violence can decrease naturally over time (e.g., natural recovery, regression to the mean). Well-controlled trials with men in various age groups are needed to confirm that alcohol treatment alone can have an impact on IPV. In addition, while the majority of couples-based alcohol treatment studies measured bi-directional violence, partner substance use was a common exclusion criteria thus the extent to which problematic alcohol use by both partner contributes to IPV, remains untested from the current evidence base.
The more recent treatment studies that combined IPV and alcohol interventions used stronger designs though several had methodological limitations. These studies focused on the effects of the addition of an IPV component to addictions treatment and/or addictions component to IPV treatment to examine the combined effect of addressing both alcohol and IPV. These studies were able to demonstrate significant reductions in alcohol consumption and IPV (compared to the control condition) but these effects were not sustained and none of the studies tested the relationships between reductions in drinking and reductions in IPV. The study of dating couples, [bib_ref] A brief motivational intervention for physically aggressive dating couples, Woodin [/bib_ref] though unable to show a link between reductions in alcohol use and physical aggression, suggests that brief interventions with younger, non-dependent adult populations are worthy of further study using larger samples because these are low cost interventions and address the population most at risk in many countries. The randomized controlled trial in which a brief alcohol intervention was added to a batterer program [bib_ref] Randomized clinical trial examining the incremental efficacy of a 90-minute motivational alcohol..., Stuart [/bib_ref] is also important despite changes not being sustained because it is the first of its kind illustrating the potential impact of addressing alcohol within the context of addressing IPV perpetration, an area that has received little attention from the IPV prevention field. This is the first systematic review to examine studies that have addressed the effects of alcohol interventions on IPV at the population, community, relationship and individual levels. These studies included a variety of research methods from across different disciplines. Despite the importance of this comprehensive approach to examining effects of alcohol interventions on IPV, the existing research on which the review is based has some significant limitations. First, the relatively small literature and the heterogeneity of the study designs precluded meta-analysis or even comparison of effects by characteristics of participants or type of intervention. Second, not all studies included alcohol consumption measures that could be used to test the central assumption that alcohol interventions affect IPV by changing alcohol consumption. Third, most population and community studies used police or hospital statistics to measure IPV which represent the most severe cases of IPV, and none of these studies was able to separate estimates of alcoholrelated and non alcohol-related IPV. Thus, one reason for the general null findings of the population and community studies we reviewed could be that a substantial proportion of incidents of IPV do not involve alcohol, with this proportion varying considerably for different countries [bib_ref] Alcohol may not cause partner violence but it seems to make it..., Graham [/bib_ref]. Finally, the search strategy focussed on English language studies and interventions found were predominately from middle/high income countries and all studies of couple and individual interventions were from the USA.
# Conclusions
Alcohol-related IPV is a complex, multi-dimensional problem much neglected in intervention and prevention research. Despite the consistent link between alcohol consumption and IPV and evidence that alcohol use contributes to increased risk and severity of IPV, our review found few studies of the effects of alcohol interventions and alcohol policy interventions on IPV where the design allowed changes in IPV to be clearly attributed to the intervention. An appropriately funded research agenda is urgently needed to investigate the potential impact of alcohol/policy interventions on IPV at the population, community, relationship and individual-level, and provide answers to the gaps in the evidence base. This includes:
(a) better theoretical models of the links between IPV and alcohol consumption, pricing and availability; (b) greater focus on those at risk in many countries, such as heavy episodic drinkers and young adults; (c) stronger designs, specifically -(i) randomized controlled trials, where possible, or studies with an appropriate comparison group/community, (ii) prospective and longitudinal designs with sufficient statistical power and (iii) designs able to test the mediating role of alcohol consumption; (d) more reliable measures distinguishing alcohol-related IPV from IPV not involving alcohol; (e) greater consistency of measurement across studies; and (f ) evaluation of interventions in low and middle income countries where the incidence of IPV is often higher and the link with alcohol stronger [bib_ref] What factors are associated with recent intimate partner violence? Findings from the..., Abramsky [/bib_ref].
[fig] Figure 1: Ecological model for understanding violence. Reproduced with permission from the World Health Organization. [/fig]
[fig] Figure 2: Selection of articles for review of alcohol and policy interventions to reduce intimate partner violence. [/fig]
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Influence of additional weight on the frequency of kicks in infants with Down syndrome and infants with typical development
| Background: Infants with Down syndrome present with organic and neurological changes that may lead to a delay in the acquisition of motor skills such as kicking, a fundamental skill that is a precursor of gait and is influenced by intrinsic and extrinsic factors. Therefore, this movement should be taken into account in early physical therapy interventions in infants. Objective: To analyze and to compare the effect of additional weight on the frequency of kicks in infants with Down syndrome and infants with typical development at 3 and 4 months of age. Method: Five infants with Down syndrome and five with typical development at 3 and 4 months of age were filmed. The experiment was divided into four experimental conditions lasting 1 minute each: training, baseline, weight (addition of ankle weight with 1/3 the weight of the lower limb), and post-weight. Results: There were significant differences between groups for all variables (p<0.05), with lower frequencies observed for infants with Down syndrome in all variables. There were significant differences between the experimental conditions baseline and post-weight (p<0.001) for both groups in the frequency of contact and success, with a higher frequency in the post-weight condition. Conclusions: The weight acted as an important stimulus for both groups, directing the kicks toward the target and improving the infants' performance in the task through repetition, however, the infants with Down syndrome had lower frequencies of kicks.HOW TO CITE THIS ARTICLESantos GL, Bueno TB, Tudella E, Dionisio J. Influence of additional weight on the frequency of kicks in infants with Down syndrome and infants with typical development. Braz J Phys Ther. 2014 May-June; 18(3):237-244. http://dx.
# Introduction
Down syndrome is caused by a trisomy of chromosome 21, with an incidence of approximately 1/700 live births 1 , and it is associated with various complex clinical phenotypes, such as a smaller size of the cerebellum and the temporal and frontal lobes [bib_ref] Neuroanatomy of Down Syndrome in vivo: a model of preclinical Alzheimer's disease, Teipel [/bib_ref] [bib_ref] Motor Development and neuropsychological patterns in persons with Down syndrome, Vicari [/bib_ref]. Some neuromotor changes are also observed, such as muscular hypotonia, joint hyperextensibility, and disturbance in the postural control and balance mechanism, which restrict the proper execution of voluntary movements [bib_ref] Síndrome de Down: etiologia, caracterização e impacto na família, Silva [/bib_ref] [bib_ref] The four ages of Down syndrome, Bittles [/bib_ref]. Moreover, infants with Down syndrome may have cognitive deficits which can influence the performance of motor tasks [bib_ref] Motor and perceptual-motor competence in children with Down syndrome: variation in performance..., Spanò [/bib_ref]. These characteristics or organic changes can lead to a delay in the acquisition of motor skills such as reaching, sitting, and kicking, which are precursors to the development of more complex skills such as crawling and walking [bib_ref] Longitudinal assessment of leg motor activity and sleep patterns in infants with..., Mackay [/bib_ref] [bib_ref] Gross motor function in children with Down syndrome: creation of motor growth..., Palisano [/bib_ref] [bib_ref] Description of the motor development of 3-12 month old infants with Down..., Tudella [/bib_ref].
The kick is one of the earliest motor behaviors, being observed from intrauterine life [bib_ref] Spontaneous kicking in fullterm and preterm infants: Are there leg asymmetries?, Piek [/bib_ref]. It is characterized by the cycle of flexion movement of the joints of one or both lower limbs, followed by extension and flexion again [bib_ref] Efeito do peso externo nos chutes espontâneos de lactentes nos primeiros dois..., Landgraf [/bib_ref]. Between the age of 1 and 4 months, there is a marked decline in the number of alternating leg movements followed by a period in which there is the emergence of unilateral kicking and, at about 4-5 months, the new patterns of bilateral coordination become more prominent [bib_ref] Spontaneous leg movements in infants with and without periventricular leukomalacia: effects of..., Vaal [/bib_ref]. Moreover, around 5 months, the infants prefer to reach to explore the environment, reducing the frequency of kicks [bib_ref] Kinematic quality of reaching movements in preterm infants, Fallang [/bib_ref]. Thus, throughout the development of this ability, infants present in-phase and out-of-phase movements and intra-and interlimb coordination, which favors increased strength and limb coordination [bib_ref] Spontaneous leg movements in infants with and without periventricular leukomalacia: effects of..., Vaal [/bib_ref]. Thus, with a more developed movement pattern, the infant is able to perform complex tasks such as crawling and walking [bib_ref] Spontaneous kicking in fullterm and preterm infants: Are there leg asymmetries?, Piek [/bib_ref] [bib_ref] Making the mobile move: constraining task and environment, Chen [/bib_ref] [bib_ref] Developmental origins of motor coordination leg movements in human infants, Thelen [/bib_ref] [bib_ref] The organization of spontaneous leg movements in newborn infants, Thelen [/bib_ref] [bib_ref] The dynamic nature of early coordination: evidence from bilateral leg movements in..., Thelen [/bib_ref].
The movement of kicking as a motor ability is influenced by the interaction between elements of the organism, environment, and task specificity [bib_ref] The dynamic nature of early coordination: evidence from bilateral leg movements in..., Thelen [/bib_ref] [bib_ref] Implications of a dynamical systems approach to understanding infant kicking behavior, Heriza [/bib_ref] [bib_ref] A quantitative analysis of spontaneous kicking in two-month-old infants, Piek [/bib_ref]. In order to verify the influence of factors extrinsic to the organism (mobile reinforcement and additional weight), some authors 15 examined the movement of kicking in healthy 4-month-olds by using a reinforcement task of kicking a touch pad which activated a mobile and adding an ankle weight of 5% the total mass of the limb to the lower limbs. For this, the authors designed the following experimental protocol: 2 minutes of baseline (spontaneous kicking), 8 minutes of acquisition (mobile reinforcement with weight), and 4 minutes of extinction (spontaneous kicking). The authors observed that the variable frequency of contact, which indicates if the infant is able to learn the task, increased during acquisition. This finding suggested that 4-month-olds could effectively accomplish the 'mobile with weight' task and these infants displayed a significant learning effect in the acquisition condition.
Another study 21 examined the influence of factors extrinsic to the organism, but in children with Down syndrome with corrected chronological age ranging from 4 to 6 months compared with typically developing children matched for chronological age and motor age, defined by the psychomotor items of the Bayley Scales of Infant Development. For this, the infants were positioned in supine and their lower limb movements were filmed for 8 minutes in four experimental conditions: control (no stimulus), verbal (verbal stimulus with caregiver), mobile (with visual stimulus of the mobile), and enriched (with verbal stimuli and touch from the caregiver). The authors found no difference between groups in the frequency of lower limb movements in any of the experimental conditions, however the infants demonstrated fewer of the more complex movements. Moreover, the authors found differences in frequency between the conditions of movement, with an increase of frequency of limb movements in the verbal condition when compared with control condition, followed by a decrease in the mobile condition, and subsequently an increase in the enriched condition. Thus, the authors concluded that the context influences the frequency of lower limb movements.
Given the above, it appears that the gradual increase in weight acts as an important stimulus for muscle strengthening and a more coordinated movement of the lower limbs, in addition to being highly correlated with the time of onset of gait, as observed in healthy infants [bib_ref] The dynamic nature of early coordination: evidence from bilateral leg movements in..., Thelen [/bib_ref] [bib_ref] A quantitative analysis of spontaneous kicking in two-month-old infants, Piek [/bib_ref]. Furthermore, these authors verify the additional influence of weight on the frequency of kicks because the additional weight alters both the environmental (gravitational torque) and organismic (moment of inertia) context of constraints [bib_ref] Making the mobile move: constraining task and environment, Chen [/bib_ref] [bib_ref] The dynamic nature of early coordination: evidence from bilateral leg movements in..., Thelen [/bib_ref].
It has already been demonstrated that factors extrinsic to the organism (mobile and verbal stimulus) influence the behavior of kick in infants with Down syndrome, however, there are no studies that have investigated the effect of additional weight in these infants. This gap in the literature justifies this work, in which the results will provide a better understanding of the motor development of infants with Down syndrome and support for physical therapy treatment in this population, favoring a more complex pattern of movement and influencing the time of onset of gait. Therefore, this study aims to analyze and compare the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the pad with and without additional weight in 3 and 4-month-old infants with Down syndrome and typical infants.
To this end, the following hypotheses were tested: 1) the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the touch pad in infants with Down syndrome will be significantly lower when compared to typical infants due to organic changes such as hypotonia and muscle weakness; 2) the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the pad will not be significantly different between 3 and 4 months of age for the group with Down syndrome due to delayed motor development; 3) these variables will be significantly lower at four months of age for the group of typical infants because it is the acquisition phase of reaching and grasping movements when infants prefer to explore the environment with their hands and the frequency of kicks decreases; 4) the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the touch pad will be significantly lower in the weight experimental condition compared to the baseline and post-weight experimental conditions due to the greater difficulty of the task; and 5) these variables will be higher in the post-weight experimental condition when compared to the baseline experimental condition because it favors learning and a more complex kick pattern.
# Method
This experimental and longitudinal study was approved by the Human Research Ethics Committee of Universidade Federal de São Carlos (UFSCar), São Carlos, SP, Brazil (Process No. 081/2006). The subjects were selected from two Basic Health Units of São Carlos, and the parents signed a consent form agreeing to the participation of the infants in the study, which included a convenience sample.
## Participants
The study included 5 infants with Down syndrome and 5 infants with typical development [fig_ref] Table 1: Demographic data of patients [/fig_ref]. The infants did not play with mobiles at home, but only with plush toys held by their caregivers. The infants with Down syndrome were not participating in any therapy programs. The typically developing group was matched for gender and chronological age with the Down syndrome (DS) group.
Infants with orthopedic or sensory changes, hearing loss or cardiac complications were excluded. The diagnosis of DS was confirmed by the medical report of cytogenetic analysis. Subject participation was interrupted if they were absent from any of the two assessments, if they showed events that could influence the results or when their parents did not want to continue.
## Procedures
The children were assessed on their 3 and 4 month birthday, considering a range of approximately ±5 days. At the Laboratory, the infants were undressed by their mother and the examiner collected the anthropometric measurements (weight and total body length, length, and circumference of thighs and legs, length and width of feet) to estimate the mass of each lower limb [bib_ref] Mass, centre of mass, and moment of inertia estimates for infant limb..., Schneider [/bib_ref].
After these procedures, infants were positioned in supine on a table developed for the study and stabilized by their shoulders by a research assistant. At the lower end of the table, there was a touch pad that when lifted by the infant's feet, activated a mobile set at a height of approximately 30 cm above the infant's face, acting as visual and auditory stimulus for the performance of kicks 12 . The touch pad position was defined by of multiplication the length of the infant's lower limb by sine 30°1 [bib_ref] The four ages of Down syndrome, Bittles [/bib_ref].
The experiment was divided into four conditions: 1) training (T) -the infant's feet were placed by the examiner on the touch pad to activate the mobile, being performed three times with each limb separately and three times with both limbs together; 2) baseline (BL) -infants were free to perform kicks and could raise the touch pad with their feet and activate the mobile; 3) weight (W) -identical to the previous condition, however the infant had an ankle weight corresponding to 1/3 the weight of the lower limb; 4) post-weight (PW) -same as BL. In each condition, 1 minute was allowed for the performance of kicks and a 30-second interval was given between conditions 2, 3, and 4 for the placement or removal of the ankle weight. The entire test lasted about 5 minutes [fig_ref] Figure 1: Temporal representation of experimental conditions [/fig_ref]. Throughout the procedure, the infants remained in an active, alert state [bib_ref] Influência do peso adicional nos chutes de lactentes pré-termo e de termo, Dionisio [/bib_ref].
To record the assessments, two JVC digital cameras were used (Model GY DV-300) mounted on tripods positioned anterolaterally to the chair, one on each side, with two light sources placed next to the cameras and directed towards the wall. After the recordings, an examiner placed a black bar over the infants' faces to conceal their identity. After viewing the infants in the video, another examiner counted the frequencies of motions according to the variables defined below. This count was performed by a single examiner.
## Variables analyzed
Kicking movement was defined as the movement of one or both lower limbs, starting from full flexion of the hip, knee, and ankle to extension and returning to the starting position, thus indicating a kicking cycle [bib_ref] Efeito do peso externo nos chutes espontâneos de lactentes nos primeiros dois..., Landgraf [/bib_ref]. From this definition, the following variables were evaluated: 1) frequency of kicks, which corresponds to the total number of kicks performed by the infant in each experimental condition; 2) frequency of foot contact with the touch pad, which represents the number of kicks with one or both feet (simultaneously or not) directed towards the touch pad that made contact with it; 3) frequency of success to raise the touch pad, which occurred when one or both feet (simultaneously or not) made contact with the touch pad, raising it and activating the mobile 12,23 .
# Statistical analysis
The Shapiro-Wilk normality test and Levene's test for homogeneity of variance were performed. Then, three-way MANOVA (group, age, and condition) followed by Tukey's post hoc was performed for each dependent variable (frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the touch pad). All p-values <0.05 were considered significant. Data were organized and tabulated using the Statistical Package for the Social Sciences (SPSS 17).
Following the above-mentioned analysis, we performed a power calculation, resulting in a power of 98% for the variable frequency of success in raising the touch pad in infants with DS when comparing the baseline and post-weight conditions at 3 months (BL -mean 2.2±0.84 and PW -mean 5.0±0.71) and a power of 97.6% at 4 months (BL -mean 2.6±1.14 and PW -mean 7.2±1.48). [fig_ref] Figure 2: Frequency of kicks of infants with Down syndrome and typical infants at... [/fig_ref].
# Results
In the latter condition, the frequency of kicks was lower (p<0.001) when compared with the post-weight condition in both groups and ages. Additionally, the frequency of foot contact with the touch pad and the frequency of success in raising the touch pad was lower in the weight condition when compared to the baseline (p<0.001) and post-weight (p<0.001) conditions. Furthermore, the frequency of contact and frequency of success in raising the touch pad in the baseline condition was lower when compared to the post-weight condition (p<0.001; [fig_ref] Figure 2: Frequency of kicks of infants with Down syndrome and typical infants at... [/fig_ref].
# Discussion
The present study analyzed and compared the frequency of kicks with and without additional weight in infants with DS and infants with typical development at 3 and 4 months of age. We found that infants with DS showed lower frequencies when compared to the group of typical infants in the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the touch pad, confirming the first hypothesis. This result can be explained by the high incidence of muscle coactivation 24 , hypotonia, and ligament laxity [bib_ref] Spontaneous leg movements of infants with Down syndrome and nondisabled infants, Ulrich [/bib_ref]. These characteristics hinder the transmission of contractile forces to the bone structures [bib_ref] What is currently known about hypotonia, motor skill development, and physical activity..., Latash [/bib_ref] and the ability to maintain concentric and eccentric contraction of the leg muscles against gravity during the task. Thus, these factors restrict the movements of infants with DS and make it difficult for them to challenge gravity and explore the environment. Furthermore, these infants can have cognitive deficits that affect the execution of motor tasks due to comprehension difficulties 6 .
Given these characteristics and their consequences, infants with DS have a delay in the acquisition of motor skills and a slower development of these skills; however, it is noteworthy that the development of these infants occurs in the same sequence as typical infants [bib_ref] Longitudinal assessment of leg motor activity and sleep patterns in infants with..., Mackay [/bib_ref] [bib_ref] Description of the motor development of 3-12 month old infants with Down..., Tudella [/bib_ref] [bib_ref] Spontaneous leg movements of infants with Down syndrome and nondisabled infants, Ulrich [/bib_ref]. Thus, the present study confirms previous studies when confirming the second hypothesis, namely, that there was no significant difference between DS infants at 3 and 4 months of age in the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the touch pad.
For the group of typical infants, significant differences were observed between ages 3 and 4 months in the variables frequency of kicks, frequency of foot contact with the touch pad, and frequency of success in raising the touch pad. It was observed that in the baseline and post-weight conditions, these variables were lower at 4 months of age because between 3 and 6 months of age, infants begin to perform reaching movements, exploring the external environment with their upper limbs [bib_ref] Developmental origins of motor coordination leg movements in human infants, Thelen [/bib_ref] [bib_ref] Proximodistal structure of early reaching in human infants, Berthier [/bib_ref] and reducing the frequency of kicks 23 . However, it was observed in the weight condition that 3-month-olds had a lower overall frequency of kicks compared to 4-month-olds because the former have less muscle strength and the additional weight made the performance of the movement and task more difficult [bib_ref] Influência do peso adicional nos chutes de lactentes pré-termo e de termo, Dionisio [/bib_ref]. These results refute the third hypothesis.
Comparing the experimental conditions, there was a decrease in the values of all variables in the weight condition for both groups at 3 and 4 months of age, confirming the fourth hypothesis. This variation may have occurred due to the adaptation of the infants to the ankle weights. Additional weight is an environmental and organismic constraint in the task context because it makes the task more difficult, forcing the infant to produce greater muscular force to overcome gravity and hindering the performance of the kicking movement thus reducing all frequencies [bib_ref] Making the mobile move: constraining task and environment, Chen [/bib_ref]. However, when this constraint is maintained for a period time, neural plasticity can occur, resulting in improved performance and/or new behavior. In other words, practice combined with feedback (in this case, the mobile and the additional weight) causes a momentary change in the performance of the task and hence the frequency of kicks. These momentary changes are reflections of neural activations that, with practice, cause neural plasticity [bib_ref] Learning by doing: action performance facilitates affordance perception, Franchak [/bib_ref]. Thus, the infant undergoes a change in motor behavior due to neural activation and alteration [bib_ref] The dynamic nature of early coordination: evidence from bilateral leg movements in..., Thelen [/bib_ref].
By comparing the baseline and post-weight experimental conditions for the variables frequency of foot contact with the touch pad, and frequency of success in raising the touch pad, it was found that infants from both groups increased the frequency of these variables in the post-weight condition. These results show that the additional weight had a positive influence on the kicking movement, providing proprioceptive and tactile information that acts as sensory cues during the performance of kicks, and after weight removal, these cues facilitate the movement, leading to an increased frequency of contact [bib_ref] The organization of reactivated memory in infancy, Hayne [/bib_ref] and success. It is noteworthy that adding weight to the lower limb also increases neural activation and improves movement patterns [bib_ref] The dynamic nature of early coordination: evidence from bilateral leg movements in..., Thelen [/bib_ref] , acting as a stimulus that activates muscle proprioceptors, leading to muscle activation and movement of the lower limbs. Moreover, weight activates mechanoreceptors that perceive it and use this information to select an adaptive pattern of the kicking movement, making it more directed towards the target [bib_ref] Influência do peso adicional nos chutes de lactentes pré-termo e de termo, Dionisio [/bib_ref]. In other words, the additional weight activates the proprioceptive and tactile receptors, sending the somatosensory information to the central nervous system with the information about the positioning of the limb, the muscle strength produced, and the presence of limb movement. Based on this information, the infant makes adjustments in muscle activation and joint positioning in order to perform the task in the most appropriate manner [bib_ref] The sensorimotor system, part I: the physiologic basis of functional joint stability, Riemann [/bib_ref]. Thus, these two mechanisms justify the increased frequency of success and contact after the weight removal in both groups. However, these results refute the fifth hypothesis because there was only an increase in the post-weight frequency of contact and success.
The added weight acts as an important stimulus for both groups, especially for infants with DS, improving the movement pattern by increasing the frequency of contact and success after weight removal. In addition to improving the infant's performance in the task, the repetition of the kicking movement combined with the ankle weight can lead to an increase in the control of movement and muscular strength and resistance required for the acquisition of new motor milestones such as gait and can also influence motor learning, and these aspects are important in clinical practice.
# Limitations
The limitations of this study were the reduced number of participants and the convenience sample, which requires caution in generalizing the results to the entire population of infants with DS. Thus, the results may be considered preliminary findings. Furthermore further studies with a larger number of participants are needed even though the study showed increased power for its main variable.
# Conclusion
Thus, it could be concluded that infants with DS have delayed development of the kicking movement when compared with typical infants and that weight acts as an important stimulus for both groups, especially for infants with DS, improving the movement pattern by increasing the frequency of contact and success after weight removal.
[fig] Figure 1: Temporal representation of experimental conditions. [/fig]
[fig] Figure 2: Frequency of kicks of infants with Down syndrome and typical infants at 3 and 4 months of age in the baseline (BL), weight (W), and post-weight (PW) conditions. [/fig]
[fig] Figure 3: Frequency of foot contact with the touch pad in infants with Down syndrome and typical infants at 3 and 4 months of age in the baseline (BL), weight (W), and post-weight (PW) conditions. [/fig]
[fig] Figure 4: Frequency of success of infants with Down syndrome and typical infants at 3 and 4 months of age in raising the touch pad in the baseline (BL), weight (W), and post-weight (PW) conditions. [/fig]
[table] Table 1: Demographic data of patients (n=10). The differences are tested using the t-test for independent samples and found significant differences between groups for the variable weight [/table]
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Nonsequential double ionization with mid-infrared laser fields
Using a full-dimensional Monte Carlo classical ensemble method, we present a theoretical study of atomic nonsequential double ionization (NSDI) with mid-infrared laser fields, and compare with results from near-infrared laser fields. Unlike single-electron strong-field processes, double ionization shows complex and unexpected interplays between the returning electron and its parent ion core. As a result of these interplays, NSDI for mid-IR fields is dominated by second-returning electron trajectories, instead of first-returning trajectories for near-IR fields. Some complex NSDI channels commonly happen with near-IR fields, such as the recollision-excitation-with-subsequent-ionization (RESI) channel, are virtually shut down by mid-IR fields. Besides, the final energies of the two electrons can be extremely unequal, leading to novel e-e momentum correlation spectra that can be measured experimentally.Interaction between intense laser fields (10 13 -10 16 W/cm 2 ) and gas-phase atoms or molecules has led to many new physical phenomena, such as high harmonic generation (HHG) 1,2 , above-threshold ionization (ATI) 3-5 , nonsequential double ionization (NSDI) 6 , attosecond pulse generation 7,8 , etc. The core physical process underlying these phenomena is the so-called recollision process 9-11 : An emitted electron, after being accelerated in the laser field for a fraction of an optical cycle, can be driven back by the oscillating laser field and recollide with its parent ion core.The most important laser parameter for the recollision process is the wavelength λ . Most work has been done with 800 nm, the fundamental wavelength of Ti:Sapphire laser systems. With the advancement of ultrafast laser technology, such as optical parametric chirped pulse amplification (OPCPA), the laser wavelength has been pushed longer into the mid-IR regime with λ > 3 μ m 12 . These wavelengths are desirable in order to achieve high enough electron recollision energies, which scales as λ 2 , for the purpose of dynamic imaging of molecular structures 13,14 or generating high energy broadband radiations 15 .We notice that up to now not much attention has been paid to the process of NSDI with longer wavelengths, especially in the mid-IR regime[16][17][18]. NSDI with near-IR wavelengths has been known to provide an excellent, probably also the simplest, platform for the study of electron correlation effects 19-36 , which are the foundation for all materials. The questions that we were curious about and trying to answer in this paper include: What do recollision and NSDI processes look like with mid-IR laser fields? Is there any substantially new physics?We will show how the obvious effect of a much higher recollision energy, which scales as λ 2 , leads to unexpected changes to the NSDI process and dynamics. For example, the electron trajectories that dominate NSDI with mid-IR laser fields are no longer the short first-returning ones. Longer second-returning trajectories may be more efficient in producing NSDI, although they usually have lower returning energies than the first-returning trajectories do. For another example, some complex NSDI channels that are common or even dominant with near-IR laser fields, such as the so-called recollision-excitation-with-subsequent-ionization (RESI) channel, are virtually shut down by the much higher recollision energy. Therefore although the fact of higher recollision energies is obvious for mid-IR laser fields, the impact of this fact on the resulting NSDI process is not, due to complex interplays between the returning electron and the remaining ion core. We will also show that the above-mentioned changes to the NSDI dynamics leave their footprints on the e-e momentum correlation spectra, which can be measured experimentally.This paper is organized as follows. We first give a brief introduction to the classical ensemble method that we use to obtain the numerical results in the following Method section. Then we present our main results in the Numerical Results section, followed by more elaborate discussions in the Discussions section. Finally a Conclusion section will be given.
# Method
The method that we use in this paper is the classical ensemble method developed by Eberly and coworkers [bib_ref] Comparing classical and quantum simulations of strong-field double-ionization, Panfili [/bib_ref] , which has been widely used to describe strong-field double ionization processes qualitatively or quantitatively [bib_ref] Slow-down collision and nonsequential double ionization in classical simulations, Panfili [/bib_ref] [bib_ref] Nonsequential double ionization as a completely classical photoelectric effect, Ho [/bib_ref] [bib_ref] Variable time lag and bachward election in full-dimension analysis of strong field..., Haan [/bib_ref] [bib_ref] Recollision excitation, electron correlation, and the production of high-momentum electrons in double..., Haan [/bib_ref] [bib_ref] Asymmetric electron energy sharing in strong-field double ionization of helium, Zhou [/bib_ref] [bib_ref] Effects of elliptical polarization on strong-field short-pulse double ionization, Wang [/bib_ref] [bib_ref] Strong field double ionization: the phase space perspective, Mauger [/bib_ref] [bib_ref] Elliptical polarization and probability of double ionization, Wang [/bib_ref] [bib_ref] Recollision dynamics and phase diagram for nonsequential double ionization with circularly polarized..., Fu [/bib_ref] [bib_ref] Classical simulation including electron correlations for sequential double ionization, Zhou [/bib_ref] [bib_ref] Angular correlation in strong-field double ionization under circular polarization, Wang [/bib_ref] [bib_ref] Dynamics of strong-field double ionization in two-color counterrotating fields, Chaloupka [/bib_ref] [bib_ref] Transition of recollision trajectories from linear to elliptical polarization, Li [/bib_ref]. Although atoms are fundamentally quantum mechanical systems and the interaction between an atom and an external laser field is described by the corresponding multi-electron time-dependent Schrödinger equation, the computational load is extremely demanding if not impossible [bib_ref] Double ionization of helium at 390 nm, Parker [/bib_ref] [bib_ref] Boosting photoabsorption by attosecond control of electron correlation, Hu [/bib_ref]. The classical ensemble method provides a computationally efficient and physically intuitive approach to gain insights of strong-field double ionization problems.
Since the details of the classical ensemble method have been explained in numerous previous publications as cited above, here we only give a brief introduction to it. The general idea is to mimic a quantum mechanical wave function using an ensemble of classically modeled atoms. Before the laser pulse is turned on, an ensemble of classically modeled atoms is first populated within the classically allowed phase space for a total energy E tot = − 1.23 a.u., which is set to be the negative sum of the first two ionization energies of the Xe atom (12.13 eV and 20.98 eV, respectively). E tot has the following form (atomic units are used unless stated otherwise) where p i and r i are the momentum and position of the i-th electron (i = 1, 2), = − r r r 2 is the relative position of the two electrons, a and b are the soften parameters [bib_ref] Numerical simulations of multiphoton ionization and above-threshold electron spectra, Javanainen [/bib_ref] [bib_ref] Model atom for multiphoton physics, Su [/bib_ref] for the nuclear Coulomb potential and electron Coulomb potential. In our calculation we have set a = 2.0 a.u. to stabilize the classical atom (otherwise the classical atom will suffer from an unphysical autoionization effect), and b = 0.1 a.u. to prevent numerical singularity [bib_ref] Transition of recollision trajectories from linear to elliptical polarization, Li [/bib_ref]. The size of the ensemble (i.e., the number of classically modeled atoms) is usually between 1 million and 10 million in our calculations.
After the initial microcanonical ensemble (i.e., every ensemble member has the same total energy) is populated, the laser electric field is turned on, and the two electrons start to move. The motions of the two electrons are governed by the time-dependent Newtonian equation of motion
[formula] = − ∇ − + + + − d r dt r a r b E t 2 1 ( ) (2) i i 2 2 2 2 12 2 2 [/formula]
where E t ( ) is the laser electric field. In our calculations here the laser field is polarized along the z direction and has a (2 + 6 + 2) trapezoidal shape, meaning that the laser pulse linearly turns on for 2 cycles, maintains full strength for 6 cycles, and then linearly turns off for 2 cycles.
The positions and momenta of the two electrons are recorded at each time step during the laser pulse. Statistics can be obtained for both experimentally measurable quantities, such as double ionization probability or end-of-pulse electron momentum correlation spectra, and theoretical quantities that cannot be measured experimentally, such as the emission times of the two electrons and the recollision time of the first-emitted electron. These theoretical quantities are very useful however to gain physical insights into the double ionization process.
# Numerical results
Probability of double ionization versus laser intensity. In this study we use two laser wavelengths, one is 800 nm (near-IR) and the other is 3200 nm (mid-IR). [fig_ref] Figure 1: Probabilities of double ionization as a function of the laser peak intensity... [/fig_ref] shows the probability of double ionization (DI) versus laser peak intensity for both the 800 nm (green squares) and the 3200 nm (red circles) laser fields. We can see that for both wavelengths, saturation of DI happens around 1.0 × 10 15 W/cm 2 . Characteristic "knee" structures of NSDI can be seen in both cases below the saturation intensity.
It is interesting to see a crossing between the two curves around 2.0 × 10 13 W/cm 2 , below which 3200 nm is more efficient in generating DI and above which 800 nm is more efficient. This is the result of competition between two factors: the returning energy and the returning flux (probability), of the first-emitted electron. The longer the wavelength, the higher the returning energy, the smaller the returning probability due to dispersion along the transverse direction (or wavepacket spreading along the transverse direction, in the quantum language, though a classical trajectory approach is used here). Below the crossing intensity around 2.0 × 10 13 W/cm 2 , the returning energy wins, therefore the DI probability is higher for 3200 nm; above this crossing intensity, the returning energy is no longer a severe limitation for 800 nm in generating DI so the returning flux wins, therefore the DI probability is higher for 800 nm.
From this traditional DI probability versus laser intensity plot, we see that NSDI is the result of the interplay between the returning electron and the remaining ion core (which determines how much returning energy is needed to kick out the second electron). This interplay indeed lies at the heart of NSDI processes.
Momentum correlation spectra of the two emitted electrons. [fig_ref] Figure 2: Electron momentum correlation spectra along the longitudinal direction [/fig_ref] shows the end-of-pulse momentum correlation between the two emitted electrons along the laser polarization direction (the z direction). For all the panels of this figure, the horizontal axis is the longitudinal momentum of one electron and the vertical axis is the longitudinal momentum of the other electron. The data were not symmetrized, so either electron could be the first-emitted electron. The left column (a, c) is for 800 nm and the right column (b, d) is for 3200 nm. The upper row (a, b) is for intensity 1.0 × 10 14 W/cm 2 and the lower row (c, d) is for intensity 4.0 × 10 13 W/cm 2 , as labeled on each panel.
Scientific RepoRts | 6:37413 | DOI: 10.1038/srep37413
One can see that for the same laser intensity, the correlation spectra are drastically different for the two wavelengths. For the high intensity (upper row), for 800 nm slightly more electron pairs are correlated (i.e., population in the 1st/3rd quadrants) than anti-correlated (i.e., population in the 2nd/4th quadrants). The ratio between the two is about 57% to 43%. In contrast, for 3200 nm the majority (80%) of electron pairs are correlated. This change is even more dramatic for the low intensity case (lower row), with 46% correlated electron pairs for 800 nm and 83% for 3200 nm.
The shapes of the spectra are also completely different for the two wavelengths. [fig_ref] Figure 2: Electron momentum correlation spectra along the longitudinal direction [/fig_ref] shows a typical V-shape (or fingerlike) structure in the 1st/3rd quadrants [bib_ref] Binary and recoil collisions in strong field double ionization of helium, Staudte [/bib_ref] [bib_ref] Correlated two-electron momentum spectra for strong-field nonsequential double ionization of He at..., Rudenko [/bib_ref]. The detailed mechanism leading to this kind of structure has been analyzed theoretically by Ye et al. in ref. [bib_ref] Classical trajectory diagnosis of a fingerlike pattern in the correlated electron momentum..., Ye [/bib_ref]. Changing the wavelength to 3200 nm, the spectrum shown in [fig_ref] Figure 2: Electron momentum correlation spectra along the longitudinal direction [/fig_ref] features a cross-shape (X-shape) structure, with most populations concentrating along the axes, meaning that the final energies of the two emitted electrons are very unequal. Similar cross-shape structure has been reported experimentally in ref. [bib_ref] Attosecond tracing of correlated electron-emission in non-sequential double ionization, Bergues [/bib_ref] with however single-cycle near-IR laser fields and also known in field-free electron impact ionization [bib_ref] Low-energy electron-impact ionization of helium, Schow [/bib_ref] [bib_ref] Energy and angular distribution of electrons from atoms and molecules by electron..., Oda [/bib_ref] [bib_ref] Differential and total cross sections for ionization of helium and hydrogen by..., Rudd [/bib_ref] [bib_ref] Electron-impact ionization doubly differential cross sections of helium, Bray [/bib_ref]. The spectrum shown in [fig_ref] Figure 2: Electron momentum correlation spectra along the longitudinal direction [/fig_ref] , with more anti-correlated electron pairs than correlated ones, is a strong indication of the RESI double ionization channel [bib_ref] Mechanism of strong-field double ionization of Xe, Sun [/bib_ref] [bib_ref] Asymmetric electron energy sharing in strong-field double ionization of helium, Zhou [/bib_ref]. Changing the wavelength to 3200 nm, however, [fig_ref] Figure 2: Electron momentum correlation spectra along the longitudinal direction [/fig_ref] indicates that RESI channel is at least greatly suppressed because the spectrum is now dominated by correlated electron pairs. From first-returning to second-returning trajectories. More direct insight about the change of recollision dynamics from 800 nm to 3200 nm can be obtained from trajectory back analyses. We post-select all DI events and trace back the positions and the velocities of the two electrons as a function of time from the beginning to the end of the laser pulse.
For each DI event, we record the time of single ionization (t SI ), the time of recollision (t r ), and the time of DI (t DI ). Here t SI is defined as the time when the energy of the first-emitted electron (including the kinetic energy, the ion-electron potential energy and half of the e-e potential energy) just becomes positive, t r is defined as the time of closest encounter between the two electrons after the departure of the first electron, and t DI is defined as the time when the energies of both emitted electrons just become positive. Statistics of these characteristic times can be obtained after analyzing all DI events.
The left column (a, c) of [fig_ref] Figure 3: Left column [/fig_ref] shows the statistical distribution of (t r − t SI ), the difference between the emission time and the later recollision time of e 1 . [fig_ref] Figure 3: Left column [/fig_ref] is for the high intensity case, with the blue dashed curve for 800 nm and the red solid curve for 3200 nm. One can see that the most probable value of (t r − t SI ) for 800 nm is peaked at about 0.75 cycles, which corresponds to the first-returning electron trajectories. For 3200 nm, however, the most probable value of (t r − t SI ) shifts to the peak at 1.25 cycles, which corresponds to the second-returning electron trajectories. The first-returning peak is greatly suppressed. Exactly the same trend appears for the low intensity case, as shown in [fig_ref] Figure 3: Left column [/fig_ref]. As will be explained later, this shift from first-returning to second-returning trajectories is the result of enhanced collision efficiency, or energy transfer between the two electrons during the recollision process. Suppression of the RESI channel. The time difference between complete DI and recollision, (t DI − t r ), is an indicator of NSDI mechanism. If this time difference is smaller than about 0.1 laser cycles, then the NSDI mechanism is recollision impact ionization (RII): the emission of the second electron is mainly due to the strong kick from the first electron. If this time difference is larger than about 0.25 cycles, then the NSDI mechanism is RESI: the first electron does not promptly kick out (though may excite) the second electron, and the second electron has to wait until the next field maximum to be pulled out by the laser field. Because recollision usually happens around field value zero, 0.25 cycles is the rough time to wait for the next field peak. Note however that there is no sharp and clear time cut to unambiguously distinguish different NSDI mechanisms.
The right column (b, d) of [fig_ref] Figure 3: Left column [/fig_ref] shows the statistical distribution of this time difference (t DI − t r ) for 800 nm (dashed blue) and 3200 nm (solid red). The thin vertical line is the 0.25-cycles mark. One can see that for both intensities, for 3200 nm this time difference is very small with peak positions < 0.1 laser cycles, indicating the prompt RII mechanism. In contrast, for 800 nm, this time difference peaks at 0.25 laser cycles for the higher intensity, and about 0.8 laser cycles for the lower intensity, indicating the dominance of the delayed RESI mechanism. Therefore changing the wavelength from 800 nm to 3200 nm, the RESI channel is greatly suppressed, leaving NSDI a simpler prompt RII process.
# Discussions
In the previous section we have shown numerical results that as the wavelength increases from 800 nm to 3200 nm, the most efficient electron trajectories to produce NSDI change from the shorter first-returning trajectories to the longer second-returning trajectories. Besides, the common or even dominant RESI channel for 800 nm is greatly suppressed for 3200 nm. These changes in NSDI mechanism will leave noticeable footprints on the experimentally measurable e-e momentum correlation spectra. In this section we discuss why these changes happen, as the wavelength increasing from 800 nm to 3200 nm. An example second-returning NSDI trajectory. To have an intuitive impression of second-returning electron trajectories for 3200 nm, we show an example in [fig_ref] Figure 4: An example second-returning NSDI trajectory for 3200 nm with peak laser intensity... [/fig_ref]. The upper panel shows the distances of the two electrons from the ion core as a function of time during the pulse, and the lower panel shows the velocities of the two electrons along the z direction (i.e., polarization direction) as a function of time. The inset on the upper panel is a zoom in the initial portion of the trajectory, enlarging the two returning events. One can see that e 1 was emitted around t SI = 1.25 cycles when the laser field was around a peak and returned for the first time around t = 2.0 cycles when the laser field was about zero value. This first returning did not kick out e 2 . From the lower panel one can see that at the time of first returning, the velocity of e 1 was about 4 a.u. Then e 1 returned for the second time around t r = 2.7 cycles and this time e 2 was kicked out rather promptly. The velocity of e 1 at the time of second returning was about 2 a.u., only half of the magnitude of that at the time of first returning, and in the opposite direction. Our statistics shows that about 52% of all NSDI trajectories are second-returning trajectories as exampled in [fig_ref] Figure 4: An example second-returning NSDI trajectory for 3200 nm with peak laser intensity... [/fig_ref] , for the intensity 1.0 × 10 14 W/cm 2 .
The returning energy of the first electron and the ionization energy of the second electron. The above example NSDI trajectory shows that although e 1 has a higher (4 times) returning energy at its first returning, it actually kicked out e 2 at its second returning. This kind of first-returning-miss-and-second-returning-hit trajectories are not uncommon for 3200 nm and they constitute the statistical distributions of (t r − t SI ) shown in [fig_ref] Figure 3: Left column [/fig_ref]. From the intuitive Simpleman's picture, it is also known that the energy of second returning is less than that of the first returning. So the question to ask is why for 3200 nm the first returning trajectories with higher returning energies are less efficient in producing NSDI (i.e., kicking out the second electron) than the second returning trajectories with less returning energies.
To find the answer to this question we record the returning energies of each returning of the first-emitted electron for both 800 nm and 3200 nm. For 800 nm and 1.0 × 10 14 W/cm 2 , the ensemble averaged first-returning energy is 0.66 a.u., and the ensemble averaged second-returning energy is 0.42 a.u. Both of them are less than the second ionization potential of Xe (i.e., the ionization potential of Xe + ), which is 0.77 a.u. . Therefore it is obvious to understand why under this wavelength and this intensity (or lower intensities) NSDI is dominated by the first-returning trajectories and the RESI mechanism.
In contrast, for 3200 nm and 1.0 × 10 14 W/cm 2 , the ensemble averaged first-returning energy is 6.13 a.u., and the ensemble averaged second-returning energy is 3.13 a.u. Both of them are much larger than the second ionization potential of Xe. However, larger returning energy also corresponds to poorer recollision efficiency, because the returning electron is so fast that it spends little time in the vicinity of the ion core and little energy can be effectively transferred to the second electron. A reduced returning energy, as long as it is sufficient to kick out the second electron, has a higher energy transfer efficiency because the returning electron can spend more time interacting with the second electron. Of course, multiple-returning electron trajectories suffer from lower returning fluxes because of longer excursion time hence more spreading along the transverse direction. A balance can be achieved to maximize the NSDI efficiency. With the laser parameters that we are using, the optimal returning order to generate NSDI is the second returning.
For 3200 nm, even when the laser intensity reduces to 4.0 × 10 13 W/cm 2 , the ensemble averaged second returning energy is 1.18 a.u., still larger than the second ionization potential. The ensemble averaged first returning energy is 3.05 a.u. Therefore even for this intensity, it is still the second-returning trajectories that lead to more efficient generation of NSDI. Extremely asymmetric energy sharing. As explained above, on the one hand mid-IR laser fields can produce returning electrons with high returning energies, much higher than the ionization potential of the second electron. On the other hand, because the first electron passes by the vicinity of the ion core very fast, the energy sharing between the two electrons is actually not as efficient as slower returnings with near-IR laser fields. These two factors combine and lead to extremely asymmetric energy sharing between the two electrons in the recollision process. The measureable effect is the cross-shaped e-e correlation spectrum shown in [fig_ref] Figure 2: Electron momentum correlation spectra along the longitudinal direction [/fig_ref] , with one electron having near-zero final momentum and the other electron having large final momentum.
To illustrate this point in a more quantitative manner, we record the energies of the two electrons "right after" (e.g., 0.03 laser cycles after) each recollision. Then we take the (absolute value of the) difference between the energies of the two electron and plot the statistical distribution of this energy difference Δ E, as shown in for 800 nm and 3200 nm, respectively. The intensity is 1.0 × 10 14 W/cm 2 . This energy difference Δ E, taken right after each recollision, is an indicator of the degree of energy sharing between the two electrons during the just happened recollision process. The smaller the value Δ E, the more sufficient the energy sharing between the two electrons, vice versa. One can see that for 800 nm, Δ E extends to a maximum value of about 1.5 a.u., whereas for 3200 nm, Δ E extends to a maximum value of about 10 a.u. Besides, for 3200 nm there is an elevated plateau structure from about 2 a.u. to 6 a.u. This asymmetric energy sharing between the two electrons during the recollision process has an obvious effect on the measurable end-of-pulse e-e momentum correlation, as shown in the lower two rows of . We divide the Δ E distributions into three parts (I, II, III) as illustrated in and plot separately the e-e correlation spectra corresponding to these three parts. The division is somehow subjective but it helps to illustrate how different degrees of recollisional energy sharing affect the end-of-pulse e-e momentum correlation. From the lower two rows of , we see that e-e correlation spectra depend critically on the symmetry or efficiency of energy sharing, and the cross-shaped structure shown in the last panel (h) is indeed the result of extremely asymmetric energy sharing.
# Conclusion
In this paper we study how the recollision and NSDI processes will change when the laser wavelength increases from near-IR to mid-IR, using a well-established classical ensemble method. We show how the obvious effect of larger returning energies of the first-emitted electrons impacts NSDI in some unexpected and interesting ways. For example, with mid-IR laser fields the longer second-returning electron trajectories can be more efficient in producing NSDI than the shorter first-returning trajectories, although the returning energies are lower. For . Distributions of Δ E, the absolute value of the energy difference between the two electrons right after (0.03 laser cycles after) each recollision, for 800 nm and 3200 nm. The intensity is 1.0 × 10 14 W/cm 2 . The distributions are divided into three regions I, II, and III, quantifying the degree of energy sharing asymmetry. End-of-pulse e-e momentum correlation spectra corresponding to the three regions of 800 nm, as labeled on each panel. End-of-pulse e-e momentum correlation spectra corresponding to the three regions of 3200 nm.
Scientific RepoRts | 6:37413 | DOI: 10.1038/srep37413 another example, mid-IR laser fields virtually shut down some complex NSDI channels which are common or even dominant with near-IR laser fields, such as the RESI channel.
We explain these unexpected results by considering the available returning energies of the first electron versus the ionization energy of the second electron. For near-IR laser fields, the available returning energies of the first electron are usually slightly higher or even lower than the ionization energy of the second electron, therefore the more energetic first-returning trajectories are favorable. For mid-IR laser fields, however, the available returning energies of the first electron are much higher than the ionization energy of the second electron. The slower second-returning trajectories can be more efficient in energy transferring during the recollision process, because the e-e interaction time is longer than the first-returning trajectories. This higher recollision efficiency compensates the lower fluxes of the second-returning trajectories due to more spreading along the transverse direction.
These changes in the NSDI mechanism will leave noticeable footprints on the experimentally measurable e-e momentum correlation spectra. For example, a cross-shaped structure with population concentrating on axes is likely to appear due to the extremely asymmetric energy sharing during the recollision process.
[fig] Figure 1: Probabilities of double ionization as a function of the laser peak intensity for the 800 nm (green squares) and 3200 nm (red circles) laser fields. The laser pulse has a trapezoidal shape with two cycles turning on, six cycles plateau, and two cycles turning off, for both wavelengths.Scientific RepoRts | 6:37413 | DOI: 10.1038/srep37413 [/fig]
[fig] Figure 2: Electron momentum correlation spectra along the longitudinal direction. P 1z and P 2z denote the momentum components of the two emitted electrons along the laser polarization (longitudinal) direction, respectively. The data were not symmetrized. The upper row (a,b) is for laser intensity 1.0 × 10 14 W/cm 2 and the lower row (c,d) is for intensity 4.0 × 10 13 W/cm 2 . The left column (a,c) is with 800 nm and the right column (b,d) is with 3200 nm. Scientific RepoRts | 6:37413 | DOI: 10.1038/srep37413 [/fig]
[fig] Figure 3: Left column (a,c): Statistical distributions of the time difference (t r − t SI ), i.e., the time difference between successful recollision and emission of the first electron, for two wavelengths and two laser intensities as labeled on the figure. Right column (b,d): Statistical difference of the time difference (t DI − t r ), i.e., the time difference between complete double ionization and recollision, for the same two wavelengths and laser intensities. [/fig]
[fig] Figure 4: An example second-returning NSDI trajectory for 3200 nm with peak laser intensity 1.0 × 10 14 W/cm 2 . Dashed blue curves are for the first-emitted electron (e 1 ) and solid red curves are for the second-emitted electron (e 2 ). The upper panel shows the distances of the two electrons from the ion core as a function of time during the laser pulse, and the lower panel shows the velocities of the two electrons as a function of time. The inset on the upper panel is a zoom in the early portion of the pulse, enlarging the two returning events. [/fig]
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Veno‐venous extracorporeal membrane oxygenation and prone ventilation for therapeutic management of COVID‐19
Background: The efficacy and safety of the combined use of veno-venous extracorporeal membrane oxygenation (ECMO) and prone ventilation are currently not known for coronavirus disease 2019 (COVID-19).Case presentation: We report two cases in which the combination of veno-venous ECMO and prone ventilation for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia were successfully carried out. Both patients had developed severe respiratory failure due to SARS-CoV-2 pneumonia, thus requiring veno-venous ECMO. Prone ventilation was also administered safely.Conclusion: Oxygenation and lung compliance gradually improved during prone ventilation, and both patients were successfully extubated. For patients with severe SARS-CoV-2 pneumonia who require veno-venous ECMO, the use of prone ventilation could be beneficial, and should be considered.
# Introduction
A T THE END of 2019, acute respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began to spread in Wuhan, China.This respiratory illness, named coronavirus disease 2019 (COVID-19), gradually spread globally, and the WHO declared COVID-19 as a pandemic in March 2020.
Prone ventilation has been reported to reduce mortality among patients with moderate-to-severe acute respiratory distress syndrome (ARDS) when used for at least 12 h daily.Furthermore, veno-venous extracorporeal membrane oxygenation (ECMO) is the standard treatment for severe ARDS.However, the efficacy of the combination of venovenous ECMO and prone ventilation is currently not known. Here, we report two cases in which the combination of veno-venous ECMO and prone ventilation was successful in treating SARS-CoV-2 pneumonia.
## Case report
## Case 1
A 61-YEAR-OLD MAN with a history of hypertension, diabetes, and chronic atrial fibrillation was admitted to a local hospital 9 days after developing a fever and tested positive for the SARS-Co-V-2. Fourteen days after the onset of symptoms, his respiratory status deteriorated, and he was intubated. Two days later, he was referred to our hospital to receive veno-venous ECMO.
The patient's vital signs on admission to our hospital were as follows: respiratory rate, 8 breaths/min; oxygen saturation, 96% under positive end-expiratory pressure (PEEP) of 14 cmH 2 O and fraction of inspiratory oxygen (FiO 2 ) of 1.0; heart rate, 129 b.p.m.; and blood pressure, 137/93 mmHg using noradrenaline 0.03 lg/kg/min and vasopressin 2 units/h. The arterial blood gas analysis results were: pH 7.158; PaO 2 , 84.0; and PaCO 2 , 64.5. Laboratory data showed elevation of N-terminal pro-brain natriuretic peptide pg/mL) and creatinine (1.47 mg/dL). Left ventricular ejection fraction was 45% by echocardiogram and the daily urine output was approximately 300 mL. Hypoxia due to heart failure or renal failure was also considered, but we determined that SARS-CoV-2 pneumonia was the leading cause of hypoxia. Therefore, we decided to treat with venovenous ECMO and continuous renal replacement therapy. Veno-venous ECMO was administered through the right internal jugular vein for blood drainage with a 25 French gauge (Fr) heparin-coated cannula, and the right femoral vein for blood return with a 20 Fr heparin-coated cannula. The procedure was carried out safely, and the patient experienced no complications. Computed tomography (CT) showed bilateral ground-glass opacities and bilateral dorsal consolidation. After hemodynamic stabilization, prone ventilation was implemented safely from day 2 to day 4 (prone position for 17 h, supine position for 7 h). The patient's PaO 2 and lung compliance were gradually improved within 72 h of prone ventilation: PaO 2 from 70.4 to 89.2 mmHg; and lung compliance from 25 mL/cmH 2 O to 32 mL/cmH 2 O. The use of ECMO was stopped on day 9. The patient was extubated on day 13.
## Case 2
Emergency medical services found a 59-year-old woman collapsed at her home. She was lethargic, and her blood pressure could not be measured. On arrival at our hospital, her vital signs were as follows: consciousness, Glasgow Coma Scale of 14 (E4V4M6); respiratory rate, 24 breaths/min; oxygen saturation, 99% with reservoir face mask at 10 L/min oxygen; blood pressure, 84/71 mmHg; heart rate, 112 b.p.m.; and body temperature, 36.8°C. The patient's laboratory data were as follows: hematocrit, 47.4%; N-terminal pro-brain natriuretic peptide, 3,325 pg/mL; creatinine, 0.82 mg/dL. The CT imaging revealed bilateral ground-glass opacities, and the patient tested positive for SARS-CoV-2. The patient had a medical history of diabetes and depression. The day after admission, the patient's respiratory status had deteriorated, and she was intubated. The arterial blood gas analysis on day 4 was as follows: pH, 7.346; PaO 2 , 64.7; and PaCO 2 , 50.7 (FiO 2 0.8, PEEP 16 cmH 2 O). The ratio of arterial oxygen partial pressure to the fractional inspired oxygen was at 80.9 and continued to decrease gradually. The patient's blood pressure was 91/68 mmHg using noradrenaline 0.1 lg/kg/min and vasopressin 2 units/ h. Left ventricular ejection fraction was 50% by echocardiogram and the daily urine output was approximately 750 mL. We decided to treat the patient with ECMO. The veno-venous ECMO was established through the right internal jugular vein for blood drainage with a 25 Fr heparin-coated cannula, and the right femoral vein for blood return with a 20 Fr heparin-coated cannula. The procedure was carried out safely, and no complications occurred. The CT imaging showed a deteriorating bilateral dorsal consolidation. Prone ventilation was administered safely from day 5 to day 7. The patient's PaO 2 and lung compliance were gradually improved within 72 h of prone ventilation: PaO 2 from 59.5 to 76.8 mmHg and lung compliance from 20 to 40 mL/cmH 2 O. The veno-venous ECMO treatment was stopped on day 9. The patient was extubated on day 13.
# Discussion
T HE GUIDELINES FOR the management of COVID-19 in mechanically ventilated adult patients with refractory hypoxemia by the Society of Critical Care Medicine and the European Society of Intensive Care Medicine suggest the use of veno-venous ECMO. The guidelines also state optimizing the ventilation and using prone ventilation for moderate-to-severe ARDS.There is no recommendation for a therapeutic combination of veno-venous ECMO and prone ventilation. Our cases show that prone ventilation is possible even when using veno-venous ECMO. The Advanced Critical Care and Emergency Center at Sapporo Medical University is a referral center for adult patients requiring ECMO in Hokkaido, Japan. The center has six beds in the intensive care unit and 22 full-time doctors. We treat approximately 10 cases of respiratory ECMO annually.
In a report of radiological date from 81 patients with COVID-19 pneumonia, CT images showed that COVID-19 pneumonia was manifested with chest abnormalities, even in asymptomatic patients, with a rapid evolution from focal unilateral to diffuse bilateral ground-glass opacities, that progressed to or coexisted with consolidations within 1-3 weeks.Therefore, prone ventilation is likely to be effective in gradually decreasing the ventral alveolar distension and dorsal alveolar collapse in COVID-19 cases. In our cases, CT images after veno-venous ECMO placement showed bilateral dorsal consolidation. These findings suggest that prone ventilation could be beneficial.
There are two main safety concerns related to the use of combined veno-venous ECMO and prone ventilation for COVID-19 treatment. The first concern is tube displacement. This problem has been discussed both in veno-venous ECMO combination therapy and prone ventilation. As the blood drainage cannula and blood return cannula of ECMO have a large diameter, in the event of displacement, the risk of a fatal bleeding complication increases. Additionally, displacement could also be fatal due to the use of a discontinued ECMO machine. However, the application of prone ventilation during veno-venous ECMO has been shown to be a safe and reliable technique when undertaken in an ECMO center by trained staff and following standard procedures.The second concern is the spread of SARS-CoV-2 as a result of an accidental disconnection of the intubation tube from the ventilator. There is a possibility that the virus persists through aerosol for a substantially long period, 7 rendering the medical personnel at risk of infection. Therefore, the disconnection of the intubation tube from the ventilator must be minimized. In order to address these concerns, we assigned qualified personnel to manage the connection between the intubation tube and the ventilator and the blood drainage and return cannulas in veno-venous ECMO when patients were shifted from the spine to prone position. There was no tube displacement in either case, and the risk of spreading SARS-COV-2 was minimized. We have used airfluidized bed when patients were shifted from the spine to prone position. By using air-fluidized bed, the shift from the spine to prone position can be carried out by four medical staff.
In conclusion, the combination therapy of veno-venous ECMO and prone ventilation is possible in patients with SARS-CoV-2 pneumonia without any complication. For severe SARS-CoV-2 pneumonia requiring ECMO, prone ventilation could be useful, as evidenced by radiographic data. Prone ventilation should be considered for use by medical personnel. |
Atomic structures of anthrax toxin protective antigen channels bound to partially unfolded lethal and edema factors
## Supplementary table 1. ef mutagenesis primers
[formula] Primer Name Sequence EF D171A Forward 5'-GACTAAATAAAAGGT-3' EF D171A Reverse 5'-TAAGAGTTTAAGCGA-3' EF D174A Forward 5'-CTTTAAATTTTTGAC-3' EF D174A Reverse 5'-TTAAGCGATGATAGT-3' Supplementary [/formula]
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Analysis of extracellular vesicle DNA at the single‐vesicle level by nano‐flow cytometry
[fig] Figure S2: Fluorescence emission spectra of SYTO 9, SYTO 13, SYTO 16, PicoGreen, SYBR Green I, and SYTO 82 without (black line) or with binding to DNA (red line) or RNA (blue line). The concentrations of RNA and DNA were both 5 μg/mL. Note: All the nucleic acid dyes were purchased from Molecular Probes. Lambda DNA (Promega, D150A) or ribosomal RNA (Thermo Fisher, R11490, component C) with [/fig]
[fig] Figure S3: nFCM analysis of the assay controls. Representative SSC and FL burst traces for (a) buffer only, (b) buffer with reagent, and (c) unstained EV preparation. [/fig]
[fig] Figure S4: Representative fluorescence burst traces of SYTO 16-stained lambda DNA before (a) and after (b) ultracentrifugation at 100,000 × g for 17 min at 4°C. 7478 and 7575 events were detected in 1 min by nFCM before and after ultracentrifugation, respectively. [/fig]
[fig] Figure S5: nFCM analysis of EV sample prepared from CCCM of HCT-15 cells by ultracentrifugation without or with Triton X-100 treatment. (a) Bivariate dot-plot of SYTO16 [/fig]
[fig] Figure S6: Representative fluorescence burst traces of SYTO 16-stained lambda DNA before (a) and after (b) 1% Triton X-100 treatment for 30 min at 4°C. 8003 and 7280 events were detected in 1 min by nFCM before and after Triton X-100 treatment, respectively. [/fig]
[fig] Figure S7: Optimization of the concentration of DNase I. EV preparations were treated with different concentrations of DNase I for 30 min at 37C, and data are represented as mean ± s.d. (n = 3). [/fig]
[fig] Figure S8: Immunofluorescent analysis of tetraspanin proteins(CD9, CD63, and CD81) for an EV preparation from CCCM of HCT-15 cells by ultracentrifugation without (a) or with (b) DNase I treatment. EVs were stained with phycoerythrin (PE)-conjugated monoclonal antibodies specific to CD9, CD63, or CD81. PE-conjugated IgG was used as the isotype control. The events without detectable SSC signal were ignored.Note: This measurement was conducted by using 532 nm laser as the excitation source.Two single-photon counting avalanche photodiodes (APDs) were used for the simultaneous detection of side scatter (SSC, bandpass filter: FF01− 524/24) and orange fluorescence (bandpass filter: FF01− 579/34) of individual particles/EVs, respectively. [/fig]
[fig] Figure S9: nFCM Analysis of CCCM of HCT-15 cells upon SYTO 16 staining. (a) Bivariate dot-plot of SYTO16 fluorescence versus SSC intensity for CCCM with no treatment (i), withDNase I digestion (ii), or with Triton X-100 lysis (iii). Note that SYTO16 staining was carried out after enzyme or detergent treatment. [/fig]
[fig] Figure S10: nFCM measurement of DNA content of single EVs in the unit of base pairs by using external DNA standard approach. (a) The bivariate dot-plot of SYTO16 fluorescence versus SSC for the EV preparation from the CCCM of HCT-15 cells by ultracentrifugation. (b) FL burst area distribution histogram for the DNA standard with three different fragment lengths upon SYTO 16 staining. (c) The correlation of the centroids of the burst areas obtained from the fitted Gaussian curve (red line in plot b) with known DNA fragment lengths. (d) The bivariate dot-plot of DNA fragment length in base pairs versus EV size upon converting the fluorescence intensity to base pairs via the calibration curve obtained in plot c and converting SSC intensity to EV size via the calibration curve built by monodispersed silica nanoparticles of know size and upon refractive index correction based on the Mie theory. Note: The data shown above can only provide an estimation of the total DNA length of single EVs owing to the different staining efficiency between EV-DNA and DNA standard. It may be impractical to accurately convert the FL intensity measured by nFCM to base pairs owing to the following reasons. 1) The success of DNA fragment sizing by flow cytometry relies upon precise, stoichiometric staining of the individual DNA fragments with fluorescent dyes. The longer the DNA fragment, the more the intercalative dye molecules bound and thus the brighter the fluorescence signal. Quantification of total fluorescence emitted by individual DNA fragments as they pass through the laser beam yields the fragment size. However, the staining efficiency of DNA fragment (how many of the intercalative binding sites offered by the DNA fragment are occupied by the intercalative dye molecules) is highly dependent on the DNA concentration, the dye concentration, and the dissociation constant of intercalation (Anal. Chem. 2005, 77, 3554-3562; Anal.Biochem. 2000, 286, 138-148). Thus, for an accurate measurement of DNA fragment length, DNA standard of known base pairs needs to be added to the sample as an internal standard. 2) For the EV isolate, cell-free DNA exists in great abundance and varies in a wide size range. Thus, it is difficult to identify the peaks of DNA standard spiked in. 3) Even though the peaks of DNA standard can be identified and the intercalating dye is membrane permeable, it is not sure whether the staining condition inside the lumen of EVs is identical to that outside of EVs. 4) The staining efficiency could be different for DNA free in solution or bound to protein complex (e.g. attached to the EV outer membrane or non-vesicular entities). [/fig]
[fig] Figure S11: Single cell analysis of HCT-15 cells cultured with 0.02% DMSO (i, negative control) or with the addition of ethynyl-modified dUTP (EdU) (ii) and then labeled with azide-AF488. A BD FACSAria IIIu cytometer with 488 nm excitation was used for the analysis, and the FITC channel (530/30 nm bandpass filter) was used for the fluorescence detection of AF488.Note: A Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (Thermo Fisher, C10632) was used in the present study. The reagents were prepared according to the manufacturer's instructions. Firstly, 0.6 μL of 10 mM EdU in DMSO or 0.6 μL DMSO (negative control) were added to 3 mL cell culture medium. The HCT-15 cells were cultured with these media at 37°C for 72 hours. Then, about 1 × 10 6 cells were collected and centrifuged at 800 g for 5 min at 4°C. Cells were resuspended in 100 μL 4% PFA and incubated at room temperature for 30 min. The PFA was washed away with 1 mL PBS by ultracentrifugation at 800 g for 5 min at 4°C. Subsequently, cells were resuspended in 100 μL of 1 saponin-based permeabilization and wash reagent (component E), incubated for 15 min at room temperature. Lastly, 500 μL click-iT reaction cocktail with Azide-AF488, copper protectant, and buffer additive mixed in PBS was added to the cells. The mixture was incubated for 30 min at room temperature and protected from light. After two washes by ultracentrifugation at 800 g for 5 min 4°C with 1 mL of 1 saponin-based permeabilization and wash reagent, the sample was resuspended in 500 L PBS for BD FACSAria IIIu cytometer analysis. [/fig]
[fig] Figure S12: The population ratios of EdU positive EVs for EVs isolated upon ultracentrifugation from the CCCM of HCT-15 cells cultured with 0.02% DMSO (reagent control), 20 μM EdU, or 20 μM EdU and upon DNase I treatment. Data are represented as mean ± s.d. (n = 3). [/fig]
[fig] Figure. S13: nFCM examination of the effectiveness of SEC in separating cell-free DNA from EVs for the EV preparation obtained from the CCCM of HCT-15 cells by ultracentrifugation. (a,b) Bivariate dot-plots of SYTO16 fluorescence versus SSC for SYTO 16-stained 6th-8th fractions of SEC and their mixture before (a) and after (b) DNase I digestion. [/fig]
[fig] Figure S14: nFCM examination of the effectiveness of density gradient ultracentrifugation in separating cell-free DNA from EVs for the EV preparation obtained from the CCCM of HCT-15 cells by ultracentrifugation. (a) The schematic flow chart for density gradient ultracentrifugation. (b) The measured density for each iodixanol fraction. (c) Normalized particle concentration of each fraction. (d) Bivariate dot-plots of SYTO16 fluorescence versus SSC for EVs isolated from different iodixanol fractions before and after DNase I treatment. [/fig]
[fig] Figure S16: Immuno-gold labelling of histone H3 in EV isolates. (a) EVs were incubated with rabbit IgG and a 10 nm gold-conjugated goat anti-rabbit IgG (H+L). (b) EVs were incubated with rabbit anti-histone H3 antibody and a 10 nm gold-conjugated goat antirabbit IgG (H+L). [/fig]
[fig] Figure S17: HCT-15 cells were stained with Alexa Fluor 488 conjugated monoclonal antibodies (MAbs) specific to histone H3 and detected by BD FACSAria IIIu Cytometer. [/fig]
[fig] Figure S18: nFCM analysis of DNA on EVs isolated from normal cell lines (CCD-18Co and NP69) and cancer cell lines (HCT-15 and C666-1) without (a) or with (b) DNase I treatment prior to SYTO 16 staining. [/fig]
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Giant Keratocystic Odontogenic Tumor of the Mandible – A Case Report
Background:The keratocystic odontogenic tumor (KCOT) is a relatively rare, benign neoplasm which develops in the maxilla or mandible, arising from the dental lamina or basal cells of the oral epithelium. It is often found incidentally and brings about late symptoms as it does not cause bone distension for a long time.Case Report:The presented case is of a young woman with a giant keratocystic odontogenic tumor of the mandible.Conclusions:Despite its rare occurrence, it must be taken into consideration in radiological and clinical diagnostics. Due to the frequent recurrence of KCOT, patients are recommended to be kept under long-term and close radiological supervision.
# Background
The keratocystic odontogenic tumor is a relatively rare, benign neoplasm which develops in the jaw or mandible, arising from the dental lamina or basal cells of the oral epithelium. It was first described by Phillipsen in 1956 and was initially named the odontogenic keratocyst (OKC). In 2005, the World Health Organization (WHO) re-classified OKC and labelled it keratocystic odontogenic tumor (KCOT). The following features of the tumor contributed to that decision: i) clinically: local malignancy with high recurrence rate (up to 60% [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref] [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref] [bib_ref] Radiological and clinical features of peripheral keratocystic odontogenic tumor, Zhu [/bib_ref] [bib_ref] Keratocystic odontogenic tumor with mural calcification: a case report, Shetty [/bib_ref] , ii) histopathologically: pullulation of the stratum basale of the epithelium into the connective tissue and absence of mitotic figures in the stratum suprabasale, iii) genetically: occurrence of mutations of the suppressor gene PATCHED 1 [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref] [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref] [bib_ref] Radiological and clinical features of peripheral keratocystic odontogenic tumor, Zhu [/bib_ref] [bib_ref] Keratocystic odontogenic tumor with mural calcification: a case report, Shetty [/bib_ref] [bib_ref] A keratocyst in the buccal mucosa with the features of keratocystic odontogenic..., Yamamoto [/bib_ref] [bib_ref] Expansile keratocystic odontogenic tumor in the maxilla: immunohistochemical studies and review of..., Byun [/bib_ref] [bib_ref] PTCH1 gene mutations in keratocystic odontogenic tumors: a study of 43 Chinese..., Guo [/bib_ref] [bib_ref] Altered expression of podoplanin in keratocystic odontogenic tumours following decompression, Zhang [/bib_ref] [bib_ref] Pigmented keratocystic odontogenic tumor: a case report with review of the literature, Ishida [/bib_ref] [bib_ref] and PCNA expression in keratocystic odontogenic tumors compared with selected odontogenic cysts, Seyedmajidi [/bib_ref] [bib_ref] Cyclooxygenase-2 expression in keratcystic odontogenic tumour decreased following decompression, Wang [/bib_ref] [bib_ref] Keratocystic odontogenic tumor with an ectopic tooth in maxilla, Bhagawati [/bib_ref]. The tumor develops more often in the mandible than maxilla, mainly in the posterior part of its body and ramus area. It occurs less frequently as an extraosseous manifestation in the gingival area, and rare cases in the malar area have also been documented [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref] [bib_ref] Radiological and clinical features of peripheral keratocystic odontogenic tumor, Zhu [/bib_ref] [bib_ref] Keratocystic odontogenic tumor with mural calcification: a case report, Shetty [/bib_ref] [bib_ref] A keratocyst in the buccal mucosa with the features of keratocystic odontogenic..., Yamamoto [/bib_ref] [bib_ref] Expansile keratocystic odontogenic tumor in the maxilla: immunohistochemical studies and review of..., Byun [/bib_ref]. KCOT may be related to the unerupted third molar [bib_ref] Expansile keratocystic odontogenic tumor in the maxilla: immunohistochemical studies and review of..., Byun [/bib_ref]. Keratocystic odontogenic tumors constitute between 2% and 11% of all mandibular neoplasms [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref] [bib_ref] Keratocystic odontogenic tumor with mural calcification: a case report, Shetty [/bib_ref]. They are more frequent in males and have their peak incidence in the third decade of life [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref] [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref]. They may develop sporadically or accompany Gorlin syndrome (nevoid basal cell carcinoma syndrome, NBCCS) [bib_ref] PTCH1 gene mutations in keratocystic odontogenic tumors: a study of 43 Chinese..., Guo [/bib_ref].
KCOTs are often found incidentally in X-ray images, they are painless and cause tooth dislocation more often than resorption, tend to grow in the mesial-distal direction (where "mesial" means directed towards the anterior midline in a dental arch) rather than vestibular-lingual, and in the mandibular ramus areas, which delays the symptoms of bone distension.
## Case report
In March 2013, a 31-year-old female patient palpated a hard nodule in the area of the angle of her left mandible. She was pregnant at the time and decided that it was caused by changes taking place in her organism. In early December, after giving birth to her child, she went to her family doctor because the nodule grew in size instead of diminishing. The GP referred her to the emergency department of the 4 th Military Hospital in Wrocław, where she was X-rayed in both posteroanterior and lateral views. The description was: "Features of reconstruction of the bone structure in the mandibular body and ramus region on the left side with considerable distension, presence of numerous cystic centres of rarefaction of bone structure and septum" [fig_ref] Figure 1: AP and lateral X-ray of the mandible [/fig_ref]. The diagnostic process was extended to include computer tomography of the facial cranium. The examination revealed locally an extensive osteolytic decrease caused by the presence of a hypodense, polycyclic lesion of approximately 10×10×4 centimetres, which did not undergo substantial enhancement after administration of intravenous contrast medium. The tumor altered topographic relations of the left-side submandibular and neck areas, and its environs featured a developed network of blood vessels. Within the neck region no enlarged lymph glands were revealed [fig_ref] Figure 2: CT of the facial cranium with contrast medium [/fig_ref].
The patient was referred to the Oral and Maxillofacial Surgery Clinic, where orthopantomography and decompression of the lesion were performed, with the resulting material examined histopathologically [fig_ref] Figure 3: Orthopantomogram of the mandible [/fig_ref]. The results of the histopathological examination revealed small fragments of connective tissue covered in epithelium paraepidermoidale with chronic inflammatory lymphoid infiltration in the stroma, without malignant lesions. The patient was scheduled for surgery and before that operation she underwent several decompression procedures. The results of the specimen cultures were always negative.
On admittance to hospital for tumor surgery, a clinical examination revealed extraorally a facial asymmetry and a painless, hard, immobile bulge covering the area of the body of the mandible and left-side submandibular area, with the skin unchanged. Intraorally, a distension of the body of the mandible was observed, extending from the mandibular ramus to the incisor teeth, as well as an active retromolar fistula discharging pus. The neighbouring mucosa was tender, and mild pain accompanied chewing. Pus sample cultures grew Escherichia coli and Streptococcus oralis.
# Discussion
The term "giant" in keratocystic odontogenic tumors is reserved for large-sized lesions which result in significant asymmetry and local dysfunction, therefore the case described here may be classified as giant -the dimensions of the tumor were 10×4×4 centimetres. The lesion is more aggressive than other odontogenic cysts. In the histopathological image it is e.g. lined with keratinized epithelium [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref] [bib_ref] Pigmented keratocystic odontogenic tumor: a case report with review of the literature, Ishida [/bib_ref].
Immunohistochemical analyses revealed that the neoplasm had an increased expression of proteins responsible for cell proliferation, i.e. Ki67, COX-2 (cyclooxygenase 2), PCNA (proliferating cell nuclear antigen), p53, and p56 (epithelial stem cell enhancer factor), BCL2 (anti-apoptotic factor). Conversely, the expression of the BAC pro-apoptotic marker was decreased. The above-mentioned proteins may be used as biological markers for future KCOT diagnostics [bib_ref] Expansile keratocystic odontogenic tumor in the maxilla: immunohistochemical studies and review of..., Byun [/bib_ref] [bib_ref] and PCNA expression in keratocystic odontogenic tumors compared with selected odontogenic cysts, Seyedmajidi [/bib_ref] [bib_ref] Cyclooxygenase-2 expression in keratcystic odontogenic tumour decreased following decompression, Wang [/bib_ref] [bib_ref] Increased expression of autophagyrelated proteins in keratocystic odontogenic tumours: its possible association..., Li [/bib_ref]. Other studies reported on an increased production of glycoprotein podoplanin by KCOT. Zhang et al. proved that after decompression (i.e. lowering the tumorinduced pressure) patients had a statistically significantly decreased level of podoplanin [bib_ref] Altered expression of podoplanin in keratocystic odontogenic tumours following decompression, Zhang [/bib_ref].
KCOT is a rare tumor which may also accompany Gorlin's syndrome (nevoid basal cell carcinoma syndrome, NBCCS). The gene responsible for the development of NBCCS is PTCH1, which codes the glycoprotein in cell membranes -TM2. In their research, Guo et al. described a close connection of TM2 mutation with the incidence of sporadic KCOT [bib_ref] PTCH1 gene mutations in keratocystic odontogenic tumors: a study of 43 Chinese..., Guo [/bib_ref].
Some atypical manifestations of the keratocystic tumor are also worth mentioning here. It may occur in an extraosseous form as a peripheral keratocystic odontogenic tumor (PKCOT) -in gums, cheek tissue and the lateral facial deep region (LFDR) [bib_ref] Radiological and clinical features of peripheral keratocystic odontogenic tumor, Zhu [/bib_ref]. Several cases of the variety known as pigmented KCOT have been documented. It owes its name to the presence of melanocyte dendrite formations without atypical features within the squamous epithelium of the tumor. This kind of KCOT mainly appears in young females (average age 18) [bib_ref] Pigmented keratocystic odontogenic tumor: a case report with review of the literature, Ishida [/bib_ref]. Shetty et al. described a case of KCOT with mural calcification [bib_ref] Keratocystic odontogenic tumor with mural calcification: a case report, Shetty [/bib_ref] , whereas Patil et al. -the occurrence of "spider-like bodies" in KCOT's inflammatory cells [bib_ref] A keratocyst in the buccal mucosa with the features of keratocystic odontogenic..., Yamamoto [/bib_ref].
The tumor tends to be found incidentally because it shows no early symptoms and is painless. Its characteristic feature is growth in the mesial-distal rather than vestibularlingual direction, and upwards in the mandibular ramus areas, which delays the symptoms of bone distension. Tooth resorption is rare, tooth dislocation is more common. Large tumors invade soft tissue after damaging the compact external bone lamella. Keratocystic tumors are seldom large-sized -only one study in the quoted literature described three cases of giant (i.e. 7-10 centimetres) mandibular tumors [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref]. The dimensions of our patient's lesion were 10×4×4 centimetres.
In radiological examinations, a KCOT may be manifested in various ways. Initially, it is shown as an oval or round centre characterized by a unilocular radiolucency. The radiolucency may be polycyclic, less often multi-locular, with an osteosclerotic rim. A third molar may be found within the tumor area. CT images show hypodense lesions reflecting keratin mass in the tumor. MR examinations reveal a heterogeneous signal -medium in T1-weighted images, high in T2-weighted images, contrast enhancement revealing only a thin perimeter of the lesion. CT and/or MR examinations may reveal infiltration of the maxillary sinus, orbit, pterygopalatine fossa and infratemporal fossa.
KCOT's differential diagnosis includes: follicular cyst when an unerupted third molar is impacted in the tumor area; ameloblastoma, odontogenic myxoma and pericoronitis of the impacted third molar. Methods of treatment of KCOT include: decompression, marginal resection, en bloc resection and adjuvant therapy, i.e. cryotherapy, peripheral ostectomy and administering Carnoy's solution after surgery [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref] [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref]. Adjuvant therapies are used in order to reduce the relapse rate. KCOT's recurrence ratio is high, up to 60% [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref] [bib_ref] Giant keratocystic odontogenic tumor: three cases and literature review, Guimarães [/bib_ref] [bib_ref] Radiological and clinical features of peripheral keratocystic odontogenic tumor, Zhu [/bib_ref] [bib_ref] Keratocystic odontogenic tumor with mural calcification: a case report, Shetty [/bib_ref]. In their study, Campos, Telles et al. proved that marsupialisation before the final enucleation of the tumor causes its fibrous capsule to thicken and modifies its epithelial lining, thus greatly facilitating the enucleation [bib_ref] Morphometric evaluation of keratocystic odontogenic tumor before and after marsupialization, Telles [/bib_ref]. The size of our patient's tumor as well as the lack of unambiguous histopathological diagnosis before surgery influenced the choice of therapy -hemimandibulectomy with simultaneous reconstruction by means of an autologous graft from the ala of the ilium and a titanium implant were performed. The patient will now undergo regular control examinations, including radiological examination, in the Oral and Maxillofacial Surgery Clinic.
# Conclusions
Keratocystic odontogenic tumors (KCOTs) are rare tumors which most frequently develop in the mandible. They are often found incidentally. Untreated, they may lead to dysfunctions of the organ and significant asymmetry of the face. Despite their rare occurrence, they must be taken into consideration in radiological and clinical diagnostics. Due to scarcity of relevant Polish publications, we decided to present the issue. Due to the frequent recurrence of KCOT, also after radical surgery, patients are recommended to be kept under long-term and close radiological supervision.
[fig] Figure 1: AP and lateral X-ray of the mandible. Features of reconstruction of bone structure and ramus area on the left side with considerable distension, presence of numerous cystic centres of rarefaction of bone structure and septum. [/fig]
[fig] Figure 2: CT of the facial cranium with contrast medium. Extensive osteolytic decrease caused by the presence of a hypodense, polycyclic lesion of approximately 10×10×4 centimetres not undergoing a substantial enhancement on administering intravenous contrast medium.The patient underwent hemisection of the mandible with exarticulation of the mandibular condyle from the left temporomandibular joint, reconstruction of the left side of the mandible and left temporomandibular joint by means of autogenic bone grafting from the left ala of the ilium and a titanium implant(Figure 4).The left neck lymphatic glands were examined histopathologically, as well as the lower left alveolar nerve, half of the mandible with the mandibular condyle and tumor, part of the joint capsule of the temporomandibular joint, and the bone of the stump of the mandible. The diagnosis was keratocystic odontogenic tumor of the mandible. The postsurgical process went without complications. [/fig]
[fig] Figure 3: Orthopantomogram of the mandible. Features of reconstruction of bone structure and ramus area on the left side with considerable distension, presence of numerous cystic centres of rarefaction of bone structure and septum. [/fig]
[fig] Figure 4: Condition after hemiresection of the mandible with exarticulation of the mandibular condyle from the left temporomandibular joint, reconstruction of the left side of the mandible and left temporomandibular joint by means of autogenic bone grafting from the left ala of the ilium and a titanium implant. [/fig]
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Rapid subcutaneous progression after immunotherapy in pretreated patients with metastatic carcinoma: two case reports
There is heterogeneity in cancer patients' responses to immune checkpoint inhibitors (ICIs), including hyperprogression, which is very rapid tumor progression following immunotherapy, and pseudoprogression, which is an initial increase followed by a decrease in tumor burden or in the number of tumor lesions. This heterogeneity complicates clinical decisions because either premature withdrawal of the treatment or prolonged ineffective treatment harms patients. We presented two patients treated with ICIs with heterogeneous responses. One patient had Merkel cell carcinoma in the right thigh, and the other had nasopharyngeal squamous carcinoma. The first patient was treated with sintilimab and the second with sintilimab combined with abraxane. In the first patient, subcutaneous lesions grew substantially after the first cycle of treatment with sintilimab. In the second patient, subcutaneous lesions grew gradually after the second cycle of treatment with sintilimab combined with abraxane. In both cases, biopsy examination confirmed that newly emerged lesions were metastases of the primary tumor. These two cases remind clinicians that when subcutaneous nodules appear after treatment with ICIs, pathological biopsy is needed to determine the nature-pseudoprogression or rapid progression-of the disease course.
# Introduction
Immune checkpoint inhibitors (ICIs), an effective treatment strategy in multiple solid tumor types, have revolutionized cancer therapy. [bib_ref] Lessons learned from the blockade of immune checkpoints in cancer immunotherapy, Li [/bib_ref] During treatment with ICIs, some patients experience an initial increase in the size of tumor lesions or the appearance of new lesions that show necrosis or inflammatory cell infiltration on biopsy pathological examination and which subsequently subside. This unconventional clinical response is recognized as pseudoprogression and could be misclassified as disease progression. [bib_ref] Pseudoprogression and immune-related response in solid tumors, Chiou [/bib_ref] In addition to pseudoprogression, another response during checkpoint blockade, named tumor flare or hyperprogression (HPD), is characterized by dramatic progression that would outpace the expected rate of growth in the absence of ICIs, according to evidence primarily from prior imaging scans. [bib_ref] A case of non-small cell lung cancer with possible "disease flare" on..., Chubachi [/bib_ref] [bib_ref] Hyperprogressive disease is a new pattern of progression in cancer patients treated..., Champiat [/bib_ref] Patients with hyperprogression have a more deleterious time course than they might have had with other therapies, or even in the absence of therapy. 5 A study described by Champiat et al. reported that "hyper-progressive disease" was found in 9% of patients treated with ICIs. [bib_ref] Hyperprogressive disease is a new pattern of progression in cancer patients treated..., Champiat [/bib_ref] Several approaches have been used to define hyperprogression, namely tumor growth rate (TGR), tumor growth kinetics (TGK), and time to treatment failure (TTF). [bib_ref] Hyperprogression: a novel response pattern under immunotherapy, Han [/bib_ref] As immunotherapeutics, such as anti-programmed death (PD)-1/PD ligand (PD-L)1 agents, are widely available, clinicians face a great challenge in accurately evaluating hyperprogression during immunotherapy. [bib_ref] Definition, incidence, and challenges for assessment of hyperprogressive disease during cancer treatment..., Park [/bib_ref] Here, we presented two patients with metastatic carcinoma, one who developed rapid subcutaneous progression, and one who developed hyperprogression, after initiation of ICIs. This case report was written in accordance with the CARE guidelines. [bib_ref] The CARE guidelines: consensus-based clinical case reporting guideline development, Gagnier [/bib_ref] Written consent for treatment and publication was obtained from both patients. Ethics approval was obtained from the Review Board of Beijing Fengtai You'anmen Hospital.
## Case presentation
## Rapid subcutaneous progression after receiving an ici
A 73-year-old man, a smoker, was diagnosed with Merkel cell carcinoma of the right thigh in February 2019. He was treated with etoposide and cis-platinum until May 2019. Computed tomography (CT) demonstrated a good response in the primary lesion. However, new metastases developed in the lymph nodes in the abdomen and bilateral inguinal regions, and radiotherapy was performed in July 2019. One month later, the disease progressed, with a dramatic increase in the neuronspecific enolase (NSE) level from 13.63 mg/ mL (at diagnosis) to 224.0 mg/mL. The NSE level is a valuable biomarker in Merkel cell carcinoma because the level can distinguish responders from non-responders during immunotherapy. [bib_ref] The clinical utility of neuron-specific enolase serum levels as a biomarker for..., Van Veenendaal [/bib_ref] A cycle of irinotecan and cis-platinum was administrated after inducing grade 4 bone marrow suppression. This cycle was followed by sintilimab at a dose of 200 mg q 21 days, in September 2019. Two days after the first cycle of sintilimab, new subcutaneous lesions appeared in the patient's right leg [fig_ref] Figure 1: Hyperprogression after immunotherapy in a patient with Merkel cell carcinoma [/fig_ref]. Three weeks after the first cycle, the NSE level decreased to 203.9 mg/mL, and the subcutaneous lesions in the right leg grew substantially [fig_ref] Figure 1: Hyperprogression after immunotherapy in a patient with Merkel cell carcinoma [/fig_ref]. Another cycle of sintilimab was administrated in October 2019, and the NSE decreased to 111.9 mg/mL 10 days later. The lymph nodes tumors in the abdomen and bilateral inguinal regions responded to the first cycle of sintilimab, decreasing from 12 cm  2 cm to 6 cm  1 cm in size (abdomen and right inguinal region) and from 2 cm  1 cm to 1 cm  1 cm (left inguinal region). However, the tumors in these regions progressed 1 month after the second cycle of sintilimab. Immunohistology (IHC) revealed the following: CAM5.2 (þ), cluster of differentiation (CD)20 (À), CD21 (À), CD3 (À), cytokeratin (CK) (þ), CK18 (þ), human melanoma black (HMB)-45 (À), Ki-67 (95%), Melan-A (À), S-100 (À), CD56 (þ), chromogranin A (CgA) (þ), synaptophysin (Syn) (þ), CD99 (À), and thyroid transcription factor (TTF)-1 (À). Pathological analysis of biopsy samples revealed that the subcutaneous nodules in the patient's right leg were not caused by pseudoprogression but were actually metastatic lesions from the primary tumor. Palliative treatment was provided after these findings. The patient died in November 2019 due to bacterial aspiration pneumonia.
## Subcutaneous progression after receiving an ici
A 51-year-old man with no family history of cancer was diagnosed with nasopharyngeal squamous carcinoma in January 2014. He received radiotherapy (70 Gy/35 fractions) until May 2014, and magnetic resonance imaging (MRI) demonstrated a good response in the primary lesion. However, in December 2018, the patient developed continuous pain in the oropharynx. MRI demonstrated a large enhancing mass in the oropharynx and laryngopharynx. He received four cycles of nimotuzumab combined with paclitaxel and cis-platinum until April 2019. The efficacy evaluation of this treatment was stable disease. Four months later, the disease progressed again. Targeted sequencing was performed and revealed a blood tumor mutational burden (TMB) of 17.75 mut/Mb. Two cycles of abraxane combined with sintilimab were administrated, and subcutaneous nodules subsequently appeared [fig_ref] Figure 2: Hyperprogression after immunotherapy in a patient with nasopharyngeal squamous carcinoma [/fig_ref]. The primary tumor responded to abraxane and sintilimab (Supplementary [fig_ref] Figure 1: Hyperprogression after immunotherapy in a patient with Merkel cell carcinoma [/fig_ref]. IHC of the biopsy tissue from the new subcutaneous nodules revealed the following: CK (þ), CK5 (þ), P40 (þ), Ki67 (80%), desmin (Des) (À), myogenic determination gene (MyoD1) (À), myogenin (À), S-100 (À), Melan-A (À), and CD68 (À), indicating that the new subcutaneous nodules were metastases from the primary lesion. Palliative treatment was provided thereafter, and unfortunately, the patient died in January 2020 owing to exhaustion.
# Discussion
ICIs, such as monoclonal antibodies targeting cytotoxic T-lymphocyte-associated antigen-4 and PD-1, are profoundly changing cancer patient management. To date, two PD-1 inhibitors (nivolumab and pembrolizumab) and three PD-L1 inhibitors (atezolizumab, avelumab, and durvalumab) have been approved by the US Food and Drug Administration for various indications. [bib_ref] Safety and activity of pembrolizumab in patients with locally advanced or metastatic..., Plimack [/bib_ref] [bib_ref] Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic..., Seiwert [/bib_ref] [bib_ref] Safety and efficacy of durvalumab (MEDI4736), an anti-programmed cell death ligand-1 immune..., Massard [/bib_ref] [bib_ref] Safety and efficacy of nivolumab and standard chemotherapy drug combination in patients..., Kanda [/bib_ref] Sintilimab is another anti-PD-1 inhibitor that was approved by National Medical Products Administration (NMPA) for treating lymphoma and lung cancer and which has also been actively tested in other types of cancers. [bib_ref] Another brick in the wall: sintilimab plus chemotherapy in advanced lung cancer, Lam [/bib_ref] [bib_ref] Recent updates on sintilimab in solid tumor immunotherapy, Liu [/bib_ref] Responses to ICIs appear to be heterogeneous. Pseudoprogression and hyperprogression present as similar initial responses to ICIs, yet they have different prognoses. However, the underlying mechanisms for the heterogeneity in response to ICIs remain largely unknown. Sarfaty et al. reported a case of subcutaneous pseudoprogression in lung squamous cell carcinoma that was treated with nivolumab previously. [bib_ref] Not only for melanoma. Subcutaneous pseudoprogression in lung squamous-cell carcinoma treated with..., Sarfaty [/bib_ref] The current report presented two advanced cancer cases with rapid progression after ICIs, emphasizing the importance of timely pathological biopsy to determine the nature of newly-developed masses and appropriate therapeutic strategies. Another concern we raised relates to the current definition of HPD. HPD is defined as tumors with a !two-fold increase in the experimental TGR compared with the reference TGR. Various HPD definitions have been proposed in addition to TGR, [bib_ref] Tumour growth rates and RECIST criteria in early drug development, Gomez-Roca [/bib_ref] namely TGK ratio, [bib_ref] Tumour growth kinetics assessment: added value to RECIST in cancer patients treated..., Tourneau [/bib_ref] early tumor burden increase and time to treatment failure, [bib_ref] Hyperprogressors after immunotherapy: analysis of genomic alterations associated with accelerated growth rate, Kato [/bib_ref] and increased ratio of measurable lesions. [bib_ref] Incidence and clinical implications of a new definition of hyperprogression (HPD) with..., Matos [/bib_ref] However, the implementation of these assessments is challenging, clinically, as the calculation of TGR and TGK ratio requires intensive measurements and multiple time point calculations. 7,21 A consensus statement and easy-to-use HPD criteria would greatly benefit cancer patients with tumor growth acceleration during immunotherapy. In conclusion, our case report shows the importance of pathological biopsy and standardization of HPD criteria for patients receiving immunotherapy.
## Declaration of conflicting interest
Ruoying Yu is an employee of Nanjing Geneseeq Technology Inc. The remaining authors declare that there is no conflict of interest.
# Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
## Orcid id
Ruoying Yu https://orcid.org/0000-0003-1560-239X
# Supplemental material
Supplemental material for this article is available online.
[fig] Figure 1: Hyperprogression after immunotherapy in a patient with Merkel cell carcinoma (a) Hyperprogression indicated by subcutaneous metastatic lesions. (b) IHC (Â20) staining of the subcutaneous metastatic lesions showed CD56 and SYN positivity. Normal tissue from the same patient was obtained and used as a negative control for IHC staining. IHC, immunohistochemistry; CD, cluster of differentiation; SYN, synaptophysin; HE, hematoxylin and eosin. [/fig]
[fig] Figure 2: Hyperprogression after immunotherapy in a patient with nasopharyngeal squamous carcinoma (a) Hyperprogression indicated by subcutaneous metastatic lesions. (b) IHC (Â20) staining of the subcutaneous metastatic lesions showed P40 positivity. Normal tissue from the same patient was obtained and used as a negative control for IHC staining. IHC, immunohistochemistry; HE, hematoxylin and eosin. [/fig]
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Imaging of brain glucose uptake by PET in obesity and cognitive dysfunction: life-course perspective
The prevalence of obesity has reached epidemic proportions and keeps growing. Obesity seems implicated in the pathogenesis of cognitive dysfunction, Alzheimer's disease and dementia, and vice versa. Growing scientific efforts are being devoted to the identification of central mechanisms underlying the frequent association between obesity and cognitive dysfunction. Glucose brain handling undergoes dynamic changes during the life-course, suggesting that its alterations might precede and contribute to degenerative changes or signaling abnormalities. Imaging of the glucose analog 18 F-labeled fluorodeoxyglucose ( 18 FDG) by positron emission tomography (PET) is the gold-standard for the assessment of cerebral glucose metabolism in vivo. This review summarizes the current literature addressing brain glucose uptake measured by PET imaging, and the effect of insulin on brain metabolism, trying to embrace a life-course vision in the identification of patterns that may explain (and contribute to) the frequent association between obesity and cognitive dysfunction. The current evidence supports that brain hypermetabolism and brain insulin resistance occur in selected high-risk conditions as a transient phenomenon, eventually evolving toward normal or low values during life or disease progression. Associative studies suggest that brain hypermetabolism predicts low BDNF levels, hepatic and whole body insulin resistance, food desire and an unfavorable balance between anticipated reward from food and cognitive inhibitory control. Emerging mechanistic links involve the microbiota and the metabolome, which correlate with brain metabolism and cognition, deserving attention as potential future prevention targets.
# Introduction
The prevalence of obesity has reached epidemic proportions and is growing in most world regions. Obesity is a risk factor for a series of chronic morbidities, the most frequent being type 2 diabetes. According to the World Health Organization (WHO), overweight and obesity have been estimated to account for about 65-80% of new cases of type 2 diabetes (http ://ww w.eur o.who .int/ en/he alth-topic s/non commu nicab le-di sease s/dia betes /data -and-stati stics ). Obesity and type 2 diabetes have been implicated in the pathogenesis of cognitive these same countries. Nearly 60% of Europeans and North Americans are affected, and China hosts 20% of the world overweight population (http ://ww w.eur o.who .int/ en/ he alth-topic s/non commu nicab le-di sease s/obe sity/ dataand-s tatis tics) . These concepts are summarized in.
The risk of obesity is settled, at least in part, during the earliest phases of life, as the brain undergoes its major development, involving a five-fold volumetric spurt in the first 5 years of life. Several early-life factors may affect the propensity of infants to become obese children, adolescents, and adults. These include maternal exposures (obesity, diabetes, stress), mode of delivery (e.g. C-section), early nutrition. Interestingly, these early risk factors have been also associated with a greater risk of premature cognitive decline, from early life to elderly age.
Growing scientific efforts are being devoted to the identification of central mechanisms underlying the mutual reinforcement exerted by obesity on cognitive dysfunction and vice versa. Both have been associated with morphological reductions in brain volume, relating to systemic dysmetabolism and insulin resistance and/or to the degree of cognitive impairment. Glucose is the most important brain fuel and central signaling factor. Glucose brain handling undergoes dynamic changes during the life-course, suggesting that its alterations might precede and contribute to degenerative changes or signaling abnormalities. Imaging of the glucose analogF-labeled fluorodeoxyglucose ( 18 FDG) by positron emission tomography (PET) is the gold standard for the assessment of cerebral glucose metabolism in vivo. In this manuscript, we review evidence on brain glucose uptake measured by PET imaging, trying to embrace a life-course vision in the identification of patterns that may explain (and contribute to) the frequent association between obesity and cognitive dysfunction.
## Brain glucose metabolism and pet imaging
The brain relies primarily on glucose as its main energy source. Given its elevated utilization of circulating glucose, the brain may contribute to regulate peripheral glucose levels in a direct manner, by subtracting glucose from blood. Beyond nourishing the tissue, glucose is a signaling factor, informing central regulatory circuits on the metabolic and feeding conditions of the body. In the brain, glucose stimulates the production of The three circles in panel A show the current prevalence of overweight (left) or dementia (middle) in Europe, and (on the right) the estimated aging of the European population in 2050, further promoting these conditions. Panel B (from data presented by Laurie Brown at the National Dementia Congress, Melbourne 2014 anddepicts the prevalence of dementia by BMI status, across age categories. rewarding neurotransmitters, thereby contributing to appetite control and more in general to mental well-being. Brain sensing translates in the regulation of metabolically relevant organs and processes, including insulin secretion by pancreatic beta-cells, hepatic glucose productionand fatty acid release by adipose tissue lipolysis. This partly occurs via afferent and efferent nerves. In turn, insulin can cross the blood-brain barrier to tune central control, and fatty acids can reach the brain, representing its main structural constituent. The characterization of brain glucose uptake in groups of individuals of progressive ages has shown important changes occurring throughout the lifespan. Supporting the five-fold expansion in gray matter observed in the first 5 years of life (6), brain glucose uptake undergoes a >fourfold increase from birth to pre-scholar and early-scholar periods. This is followed by a relatively rapid decline, already from the age of 7-8 years and throughout the teenage period, reducing brain glucose uptake by 20-30% of its maximum, as opposed to only 10% gray matter losses. A brain volume reduction of 30% is achieved in late adulthood, a time in which glucose uptake has fallen by 60% from its maximum. These time trends suggest that functional, namely metabolic changes are larger and detectable in advance of their morphological counterpart. It is therefore not surprising that FDG-PET imaging provides the earliest biomarker predicting neurodegenerative disease, as compared to other imaging (amyloid PET, magnetic resonance) or clinical modalities (signs and symptoms, cerebrospinal fluid markers). Compared to alternative methods to assess brain glucose metabolism in vivo, including arterial-venous catheterization or micro-dialysis, PET imaging is minimally invasive, and provides regional information, best reflecting the functional diversification between highly specialized brain areas. Once injected in a study subject,FDG undergoes extraction by the brain and body tissues in proportion to their requirements. Graphicaland compartmental modelshave been widely used to translate tissue and blood concentrations into biologically meaningful rate constant values, describing the transfer ofFDG from blood to brain cells and its subsequent phosphorylation. The fractional tissue extraction rate constant describes the relative amount (or percentage) ofFDG that is extracted from the circulation in a unit of time and tissue volume (or mass). In order to convert this factor into an absolute rate of glucose uptake, one has to account for the organ-specific lumped constant and the level of circulating glucose. The former corrects for different affinities of transporters and enzymes (hexokinases) for glucose compared toFDG, whereas the latter translates fractional (%) into absolute rates of glucose influx into the target tissue (e.g. μmol/min/g). Hence, the actual amount of glucose entering the tissue is given by the product of fractional extraction rate constant of 18 FDG and plasma glucose level, divided by the lumped constant term. Though the term glucose uptake may have been used interchangeably in the literature to define either absolute (glucose) or relative ( 18 FDG) tissue influxes, there is an important conceptual and numerical difference between these two processes. In neurodegenerative diseases, the extraction is frequently reported, with fewer studies quantifying absolute brain glucose uptake rates. The opposite is true in obesity studies. In this review, we primarily refer to the absolute value.
## Homeostatic regulation of brain glucose metabolism in obesity
In humans, brain glucose metabolism has been studied in the fasting state or during euglycemic insulin stimulation. In adults, data from PET imaging studies indicate that the brain accounts for at least 50% of whole-body glucose disposal during the fasting state and 10-20% during euglycemic insulin stimulation, supporting an important direct role of this organ in affecting glycemia. Studies have compared obese and lean individuals, as well as patients with glucose intolerance and normo-tolerant BMI-matched controls. The evidence from these studies indicates that fasting insulin levels may serve to maintain a physiological tone of brain glucose uptake, since the inhibition of insulin secretion suppresses glucose uptake values in healthy individuals, whereas the elevation of insulinemia toward postprandial levels does not further affect cerebral glucose uptake compared to fasting values in non-obese healthy subjects.
Compared to lean, obese subjects showed higher fasting metabolism in the parietal somatosensory cortex regions where sensation of the mouth, lips and tongue are located, and in regions regulating executive function. Other studies could not find differences between fasted lean and obese individuals, but observed elevated uptake of glucose in most brain regions during insulin stimulation in glucose-intolerant or morbidly obese patients. In the latter, the elevation in brain glucose uptake was associated with greater endogenous glucose release (indicative of hepatic insulin resistance), and lower peripheral glucose consumption (primarily reflecting skeletal muscle insulin resistance), suggesting that brain hypermetabolism is the expression of central insulin resistance. In the same study, high brain glucose uptake was also shown to negatively predict the improvement in glucose levels occurring after weight loss in morbidly obese patients, thus contrasting one of the most relevant clinical benefits of bariatric surgery, that is, the recovery from type 2 diabetes. Consistent with the above evidence, the injection of glucose in cerebral ventricles caused a suppression of hepatic glucose production in healthy mice, but a 4-week infusion failed to provoke any effect in type 2 diabetic rats. Controlled studies in pigs replicate the human findings, by showing no brain glucometabolic response to euglycemic hyperinsulinemia (compared to fasting state) in control pigs, as opposed to an elevated response in pigs fed a high-fat diet. Studies in Zucker fatty or diabetic rats have shown that their brain glucose uptake is chronically elevated under fasting and glucose loading conditions, lacking the excursion that normally signals the transition from a fasted to fed state. We also reported that young pre-obese Zucker fatty rats, showing normal body weight but impaired glucose tolerance, already manifest brain hypermetabolism during a glucose tolerance test. Overall, the available evidence in human and animal studies, as exemplified in , suggests that a chronic overexposure of the brain to glucose may interrupt relevant feedback loops.
## Hedonic regulation of brain glucose metabolism in obesity
Glucose can stimulate the production of rewardpromoting neurotransmitters in the brain. Few studies have used FDG PET imaging to address the involvement of brain glucose uptake in the hedonic regulation of appetite and body weight, which is an important underlying element in the pathogenesis of obesity. One early study suggested that an enhanced metabolic activity in regions involved with sensory processing of food in obese subjects could make them more sensitive to the rewarding properties of preferred foods, contributing to excessive food consumption. Upon sensory (visual, taste, olfactory) stimulation with palatable food, all brain regions experience a rise in glucose uptake, compared to neutral sensing. Gender differences were noted in this response, since women showed greater metabolic activation than men
[formula] A C D B E Figure 2 [/formula]
The figure summarizes the patterns of brain glucose metabolism described in this review. Panel A shows that the development of obesity in a genetic rodent model (Zucker rat) is characterized by brain hypermetabolism both in fasting condition (dashed lines) and during oral glucose tolerance test (solid line). Panel B illustrates the effect of exposure to maternal obesity, resulting in a hypermetabolic brain response to isoglycemic insulin stimulation (solid line) in very early life, and mild brain hypermetabolism in fasting conditions (dashed lines). Panel C shows the progressive increase of brain glucose uptake in response to food presentation in inhibitory control regions (open circles) and in reward related regions (closed circles) from normal weight women to women with obesity without and with food addictionin adult age. In panel D, a progressive increment in fasting brain glucose uptake from normal weight mice with Alzheimer's disease (AD, blue line), to normal weight mice without AD (green line), obese mice with AD (purple line) and obese mice without AD (red line) is shown. Based on the above observations, panel E provides a simulation of how cognitive disease, with or without obesity, may modify (black lines) the physiological time-course (green line, (24) of brain metabolism over life.
in several brain regions, and minimal response to an inhibitory control cognitive task against their preferred food stimulation, as opposed to men in whom the cognitive task diffusely suppressed brain metabolism (right insula, right striatum, amygdala, hypothalamus, anterior cingulate, hippocampus, parahippocampal gyrus, orbitofrontal cortex, cerebellum) and hunger ratings. Unfortunately, the study did not involve obese patients.
We have recently hypothesized that the magnitude and regional distribution of hedonic food responses might differ between similarly obese individuals, opening the opportunity for mechanistic based stratification, and personalized management of obese patients. In order to prove this concept, we studied two groups of obese women, who had similar metabolic profile and BMI, but different severity of food addiction symptoms, as based on the DMS Yale Food Addiction Scale. Compared to women with few symptoms, the more severely affected group showed greater brain metabolic activation in response to palatable versus neutral food cues, especially in rewardrelated regions, and less in inhibitory control regions. A lower response in inhibitory regions was predictive of a greater number of symptoms and hunger, which recalls the negative association described by others between BMI and prefrontal metabolism. Brain glucose responses and hunger ratings were reduced after modest dietinduced weight loss in the group with a food addicted profile, whereas no change was observed in control women. These proof-of-principle results strengthen the evidence of different obesity types, showing distinct metabolic brain reactions to food cues and to inhibitory control. Notably, the observation that the normalization of brain (hyper)metabolism during food sensing occurred together with a decreased perception of hunger was common to the acute cognitive inhibitory task in menor the more prolonged diet-control effort in women. Animal studies exploring brain glucose uptake during hedonic (olfactory) stimulation with palatable food (bacon) show that caloric restriction, food desire and (Zucker) genotype influence central glucose metabolic responses via a complex interplay, especially in the hippocampus (a brain region that is increasingly involved in food behaviors) and superior colliculus (a brain region that modulates the saliency value of food reinforcers) (66). exemplifies the above observations, collectively suggesting that (a) brain metabolism relates to perceived hunger but does not respond similarly to palatable foods and food restriction in all individuals; (b) gender, obesity, ability to cognitively control, food addiction symptoms (shown in humans), and obesity-prone genotype versus food accessibility (reported in rodents) contribute to differentiate brain glucose responses; (c) more studies are definitely needed to understand to what extent the amount of glucose entering brain regions involved in homeostatic, hedonic, and cognitive control can modulate feeding behavior; (d) better understanding and account of the above interactions is likely to lead to tailored weight control interventions.
## Brain metabolism as common feature in obesity and neurodegenerative disease
In the context of obesity research, the literature summarized above indicates that high cerebral metabolic rates occur in obese and glucose-intolerant subjects during fasted or homeostatic stimuli (i.e. hyperinsulinemia during eu-or hyperglycemia). Brain glucose uptake is normally increased in response to hedonic stimuli in lean individuals, especially in regions related with food sensing and reward. This effect is particularly pronounced in obese food addicted women in the whole brain, though more markedly in regions related with food sensing and reward, and in the hypothalamus. The current evidence also supports the concept that insufficient metabolic hyperactivation in orbitofrontal inhibitory regions may predict greater food dependency, possibly because the degree of hypermetabolism observed in sensing and reward-related regions is not sufficiently balanced by areas of executive function. The observation that an inhibitory cognitive task reduces brain glucose uptake in all regions, resulting in lower hunger ratings, further supports the existence of a metabolic network in which cognition prevails (metabolically) over anticipated reward. Interestingly, one study showing that BMI and cognition were inversely related (65) highlighted that low frontal glucose metabolism was associated with both BMI (negative) and cognitive function (positive), involving domains beyond inhibitory glucose metabolism, for example memory and recall, executive function, verbal and non-verbal intelligence quotients. So far, we are left with the dual hypothesis that obesity may independently affect frontal metabolism and cognition or that dysfunctional prefrontal metabolism due to for example cognitive disease may fail to inhibit overeating, contributing to obesity. Animal studies support both possibilities. On one side, lesions of the hippocampus lead to overeating, and mouse models of Alzheimer's disease (AD) type pathology show overeating compared to controls. On the other hand, high-fat feeding in these genetically predisposed animals accelerates neurodegeneration and cognitive decline. Metabolically, a high-fat diet elevated brain glucose uptake in middleaged mice, but this elevation was blunted in the AD type model and followed by a remarkable metabolic and cognitive fall throughout aging. Exposure to a high-fat diet or maternal obesity during early life development predicts premature cognitive decline and obesity along the lifespan in humans. In a minipig model, such exposure led to a marked elevation in brain glucose uptake in the initial post-natal period, due to hyperglycemia, followed by a rapid decline resulting in brain hypometabolism in later life stages. Accordingly, brain glycogen levels were depleted in adult offspring born to high-fat diet mothers.
The above studies suggest that, as we attempt to establish cerebral glucose metabolism as predictor or hallmark of disease, it is fundamental to consider that brain glucose uptake may vary depending on brain regions, disease type, staging, duration and severity. This is supported by for example mouse models of AD that are characterized by different temporal patterns of disease development, as brain metabolism was found to be deficient in animals showing advanced neurodegeneration and cognitive decline (71) already at 7 months of age, but not in 3xTg mice showing only mild signs of dysfunction and structural loss at 8 months of age. Along these spectra, brain hypermetabolism can appear as an early trait, occurring during early-or mid-life periods and vanishing along aging or advanced disease or after prolonged high-fat diet exposure. Reinforcing this conclusion, a study in patients with different degrees of cognitive impairment has shown that cerebral glucose metabolism was high in subjects with mild disease, and low in advanced disease compared to healthy controls. The authors speculate that brain hypermetabolism may serve as transient compensatory reaction to the initial neurodegenerative insult, but is progressively replaced by hypometabolism, as tissue loss becomes more severe in the chronic situation. The authors surmise that in spite of being compensatory, the initial glucose excess may overstimulate and exhaust neural networks, accelerating the degenerative process. The same compensatory theory was suggested to explain the preservation of cognitive function in a study in morbidly obese women, showing brain hypermetabolism. Again these authors refer to the initial phase of neurodegeneration, in which inhibitory synapses are first destroyed, and excitatory synapses prevail with increased local activity, which may justify brain hypermetabolism in obese patients. illustrates the above concepts, providing a hypothetical time-course of brain glucose uptake along the progression of life, in relation to cognitive disease with and without obesity.
As potential mechanisms whereby an excessive uptake of glucose by the brain can result in a simultaneous dysregulation of appetite, systemic metabolism and cognitive function, brain-derived neurotrophic factor (BDNF) stems as a credible candidate. BDNF has been implicated in the pathogenesis of obesity, type 2 diabetes, and neurodegeneration. BDNF deficiency is a recognized correlate of memory impairment and has been described in patients with obesity and type 2 diabetes. The administration of BDNF in animal models was shown to improve the control of food intake and weight loss, peripheral glucose homeostasis, neuropreservation and cognition. Few studies have addressed the relationship linking BDNF production and brain glucose uptake. Elegant human experiments, using arterial-venous catheterization across the brain, have documented that BDNF release by the human brain is suppressed by brain glucose overexposure, that is, high blood glucose levels during a hyperglycemic clamp. In Zucker fatty rats, we have shown that brain glucose uptake, as measured by FDG PET imaging correlates inversely with circulating levels of BDNF. The relationship was stronger in young, pre-obese (but already glucose intolerant) animals, and we confirmed the dependency of BDNF on glucose levels already in human fetal cord plasma. Since the latter were in turn dependent on maternal glycemia at the time of delivery, we suggested that the establishment of optimal glycemic conditions at the time of birth might be a unique opportunity to protect cognitive and metabolic health.
## Emerging mechanistic hypotheses leading to preventive perspectives
Animal studies have shown that some of the actions of glucose in the brain occur only in the presence of concurrent insulin delivery. Secreted insulin can reach the brain and its receptors are present in most brain regions, as reviewed by Blazquez et al. and Ghasemi et al.. Insulin is a growth and metabolic regulator, whose cerebral actions have been implicated in the pathogenesis of obesity (suppression of appetite, regulation of adipose tissue lipolysis and lipogenesis, type 2 diabetes (modulation of endogenous glucose production and glucose levels, and of pancreatic insulin secretion, and Alzheimer's disease. Most of these actions are lost in experimental models of obesity induced by high fat feeding. The role of insulin on brain glucose metabolism remains to be fully established. As detailed in the previous sections, compared to fasting, the induction of euglycemic hyperinsulinemia does not provoke any change in brain glucose uptake in healthy individuals, whereas it elicits a positive response in obese or glucose-intolerant patients. However, it is of note that the euglycemic-hyperinsulinemic clamp technique does not allow to dissect the direct role of insulin on the brain from the many peripheral actions occurring concomitantly. It is also important to recognize that insulin clamp studies prevent the reduction of glucose levels, which is the most important insulin effect in physiological conditions. Intranasal insulin injections have been recently used as a way to dissect central insulin roles, with translational perspectives in humans. Animal studies have shown that when insulin is delivered to the brain via the intranasal route, brain glucose uptake is reduced, mainly due to a centrally mediated hypoglycemic effect of the hormone. According to this, brain insulin resistance can be defined as failure of insulin to suppress brain glucose uptake, which is coherent with the finding of high glucose uptake under human euglycemic insulin clamp conditions. Comparing healthy lean mice of different ages, we observed that the central hypometabolic action of insulin was significant in adult and old mice, but was not present in early post-natal life. It is plausible that brain glucose suppressing signals are not yet operative in the first period of life, to ensure sufficient energy provisions during this demanding phase of rapid brain growth. Interestingly, mice born to high fat fed dams showed brain hypersensitivity to insulin in this early period and brain insulin resistance during (mid-life) adulthood, and minipigs born to high fat fed sows showed greater brain-specific insulin receptor density few days post-natally, followed by deficiency in insulin receptors and insulin-dependent glucose transporters (GLUT4) in the cortex and hypothalamus. Also the combination of high fat dieting and AD type pathology in mice is characterized by an absent response of brain glucose metabolism to intranasal insulin. These mice showed similar brain hypermetabolism during fasting and during insulin stimulation. Again this trait was clearly detectable during mid-life, but not after aging. In these mice, the chronic administration of intranasal insulin, starting in early adulthood resulted in a normalized mid-life brain metabolism and insulin response, together with a full preservation of cognitive function and hippocampus size throughout the lifespan. Insulin therapy also reduced food intake, body weight and peripheral glucose levels. Brain PET imaging studies with intranasal insulin in obese humans are lacking. In non-obese patients with clinically confirmed symptomatic cognitive disease, the chronic administration of intranasal insulin improved memory and slowed the decline in brain metabolism linked to tissue degeneration. Subsequent observations by the same authors indicate that the insulin formulation may importantly interfere with the outcome, as the effects of rapid insulin on cognition could be reproduced, whereas long-acting insulin was less effective and not consistent; unfortunately, PET imaging was not carried out in these comparative studies. In the field of obesity, acute intranasal insulin studies addressing functional responses by magnetic resonance imaging, systemic insulin sensitivity or hunger ratingssupport the suppressive effects of intranasal insulin on peripheral glucose levels, hypothalamic activity, and appetite control in humans. These metabolic effects await for chronic intervention trials to be tested for longer term efficacy and safety in obese patients.
Omics technologies represent another field of intensive investigation due to their potential to identify and/or confirm early biomarkers and mechanistic links related to obesity and neurodegenerative disease. Among them, the study of the microbiome emerges as a promising candidate for treatment, since pioneer trials with probiotics or fecal transplant have shown some benefit in dysmetabolic and Alzheimer's patients, and probiotics have been safely administered to pregnant women or infants, opening an opportunity of primary prevention. Many studies have shown differences in microbiota composition between lean and obese individuals. Seminal studies have produced first evidence in human Alzheimer's pathology. In one animal study in which brain glucose metabolism was measured, we have shown that microbiota composition differs in mice fed a high fat diet and in mice with genetically determined AD type pathology in the early course of the disease, leading to additive effects when the two conditions are combined. We have also documented that fecal and serum metabolomes were different between groups and that numerous metabolites correlated with relative bacteria abundances. Some of these bacteria were associated with cognitive dysfunction and brain metabolism. Emerging bacteria patterns include depletion of Bifidobacterium, and overabundance Turicibacteraceae, Christensenellaceae, Anaeroplasmataceae and Ruminococcaceae. Emerging metabolites were amino acids (leucine, isoleucine, glycine), ketone bodies, lactate, TMA-TMAO, and inflammatory indicators, all present in greater concentrations in diseased cases, together with a reduction in fatty acids. It is of translational interest that several of these metabolites were recently reported to be elevated in morbidly obese humans in association with an elevation in brain glucose metabolism.
Bariatric surgery represents a model of treatment in which all the elements above improve in combination. It can therefore substantiate their involvement, though it does not dissect and provide in itself a causeeffect demonstration for each factor. Interestingly, bariatric surgery leads to an amelioration in cognitive function, appetite regulation and weight loss. Brain glucose hypermetabolism and the related circulating metabolites are normalized after the intervention. Transplantation of human microbiota following bariatric surgery into mice suggests that bacterial changes induced by the gastrointestinal operation are partly responsible for the effects of surgery on circulating metabolites. Though the gut-brain axis is focus of intensive investigation, and the microbiota can potentially influence brain development, structure and function by several mechanisms, a direct effect of bacteria or their metabolic products on brain glucose metabolism has not been yet clarified in the published literature.
## Technical considerations
There are a number of technical differences between PET imaging studies, involving image acquisition and processing that might influence the interpretation of results in the reports quoted in this review; the available information is summarized in. First, dynamic scans allow to follow the progressive concentration ofFDG radioactivity in the brain over time (time-activity curve) starting from tracer injection, whereas static images provide a single-activity value, usually delayed from injection to avoid the initial blood flow-dependent phase, and primarily reflect the accumulation of 18 FDG that is induced by metabolic needs. Dynamic images allow sophisticated mathematical for example graphical and compartmental modeling (based on the changing levels of tissue to blood activities over time, to generate one or multiple rate constants) compared to simpler approaches and parameters that can be reliably obtained by static images (e.g. the ratio of tissue to injected dose per unit of body weight, or -if an input function is available -the ratio of tissue to integrated blood activity). In these ways, dynamic and static scanning modes provide different but tightly correlated absolute values of brain glucose uptake and are both widely accepted and valid. However, when complex modeling (suited to dynamic imaging) is forcefully applied to a static image in the attempt to go beyond the simple parameters it can provide, several assumptions are required, including the use of a set of predefined and fixed rate constants, introducing some degree of arbitrariness in the results. Interestingly, this method led to low absolute frontal glucose uptake values in obese individuals, as opposed to either normal or tendentially elevated values in conservative approaches. Second, the input function (i.e. the tracer concentration that is available in blood for tissue extraction, as used in the above computations) should ideally be obtained by arterial blood sampling. To circumvent this invasive procedure, several groups have replaced arterial with arterialized (by heating pad or box) venous blood, which is accepted and common practice in metabolic (non-imaging) insulin clamp studies since their origin. In theory, oxygen saturation should be measured to confirm proper arterialization of venous blood; de facto, this measurement is rarely pursued. In few studies in, an image-derived input function (from cardiac cavities or carotid images) has been used, to avoid any frequent blood sampling, especially during the peak-phase after tracer injection, with few cross-check blood samples needed. In other few cases, normalization of regional to whole brain values was done in place of any mathematical modelingor in addition to the quantification of brain glucose uptake. Normalization requires caution as it can introduce a degree of cross-dependency and amplification of results. Third, different scanners and reconstruction methods are likely to lead to different absolute values. In recent years, iterative reconstruction methods (approximating the real image by an iterative process) have replaced the (raw data-driven) filtered-backprojection, leading to images that are of superior visual quality, but less consistently accurate. PET scanner and reconstruction affect image resolution, influencing the extent of partial volume/ spill-over effects (contamination from surrounding tissue) in brain sub-regions whose size is close to or lower than the resolution. Though this error can be satisfactorily corrected in post-processing, such operation was not clearly reported in moststudies. Fourth, reconstructed images reflect radioactivity concentrations in kBq/cc, which can be converted into metabolic maps, by applying mathematical modeling to each image voxel P Iozzo and M A Guzzardi Brain glucose in obesity and cognitive decline R177 8:11. Then, statistical analyses can be done automatically on a voxel-by-voxel basis, after brain alignment to a reference space (statistical parametric mapping (SPM)) across all brain voxels. In this multiple comparisons approach it is important to correct for the risk of false positive findings, by applying e.g. familywise error or false discovery rate corrections. To reduce the impact of interdependency, one may restrict these corrections to a selected number of voxels in a given target region (small volume correction) or extract values from few larger regions of interest, corresponding to anatomically or functionally meaningful areas of the brain, and/or utilize non-SPM based analysis. Original images (kBq/ cc) can also be used to extract tissue time-activity-curves from brain subregions and apply mathematical modeling outside of the image domain (non-parametric image and non-SPM based analysis), reducing the noise related to the modeling of time-activity-curves in each very small voxel, though introducing some degree of operator dependency in the definition of the region of interest. Deeper technical insights are beyond the scope of this review, and can be found in, focusing on the relevance of a priori validation in PET data simplification, and in, covering statistical concepts that are common to MRI, PET, and other neuroimaging modalities.
# Conclusions
In this review, we have addressed brain glucose metabolism determinations by FDG PET imaging, as a potential unifying marker in the synergy linking obesity and cognitive disease. provides a graphic representation of the following conclusions, as supported by the current evidence: (a) brain glucose metabolism undergoes changes across the lifespan, increasing in first life years and declining from late childhood through aging in the normal situation; (b) compared to healthy individuals, brain glucose metabolism is high during mid-life in adults with obesity or with mild cognitive dysfunction and in animal models of AD pathology fed a high-fat diet or animal models of metabolic syndrome, as well as during early life days in offspring born to obese mothers; (c) most observations indicate that brain hypermetabolism may be a transient phenomenon, eventually evolving toward normal or low values during life or disease progression; (d) associative studies suggest that brain hypermetabolism predicts low BDNF levels, hepatic and whole body insulin resistance, food desire and an unfavorable balance between anticipated reward and cognitive control; (e) the effect of (intranasal) insulin is to reduce brain glucose exposure, that is, brain insulin resistance manifests as failure of insulin to achieve this suppression; (f) according to this definition, brain insulin resistance occurs in obese individuals, in mice born to obese mothers, and in mice with AD pathology when combined with high fat feeding; (g) emerging mechanistic links, showing summative alterations in AD, obesity and high-fat diets involve the microbiota and the metabolome, which correlate with brain metabolism and cognition. Though brain hypermetabolism is considered a potential compensatory reaction against initial brain damage, its consequences may still be deleterious. Research priorities in this area require clarification of whether brain hypermetabolism, and its reversal play a causative or protective role in obesity, neurodegeneration and their combination, and whether metabolites or bacteria or hormones (insulin and beyond) can be an effective strategy to prevent or mitigate the risk of neurodegeneration.
## Figure 3
The central panel highlights that high brain glucose uptake occurs in selected stages of metabolic and neurodegenerative diseases, and may be offensive and/or defensive. The right panel addresses conditions that might be fostered by brain hypermetabolism, whereas the left panel lists early phenomena that might be promising prevention targets.
## Early
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