Reverse engineering dynamic temporal models of biological processes and their relationships.
Biological processes such as circadian rhythms, cell division, metabolism, and development occur as ordered sequences of events. The synchronization of these coordinated events is essential for proper cell function, and hence the determination of critical time points in biological processes is an important component of all biological investigations. In particular, such critical time points establish logical ordering constraints on subprocesses, impose prerequisites on temporal regulation and spatial compartmentalization, and situate dynamic reorganization of functional elements in preparation for subsequent stages. Thus, building temporal phenomenological representations of biological processes from genome-wide datasets is relevant in formulating biological hypotheses on: how processes are mechanistically regulated; how the regulations vary on an evolutionary scale, and how their inadvertent disregulation leads to a diseased state or fatality. This paper presents a general framework (GOALIE) to reconstruct temporal models of cellular processes from time-course gene expression data. We mathematically formulate the problem as one of optimally segmenting datasets into a succession of "informative" windows such that time points within a window expose concerted clusters of gene action whereas time points straddling window boundaries constitute points of significant restructuring. We illustrate here how GOALIE successfully brings out the interplay between multiple yeast processes, inferred from combined experimental datasets for the cell cycle and the metabolic cycle.


Prevention Measures for COVID-19 and Changes in Kawasaki Disease Incidence.
Kawasaki disease is suspected to be triggered by previous infection. The prevention measures for coronavirus disease 2019 (COVID-19) have reportedly reduced transmission of certain infectious diseases. Under these circumstances, the prevention measures for COVID-19 may reduce the incidence of Kawasaki disease. We conducted a retrospective study using registration datasets of patients with Kawasaki disease who were diagnosed in all 11 inpatient pediatric facilities in Yamanashi Prefecture. The eligible cases were 595 cases that were diagnosed before the COVID-19 pandemic (from January 2015 through February 2020) and 38 cases that were diagnosed during the COVID-19 pandemic (from March through November 2020). Incidence of several infectious disease were evaluated using data from the Infectious Disease Weekly Report conducted by the National Institute of Infectious Diseases. Epidemics of various infectious diseases generally remained at low levels during the first 9 months (March through November 2020) of the COVID-19 pandemic. Moreover, the incidence of COVID-19 was 50-80 times lower than the incidence in European countries and the United States. The total number of 38 cases with Kawasaki disease for the 9 months during the COVID-19 pandemic was 46.3% (-3.5 standard deviations [SDs] of the average [82.0; SD, 12.7 cases] for the corresponding 9 months of the previous 5 years. None of the 38 cases was determined to be triggered by COVID-19 based on their medical histories and negative results of severe acute respiratory syndrome coronavirus 2 testing at admission. These observations provide a new epidemiological evidence for the notion that Kawasaki disease is triggered by major infectious diseases in children.


Novel heteroleptic Ru(II) complexes: synthesis, characterization and application in dye-sensitized solar cells.
Six new heteroleptic Ru(II) complexes (MC118, MC120-123 and MC126), of general formula Ru(L1)(L2)(NCS)2, where L1 and L2 are respectively dicarboxylated and π-conjugated functionalized (dissymmetric) bipyridine ligands, were designed, synthesized and applied as sensitizers in dye-sensitized solar cells. These complexes were characterized both experimentally and theoretically, showing promising optical properties, with higher extinction coefficients compared to the prototypical N719 dye. When employed in working devices, in combination with a liquid I(-)/I3(-) redox electrolyte, the investigated sensitizers have shown power conversion efficiencies between 6.0 and 8.0%, comparable with the reference N719 dye tested under similar fabrication and evaluation conditions.


Measuring physical activity in older adults: calibrating cut-points for the MotionWatch 8(©).
Given the world's aging population, the staggering economic impact of dementia, the lack of effective treatments, and the fact a cure for dementia is likely many years away - there is an urgent need to develop interventions to prevent or at least delay dementia's progression. Thus, lifestyle approaches to promote healthy aging are an important line of scientific inquiry. Good sleep quality and physical activity (PA) are pillars of healthy aging, and as such, are an increasing focus for intervention studies aimed at promoting health and cognitive function in older adults. However, PA and sleep quality are difficult constructs to evaluate empirically. Wrist-worn actigraphy (WWA) is currently accepted as a valid objective measure of sleep quality. The MotionWatch 8(©) (MW8) is the latest WWA, replacing the discontinued Actiwatch 4 and Actiwatch 7. In the current study, concurrent measurement of WWA and indirect calorimetry was performed during 10 different activities of daily living for 23 healthy older adults (aged 57-80 years) to determine cut-points for sedentary and moderate-vigorous PA - using receiver operating characteristic curves - with the cut-point for light activity being the boundaries between sedentary and moderate to vigorous PA. In addition, simultaneous multi-unit reliability was determined for the MW8 using inter-class correlations. The current study is the first to validate MW8 activity count cut-points - for sedentary, light, and moderate to vigorous PA - specifically for use with healthy older adults. These cut-points provide important context for better interpretation of MW8 activity counts, and a greater understanding of what these counts mean in terms of PA. Hence, our results validate another level of analysis for researchers using the MW8 in studies aiming to examine PA and sleep quality concurrently in older adults.


Mortality attributable to rheumatic heart disease in the Kimberley: a data linkage approach.
Acute rheumatic fever (ARF) and its sequelae, rheumatic heart disease (RHD) are now uncommon in the general Australian population. However, these preventable and treatable diseases continue to affect Aboriginal Australians disproportionately, especially in remote communities. In the Kimberley region of Western Australia (WA), the prevalence of RHD is approximately 1% among Aboriginal residents. Yet an accurate and comprehensive picture of RHD-related mortality is lacking. This study aims to determine the mortality burden attributable to ARF and RHD in the Kimberley using linked hospitalisation and death registry data. A retrospective cohort study was undertaken comprising all Kimberley residents with a WA hospital admission for ARF or RHD between 1970 and 2010, linked with the WA Death Register. We manually classified RHD-attributable deaths ('definite' or 'probable') to determine mortality burden. Hospitalisation prior to death, including valvular surgery was also ascertained. There were 35 RHD-attributable deaths in the Kimberley between 1990 and 2010, with 94% occurring in Aboriginal people. Their median age of death was 40 years. The age-standardised RHD annual death rate was 15.6 per 100 000 with a total of 1100 premature years of life lost before age of 75 within this group. Conventional International Classification of Diseases-generated mortality data underestimated mortality burden. RHD remains a significant cause of premature mortality for Aboriginal people in the Kimberley, with mortality rates unmatched in the general Australian population since the first half of the 20th century. Efforts to reduce progression of this disease through RHD Register and Control Programs are crucial alongside action to address underlying socioeconomic and environmental inequities.


Nivolumab plus ipilimumab or nivolumab alone versus ipilimumab alone in advanced melanoma (CheckMate 067): 4-year outcomes of a multicentre, randomised, phase 3 trial.
Previously reported results from the phase 3 CheckMate 067 trial showed a significant improvement in objective responses, progression-free survival, and overall survival with nivolumab plus ipilimumab or nivolumab alone compared with ipilimumab alone in patients with advanced melanoma. The aim of this report is to provide 4-year updated efficacy and safety data from this study. In this phase 3 trial, eligible patients were aged 18 years or older with previously untreated, unresectable, stage III or stage IV melanoma, known BRAFV600 mutation status, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients were randomly assigned 1:1:1 to receive intravenous nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks for four doses, followed by nivolumab 3 mg/kg every 2 weeks, or nivolumab 3 mg/kg every 2 weeks plus placebo, or ipilimumab 3 mg/kg every 3 weeks for four doses plus placebo. Randomisation was done via an interactive voice response system with a permuted block schedule (block size of six) and stratification by PD-L1 status, BRAF mutation status, and metastasis stage. The patients, investigators, study site staff, and study funder were masked to the study drug administered. The co-primary endpoints were progression-free survival and overall survival. Efficacy analyses were done on the intention-to-treat population, whereas safety was assessed in all patients who received at least one dose of study drug. The results presented in this report reflect the 4-year update of the ongoing study with a database lock date of May 10, 2018. This study is registered with ClinicalTrials.gov, number NCT01844505. Between July 3, 2013, and March 31, 2014, 945 patients were enrolled and randomly assigned to nivolumab plus ipilimumab (n=314), nivolumab (n=316), or ipilimumab (n=315). Median follow-up was 46·9 months (IQR 10·9-51·8) in the nivolumab plus ipilimumab group, 36·0 months (10·5-51·4) in the nivolumab group, and 18·6 months (7·6-49·5) in the ipilimumab group. At a minimum follow-up of 48 months from the date that the final patient was enrolled and randomised, median overall survival was not reached (95% CI 38·2-not reached) in the nivolumab plus ipilimumab group, 36·9 months (28·3-not reached) in the nivolumab group, and 19·9 months (16·9-24·6) in the ipilimumab group. The hazard ratio for death for the combination versus ipilimumab was 0·54 (95% CI 0·44-0·67; p<0·0001) and for nivolumab versus ipilimumab was 0·65 (0·53-0·79; p<0·0001). Median progression-free survival was 11·5 months (95% CI 8·7-19·3) in the nivolumab plus ipilimumab group, 6·9 months (5·1-10·2) in the nivolumab group, and 2·9 months (2·8-3·2) in the ipilimumab group. The hazard ratio for progression-free survival for the combination versus ipilimumab was 0·42 (95% CI 0·35-0·51; p<0·0001) and for nivolumab versus ipilimumab was 0·53 (0·44-0·64; p<0·0001). Treatment-related grade 3-4 adverse events were reported in 185 (59%) of 313 patients who received nivolumab plus ipilimumab, 70 (22%) of 313 who received nivolumab, and 86 (28%) of 311 who received ipilimumab. The most common treatment-related grade 3 adverse events were diarrhoea in the nivolumab plus ipilimumab group (29 [9%] of 313) and in the nivolumab group (nine [3%] of 313) and colitis in the ipilimumab group (23 [7%] of 311); the most common grade 4 adverse event in all three groups was increased lipase (15 [5%] of 313 in the combination group, ten [3%] of 313 in the nivolumab group, and four [1%] of 311 in the ipilimumab group). Serious adverse events were not analysed for the 4-year follow-up. In total for the study, there were four treatment-related deaths: two in the nivolumab plus ipilimumab group (one cardiomyopathy and one liver necrosis), one in the nivolumab group (neutropenia), and one in the ipilimumab group (colon perforation). No additional treatment-related deaths have occurred since the previous (3-year) analysis. The results of this analysis at 4 years of follow-up show that a durable, sustained survival benefit can be achieved with first-line nivolumab plus ipilimumab or nivolumab alone in patients with advanced melanoma. Bristol-Myers Squibb.


Salivary testosterone is related to self-selected training load in elite female athletes.
Testosterone has been related to improved acute neuromuscular performance in athletic populations. It is our contention that testosterone may also contribute to improved volitional motivation and, when monitored longitudinally, may provide one proxy marker for readiness to perform. Twelve female netball players provided saliva samples prior to five standardized training sessions in which they completed a maximal-distance medicine ball throw, and then 3 sets of bench press and then back squat using a self-selected load perceived to equal a 3-repetition maximum load. Additional repetitions were encouraged when possible and total voluntary workload was calculated from the product of the load lifted and repetitions performed. Relative salivary testosterone levels as a group were correlated with bench press (r=0.8399; p=0.0007) and squat (r=0.6703; p=0.0171) self-selected workload, as well as maximal medicine ball throw performance (r=0.7062; p=0.0103). Individual salivary testosterone, when viewed relatively over time, demonstrated strong relationships with self-selected workloads during an in-season training period in female netball players. As such, daily variations in testosterone may provide information regarding voluntary training motivation and readiness to perform in elite athletic populations. Psychological and behavioral aspects of testosterone may have the potential to enhance training adaptation by complementing the known anabolic and permissive properties of testosterone.


Stb1 collaborates with other regulators to modulate the G1-specific transcriptional circuit.
G(1)-specific transcription in the budding yeast Saccharomyces cerevisiae depends upon SBF and MBF. Whereas inactivation of SBF-regulated genes during the G(1)/S transition depends upon mitotic B-type cyclins, inactivation of MBF has been reported to involve multiple regulators, Nrm1 and Stb1. Nrm1 is a transcriptional corepressor that inactivates MBF-regulated transcription via negative feedback as cells exit G(1) phase. Cln/cyclin-dependent kinase (CDK)-dependent inactivation of Stb1, identified via its interaction with the histone deacetylase (HDAC) component Sin3, has also been reported to inactivate MBF-regulated transcription. This report shows that Stb1 is a stable component of both SBF and MBF that binds G(1)-specific promoters via Swi6 during G(1) phase. It is important for the growth of cells in which SBF or MBF is inactive. Although dissociation of Stb1 from promoters as cells exit G(1) correlates with Stb1 phosphorylation, phosphorylation is only partially dependent upon Cln1/2 and is not involved in transcription inactivation. Inactivation depends upon Nrm1 and Clb/CDK activity. Stb1 inactivation dampens maximal transcriptional induction during late G(1) phase and also derepresses gene expression in G(1)-phase cells prior to Cln3-dependent transcriptional activation. The repression during G(1) also depends upon Sin3. We speculate that the interaction between Stb1 and Sin3 regulates the Sin3/HDAC complex at G(1)-specific promoters.


A personalized platform identifies trametinib plus zoledronate for a patient with KRAS-mutant metastatic colorectal cancer.
Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor's genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease's genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer.


Cdc20 is required for the post-anaphase, KEN-dependent degradation of centromere protein F.
Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply 'switch' upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.


Predictors and Measures of Adherence to Core Treatments for Osteoarthritis.
Adherence has been proposed as a major barrier to implementing and maintaining the benefits of osteoarthritis (OA) core treatments and is influenced by many factors. Although there are reasonable data to support factors influencing adherence to exercise/physical activity in knee/hip OA populations, there is less research examining alternative interventions, or in the hand OA population. This problem is complicated by the lack of gold-standard measurement of adherence for core osteoarthritis treatments. The predictors of treatment adherence are not well understood, and findings are contradictory. Strategies incorporating behavior change techniques should be implemented to improve and maintain long-term adherence.


Endocytic pathway and resistance to cholesterol depletion of cholesterol derived cationic lipids for gene delivery.
Cholesterol-based cationic lipids have been widely used because of biocompatibility and serum resistance. However, the reason for the effectiveness of cholesterol-based cationic lipids remains unclear. We compared the transfection route of CHOL-E, a cholesterol-based cationic lipid having an amine head and an ether linker, with that of DOTAP. The luciferase assay with chemical inhibitors and microscopic observation of pathway markers revealed that clathrin mediated endocytosis is the main pathway for CHOL-E and DOTAP. However, CHOL-E showed resistance to cholesterol depletion by methyl-β-cyclodextrin. Furthermore, CHOL-E recovered the transfection efficiency of DOTAP from cholesterol depletion. These results suggested that superior transfection of CHOL-E might be partly derived from effects on the cell membrane.


Tenant outcomes in supported housing and community residences in New York City.
This study examined whether outcomes in housing, clinical status, and well-being of persons with severe mental illness and a history of homelessness differ between those in supported housing and those in community residences, two housing arrangements that substantially differ in the level of independence that is offered to its tenants. A quasi-experimental 18-month follow-up study was conducted with 157 persons newly entering supported housing and community residences. The housing models accepted persons with similar illness characteristics and homelessness histories, so that the inability to randomly assign tenants to housing types could be compensated for by propensity scoring methods. Tenure in housing was examined by using survival models. Analyses of other outcomes used hierarchical linear and regression models in both intent-to-treat (N=139) and true-stayer (N=80) analyses. Tenure in housing did not differ by housing type. Substantial proportions of tenants in both models remained housed during the follow-up period. Tenants in supported housing reported greater housing satisfaction in terms of autonomy and economic viability. Over time some tenants in supported housing reported greater feelings of isolation. Independent of housing type, symptoms of depression or anxiety at housing entry increased the risk of poorer outcomes. The models of supported housing were viable portals of entry into community housing for homeless persons, even for consumers with characteristics indicating that they would have been more likely to be placed in community residences. The results suggest that greater clinical attention should be paid to persons who exhibit depression or anxiety when entering housing.


Critiquing Protein Family Classification Models Using Sufficient Input Subsets.
In many application domains, neural networks are highly accurate and have been deployed at large scale. However, users often do not have good tools for understanding how these models arrive at their predictions. This has hindered adoption in fields such as the life and medical sciences, where researchers require that models base their decisions on underlying biological phenomena rather than peculiarities of the dataset. We propose a set of methods for critiquing deep learning models and demonstrate their application for protein family classification, a task for which high-accuracy models have considerable potential impact. Our methods extend the Sufficient Input Subsets (SIS) technique, which we use to identify subsets of features in each protein sequence that are alone sufficient for classification. Our suite of tools analyzes these subsets to shed light on the decision-making criteria employed by models trained on this task. These tools show that while deep models may perform classification for biologically relevant reasons, their behavior varies considerably across the choice of network architecture and parameter initialization. While the techniques that we develop are specific to the protein sequence classification task, the approach taken generalizes to a broad set of scientific contexts in which model interpretability is essential.


Oligodendrocyte progenitor programming and reprogramming: Toward myelin regeneration.
Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. Immunosuppression therapy has a limited efficacy in MS patients, arguing for a paradigm shift to strategies that target OL lineage cells to achieve myelin repair. The inhibitory microenvironment in MS lesions abrogates the expansion and differentiation of resident OL precursor cells (OPCs) into mature myelin-forming OLs. Recent studies indicate that OPCs display a highly plastic ability to differentiate into alternative cell lineages under certain circumstances. Thus, understanding the mechanisms that maintain and control OPC fate and differentiation into mature OLs in a hostile, non-permissive lesion environment may open new opportunities for regenerative therapies. In this review, we will focus on 1) the plasticity of OPCs in terms of their developmental origins, distribution, and differentiation potentials in the normal and injured brain; 2) recent discoveries of extrinsic and intrinsic factors and small molecule compounds that control OPC specification and differentiation; and 3) therapeutic potential for motivation of neural progenitor cells and reprogramming of differentiated cells into OPCs and their likely impacts on remyelination. OL-based therapies through activating regenerative potentials of OPCs or cell replacement offer exciting opportunities for innovative strategies to promote remyelination and neuroprotection in devastating demyelinating diseases like MS. This article is part of a Special Issue entitled SI:NG2-glia(Invited only).


Optimizing Teamwork for Human Performance Teams: Strategies for Enhancing Team Effectiveness.
Human performance teams (HPTs) are highly capable and complex teams comprised of medical and performance professionals dedicated to supporting health and sustaining mission capabilities of the Special Operations Forces (SOF) warfighter community. As resources continue to be devoted to recruiting, hiring, and organizing HPTs, there is an increased need to support team-based capabilities, or their ability to work collaboratively and cooperatively across boundaries. In this article, we draw on existing evidence-based approaches to supporting team-based competencies to present a set of strategies designed to address barriers to cross-boundary teaming, catalyze innovation and precision of human performance optimization (HPO) service delivery, and maximize the impact of HPTs on warfighter medical and mission readiness. We begin by offering a conceptual paradigm shift that broadens the lens through which HPO intervention opportunities exist. We then explore how to promote a common understanding of the needs, performance demands, and occupational risks, which should clarify shared goals and targets for service delivery. We also discuss a refined strategy for hiring and recruiting members of HPTs, and finally, we propose opportunities for cultivating communication and collaboration across and within the HPO spectrum. By elevating HPT-based capabilities, the SOF community should be able to amplify the investment made in these invaluable resources.


Regulation of heterochromatin transcription by Snail1/LOXL2 during epithelial-to-mesenchymal transition.
Although heterochromatin is enriched with repressive traits, it is also actively transcribed, giving rise to large amounts of noncoding RNAs. Although these RNAs are responsible for the formation and maintenance of heterochromatin, little is known about how their transcription is regulated. Here, we show that the Snail1 transcription factor represses mouse pericentromeric transcription, acting through the H3K4 deaminase LOXL2. Since Snail1 plays a key role in the epithelial-to-mesenchymal transition (EMT), we analyzed the regulation of heterochromatin transcription in this process. At the onset of EMT, one of the major structural heterochromatin proteins, HP1α, is transiently released from heterochromatin foci in a Snail1/LOXL2-dependent manner, concomitantly with a downregulation of major satellite transcription. Moreover, preventing the downregulation of major satellite transcripts compromised the migratory and invasive behavior of mesenchymal cells. We propose that Snail1 regulates heterochromatin transcription through LOXL2, thus creating the favorable transcriptional state necessary for completing EMT.


Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures.
At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.


The roles of valerenic acid on BDNF expression in the SH-SY5Y cell.
The roots of Valeriana officinalis L. (Valerianaceae) are used for treating sleep disorders and/or mild nerve tension. The effect of valerenic acid on brain-derived neurotrophic factor (BDNF) has not yet been studied, although it is known that gamma-amino butyric acid A (GABAA) receptor is regulated by BDNF, which modulates the depressive-like behavior and neurogenesis. The purpose of this study is to determine the effect of V. officinalis root extract (VO), its main constituents valerenic acid (VA) and acetoxy valerenic acid (AVA) as well as valerenic acid-free (VAF), acetoxy valerenic acid-free (AVAF) extracts and increasing amounts of valerenic acid containing extracts on the BDNF expression in SH-SY5Y cell lines. The effect of methanolic extracts of VO, VA, AVA, VAF, AVAF, and the extracts whose amount of VA were increased gradually, were tested using a Human BDNF ELISA kit with 17β-estradiol as a positive control. The VO and VA extracts caused a significant (p < 0.001) increase in the BDNF expression in SH-SY5Y cells compared to control. This effect completely disappeared when cells were treated with VAF extract. AVA alone did not show any significant change in the BDNF levels. The extracts with increasing amount of VA led to a concentration- dependent effect on the cells. In conclusion, our findings suggest that the antidepressant-like effect of the VO extract is also related to BDNF expression, and that this is mainly due to the presence of VA in the extract. Removing VA from VO extract leads to a loss of activity. Moreover, the concentration of VA plays a role for BDNF expressions in SH-SY5Y cells, which demonstrates the importance of quality control on the commercially available products.


The stratigraphy of the Middle Stone Age sediments at Pinnacle Point Cave 13B (Mossel Bay, Western Cape Province, South Africa).
Pinnacle Point Cave 13B (PP13B) has provided the earliest archaeological evidence for the exploitation of marine shellfish, along with very early evidence for use and modification of pigments and the production of bladelets, all dated to approximately 164 ka (Marean et al., 2007). This makes PP13B a key site in studies of the origins of modern humans, one of a handful of sites in Africa dating to Marine Isotope Stage 6 (MIS 6), and the only site on the coast of South Africa with human occupation confidently dated to MIS 6. Along with this MIS 6 occupation there are rich archaeological sediments dated to MIS 5, and together these sediments are differentially preserved in three different areas of the cave. The sediments represent a complex palimpsest of geogenic, biogenic, and anthropogenic input and alteration that are described and interpreted through the use of a variety of macrostratigraphic, micromorphologic, and geochemical techniques. Three independent dating techniques allow us to constrain the age range of these sediments and together provide the stratigraphic context for the analyses of the material that follow in this special issue.


Intracellular delivery of a cell-penetrating SOCS1 that targets IFN-gamma signaling.
Suppressor of cytokine signaling-1 (SOCS1) is an intracellular inhibitor of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that couples interferon-gamma (IFN-gamma) signaling to the nucleus. Because several inflammatory diseases are associated with uncontrolled IFN-gamma signaling, we engineered a recombinant cell-penetrating SOCS1 (CP-SOCS1) to target this pathway. Here, we show that CP-SOCS1, analogous to endogenous SOCS1, interacted with components of the IFN-gamma signaling complex and functionally attenuated the phosphorylation of STAT1, which resulted in the subsequent inhibition of the production of proinflammatory chemokines and cytokines. Thus, controlled, intracellular delivery of recombinant CP-SOCS1 boosted the anti-inflammatory potential of the cell by restoring the homeostatic balance between pro- and anti-inflammatory signaling. This approach to controlling signal transduction has potential use for therapeutic targeting of signaling pathways associated with inflammatory diseases.


Blindness: how to assess numbers and causes?
Traditionally, blindness surveys have modelled themselves on the "gold standard" of a census and examination of a whole population. Blindness, however, is a relatively rare condition even in badly affected communities; hence, large sample sizes are required to gain adequate estimates of prevalence, particularly by cause. Three assessments of blindness prevalence and aetiology in the same communities are reported. One involved asking individuals questions concerning their visual status during a census (perceived visual status, PVD), one involved examination of all ostensibly visually disabled people presenting to a central point within each community (examination of the visually disabled, EVD), and the final assessment involved a gold standard examination of the whole population (whole community examination, WCE). In a population of 8139 the blindness prevalence was 2.7% PVS, 3.6% EVD, and 3.1% WCE. Attributed causes of blindness were not representative in the PVS except for cataract. The END yielded cause specific estimates not far from those found at WE except for a relative under-representation of glaucoma and optic atrophy. Since cataract is, by a significant margin, the most common cause of blindness in the world such a simple method as asking individuals if they are blind and what they believe to be the cause may yield adequate estimates of the problem for planning eye care strategies for this condition. Alternatively, an ophthalmologist visiting villages and examining allcomers for visual disability may provide reasonably accurate cause specific prevalence estimates without the expense of a major blindness survey.


[Esophageal sarcomatoid squamous carcinoma: report of one case].
Sarcomatoid squamous carcinoma (ESC) is a rare esophageal neoplasm, with a clinical, etiological and pathological behavior that differs from squamous cancer. From the histological point of view it has a dual configuration. The squamous epithelial component is usually limited to small areas, while the major part is constituted by mesenchymatous (sarcomatoid) polypoid tissue. Treatment is esophagectomy or total esophagogastrectomy depending on the tumor location. Early detection is critical in terms of survival. For large lesions, preoperative chemo-radiotherapy can be considered. We report a 78-year-old male presenting with dysphagia. An upper gastrointestinal endoscopy showed a tumor located below the cardia. The patient was subjected to a total esophageal and gastric resection. In a second operation, the digestive transit was reconstituted in 2 steps. The pathology report informed a sarcomatoid squamous carcinoma. After 18 months of follow up, the patient is ambulatory.


Cancer, infant mortality and birth sex-ratio in Fallujah, Iraq 2005-2009.
There have been anecdotal reports of increases in birth defects and cancer in Fallujah, Iraq blamed on the use of novel weapons (possibly including depleted uranium) in heavy fighting which occurred in that town between US led forces and local elements in 2004. In Jan/Feb 2010 the authors organised a team of researchers who visited 711 houses in Fallujah, Iraq and obtained responses to a questionnaire in Arabic on cancer, birth defects and infant mortality. The total population in the resulting sample was 4,843 persons with and overall response rate was better than 60%. Relative Risks for cancer were age-standardised and compared to rates in the Middle East Cancer Registry (MECC, Garbiah Egypt) for 1999 and rates in Jordan 1996-2001. Between Jan 2005 and the survey end date there were 62 cases of cancer malignancy reported (RR = 4.22; CI: 2.8, 6.6; p < 0.00000001) including 16 cases of childhood cancer 0-14 (RR = 12.6; CI: 4.9, 32; p < 0.00000001). Highest risks were found in all-leukaemia in the age groups 0-34 (20 cases RR = 38.5; CI: 19.2, 77; p < 0.00000001), all lymphoma 0-34 (8 cases, RR = 9.24;CI: 4.12, 20.8; p < 0.00000001), female breast cancer 0-44 (12 cases RR = 9.7;CI: 3.6, 25.6; p < 0.00000001) and brain tumours all ages (4 cases, RR = 7.4;CI: 2.4, 23.1; P < 0.004). Infant mortality was based on the mean birth rate over the 4 year period 2006-2009 with 1/6th added for cases reported in January and February 2010. There were 34 deaths in the age group 0-1 in this period giving a rate of 80 deaths per 1,000 births. This may be compared with a rate of 19.8 in Egypt (RR = 4.2 p < 0.00001) 17 in Jordan in 2008 and 9.7 in Kuwait in 2008. The mean birth sex-ratio in the recent 5-year cohort was anomalous. Normally the sex ratio in human populations is a constant with 1,050 boys born to 1,000 girls. This is disturbed if there is a genetic damage stress. The ratio of boys to 1,000 girls in the 0-4, 5-9, 10-14 and 15-19 age cohorts in the Fallujah sample were 860, 1,182, 1,108 and 1,010 respectively suggesting genetic damage to the 0-4 group (p < 0.01). Whilst the results seem to qualitatively support the existence of serious mutation-related health effects in Fallujah, owing to the structural problems associated with surveys of this kind, care should be exercised in interpreting the findings quantitatively.


Severely reduced female fertility in CD9-deficient mice.
CD9 is a widely expressed cell surface molecule that belongs to the tetraspanin superfamily of proteins. The tetraspanins CD9, KAI-1/CD82, and CD63 are involved in metastasis suppression, an effect that may be related to their association with beta1 integrins. Knockout mice lacking CD9 were created to evaluate the physiological importance of CD9. CD9-/- females displayed a severe reduction of fertility. Oocytes were ovulated but were not successfully fertilized because sperm did not fuse with the oocytes from CD9-/- females. Thus, CD9 appears to be essential for sperm-egg fusion, a process involving the CD9-associated integrin alpha6beta1.


Emerging treatment using tubulin inhibitors in advanced non-small cell lung cancer.
Tubulin inhibitors including taxanes and vinca alkaloids are important components of chemotherapy regimens used in advanced non-small cell lung cancer (NSCLC). Despite a treatment paradigm shift due to molecularly-targeted therapies and immunotherapy, a majority of patients will receive chemotherapy during their treatment course. Either used alone or in combination, tubulin inhibitors have demonstrated clinical benefits in different settings of lung cancer management. Areas covered: This review first discusses FDA-approved tubulin inhibitors for NSCLC, such as paclitaxel, docetaxel, vinorelbine, and nab-paclitaxel. The article then provides a summary of novel tubulin inhibitors, including cabazitaxel, eribulin, ixabepilone, patupilone, plinabulin, new colchicine analogues and others. It also discusses new tubulin inhibitor combinations with immunotherapy (PD-1/PD-L1 inhibitors) and molecularly-targeted therapies (e.g. anti-angiogenic agents, mTOR inhibitors, heat shock protein 90 inhibitors, MEK inhibitors, and anti-HER3 agents). Lastly, emerging data on potential resistance mechanisms and predictive biomarkers for tubulin inhibitors are explored. Expert opinion: Tubulin inhibitors will likely continue to play important roles in NSCLC management due to the advent of novel agents and combinations. Through further understanding of tumor biology, investigation of drug resistance, and development of predictive biomarkers, we will be better positioned to incorporate microtubule inhibition into patient specific treatment strategies.


The long-term survival of a patient with pancreatic cancer with metastases to the liver after treatment with the intravenous alpha-lipoic acid/low-dose naltrexone protocol.
The authors describe the long-term survival of a patient with pancreatic cancer without any toxic adverse effects. The treatment regimen includes the intravenous alpha-lipoic acid and low-dose naltrexone (ALA-N) protocol and a healthy lifestyle program. The patient was told by a reputable university oncology center in October 2002 that there was little hope for his survival. Today, January 2006, however, he is back at work, free from symptoms, and without appreciable progression of his malignancy. The integrative protocol described in this article may have the possibility of extending the life of a patient who would be customarily considered to be terminal. The authors believe that life scientists will one day develop a cure for metastatic pancreatic cancer, perhaps via gene therapy or another biological platform. But until such protocols come to market, the ALA-N protocol should be studied and considered, given its lack of toxicity at levels reported. Several other patients are on this treatment protocol and appear to be doing well at this time.


Bioassay-guided isolation of kaempferol-3-O-beta-D-galactoside with anti-inflammatory and antinociceptive activity from the aerial part of Calluna vulgaris L.
Calluna vulgaris L. (Ericaceae) is used for the treatment of various inflammatory ailments in traditional medicines. In order to evaluate this ethnobotanical information, its anti-inflammatory and antinociceptive activities were studied using in vivo experimental models in mice. The ethanolic extract of the plant was first fractionated into five extracts; namely, n-hexane, chloroform, ethyl acetate (EtOAc), n-butanol, and water fractions. Among them, the EtOAc Fr. was found to be the most effective and was further subjected to bioassay-guided fractionation and isolation procedures. After successive column chromatography applications, on Sephadex LH-20 and silica gel, a component, which is responsible for the above-mentioned activities of this species of Turkish origin, was isolated and its structure was elucidated as kaempferol-3-O-beta-D-galactoside, a common flavonol derivative by means of spectral techniques.


Spatial Proximity to Fibroblasts Impacts Molecular Features and Therapeutic Sensitivity of Breast Cancer Cells Influencing Clinical Outcomes.
Using a three-dimensional coculture model, we identified significant subtype-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells in contact with cancer-associated fibroblasts (CAF). CAF-induced gene expression signatures predicted clinical outcome and immune-related differences in the microenvironment. We found that fibroblasts strongly protect carcinoma cells from lapatinib, attributable to its reduced accumulation in carcinoma cells and an elevated apoptotic threshold. Fibroblasts from normal breast tissues and stromal cultures of brain metastases of breast cancer had similar effects as CAFs. Using synthetic lethality approaches, we identified molecular pathways whose inhibition sensitizes HER2+ breast cancer cells to lapatinib both in vitro and in vivo, including JAK2/STAT3 and hyaluronic acid. Neoadjuvant lapatinib therapy in HER2+ breast tumors lead to a significant increase of phospho-STAT3+ cancer cells and a decrease in the spatial proximity of proliferating (Ki67+) cells to CAFs impacting therapeutic responses. Our studies identify CAF-induced physiologically and clinically relevant changes in cancer cells and offer novel approaches for overcoming microenvironment-mediated therapeutic resistance. Cancer Res; 76(22); 6495-506. ©2016 AACR.


A strain gauge analysis of microstrain induced by various splinting methods and acrylic resin types for implant impressions.
The aim of this study was to investigate the level of microstrain that is exerted during polymerization of acrylic resins used for splinting during implant impressions. Two acrylic resins (GC Pattern Resin, Duralay II) and square transfer coping splinting methods were evaluated by means of strain gauge analysis. Two implants were embedded in a polyurethane block, and the abutments were positioned. Sixty specimens were prepared using two square transfer copings that were rigidly connected to each other using the acrylic resins. The specimens were randomly divided into three groups of 20 each for the splinting methods: Method 1 was a one-piece method; in method 2, the splint was separated and reconnected after 17 minutes; and in method 3, the splint was separated and reconnected after 24 hours. In each group, half the specimens were splinted with GC Pattern Resin and the other half were splinted with Duralay II. Three microstrain measurements were performed by four strain gauges placed on the upper surface of the polyurethane blocks at 5 hours after resin polymerization for all groups. The data were analyzed statistically. Both resin type and splinting method significantly affected microstrain. Interaction terms were also significant. Method 1 in combination with Duralay II produced significantly higher microstrain (1,962.1 με) than the other methods with this material (method 2: 241.1 με; method 3: 181.5 με). No significant difference was found between splinting methods in combination with GC Pattern Resin (method 1: 173.8 με; method 2: 112.6 με; method 3: 105.4 με). Because of the high microstrain generated, Duralay II should not be used for one-piece acrylic resin splinting, and separation and reconnection are suggested. For GC Pattern Resin, variations in splinting methods did not significantly affect the microstrain created.


Aedes aegypti in Ho Chi Minh City (Viet Nam): susceptibility to dengue 2 virus and genetic differentiation.
Aedes aegypti is the principal vector of dengue viruses, responsible for a viral infection that has become a major public health concern in Asia. In Viet Nam, dengue haemorrhagic fever was first detected in the 1960s and is now a leading cause of death in childhood. We studied the variability in competence of Ae. aegypti as a vector for dengue 2 virus and genetic differentiation in this mosquito species. Twenty mosquito samples collected in 1998 in Ho Chi Minh City were subjected to oral infection and isoenzyme polymorphism analysis by starch gel electrophoresis. Ae. aegypti populations from the centre of Ho Chi Minh City were genetically differentiated and their infection rates differed from those of populations from the commuter belt. These results have implications for insecticidal control during dengue outbreaks.


Genistein reduces lysosomal storage in peripheral tissues of mucopolysaccharide IIIB mice.
Mucopolysaccharidosis type IIIB (Sanfilippo syndrome) is a lysosomal storage disease caused by a genetic defect in the production of alpha-N-acetylglucosaminidase. This results in lysosomal and extracellular accumulation of the undegraded glycosaminoglycan (GAG) substrate, heparan sulphate. Affected patients show progressive CNS degeneration characterised by mental retardation, hyperactivity and seizures, with death usually in the mid teens to early twenties. Visceral organ storage is also present but is relatively mild compared to other MPS diseases storing similar substrates. No treatments currently exist for MPS IIIB. Genistein is a broad spectrum protein tyrosine kinase inhibitor which acts on several different growth factor receptors, notably EGF and IGF receptors, both of which are important for proteoglycan synthesis. Recent work has shown that genistein can reduce GAG synthesis in patients' fibroblasts in vitro and there is evidence in patients to suggest that it may be an effective substrate reduction therapy agent for MPS III. Here we have tested the dose responses of MPS IIIB mice to daily sub-chronic dosing of genistein in half log increments compared to carrier over 8 weeks. We show clear reductions in liver lysosome compartment size in both sexes and significant dose dependent improvements in total liver GAGs and hair morphology in male MPS IIIB animals following genistein treatment. Male MPS IIIB mice exhibited considerably more liver storage than females and responded better to treatment. No changes in total GAGs, lysosomal size or reactive astrogliosis in the brain cortex were observed after 8 weeks of treatment despite evidence that genistein can cross the blood brain barrier. This is the first demonstration of genistein treatment in MPS models in vivo.


Sensors of extracellular nutrients in Saccharomyces cerevisiae.
It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions. Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors. Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium. and glucose. Strikingly, the transmembrane components of several of these sensors, Ssylp, Mep2p, Snf3p. and Rgt2p, are unique members of nutrient-transport protein families. Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors. Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor. These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought. The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments.


Nonadiabatic Photochemistry Induced by Inaccessible Conical Intersections.
Conical intersections are ubiquitous in photochemical processes, where nonadiabatic transfer induces ultrafast nonradiative decay from an excited state. Although they eluded experimental detection until the 1990s, today three diagnostic attributes are generally associated with photochemical reactions through conical intersections: ultrafast electronic dynamics, negligible fluorescence, and coherent wavepacket transfer. Here, we use generalized quantum dynamics simulations to show that coherent nonadiabatic transfer of excited vibrational wavepackets can occur even without reaching the conical intersection region. Instead, the wavepacket remains distant from the conical intersection throughout. In some topographies, an energetically inaccessible conical intersection can be completely avoided, yet still induce substantial nonadiabatic transfer with ultrafast transfer efficiencies that are nearly identical to those of direct transfer through a conical intersection. These results reveal that the diagnostic properties of conical intersections are not actually specific to decay pathways traveling directly through the intersection funnel, as is the common interpretation, but can also arise from alternative pathways that do not reach the intersection. This suggests that the diagnostic features and experimental signals associated with conical intersections should be reassessed, and the concept of pathways through a conical intersection as the "paradigm of photochemistry" may need to be adjusted.


Issues related to human immunodeficiency virus transmission in schools, child care, medical settings, the home, and community. American Academy of Pediatrics. Committee of Pediatric AIDS and Committee on Infectious Diseases.
Current recommendations of the American Academy of Pediatrics (AAP) for infection control practices to prevent transmission of blood-borne pathogens, including human immunodeficiency virus (HIV) in hospitals, other medical settings, schools, and child care facilities, are reviewed and explained. Hand-washing is essential, whether or not gloves are used, and gloves should be used when contact with blood or blood-containing body fluids may occur. In hospitalized children, the 1996 recommendations of the Centers for Disease Control and Prevention (CDC) should be implemented as modified in the 1997 Red Book. The generic principles of Standard Precautions in the CDC guidelines generally are applicable to children in all health care settings, schools, child care facilities, and the home. However, gloves are not required for routine changing of diapers or for wiping nasal secretions of children in most circumstances. This AAP recommendation differs from that in the CDC guidelines. Current US Public Health Service guidelines for the management of potential occupational exposures of health care workers to HIV are summarized. As previously recommended by the AAP, HIV-infected children should be admitted without restriction to child care centers and schools and allowed to participate in all activities to the extent that their health and other recommendations for management of contagious diseases permit. Because it is not required that the school be notified of HIV infection, it may be helpful if the pediatrician notify the school that he or she is operating under a policy of nondisclosure of infection with blood-borne pathogens. Thus, it is possible that the pediatrician will not report the presence of such infections on the form. Because HIV infection occurs in persons throughout the United States, these recommendations for prevention of HIV transmission should be applied universally.


The architecture and evolution of cancer neochromosomes.
We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corrosion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.


Design of stiff, tough and stretchy hydrogel composites via nanoscale hybrid crosslinking and macroscale fiber reinforcement.
Hydrogels' applications are usually limited by their weak mechanical properties. Despite recent great progress in developing tough hydrogels, it is still challenging to achieve high values of , toughness and modulus all together in synthetic hydrogels. In this paper, we designed highly stretchable, tough, yet stiff hydrogel composites via a combination of nanoscale hybrid crosslinking and macroscale fiber reinforcement. The hydrogel composites were constructed by impregnating a 3D-printed thermoplastic-fiber mesh with a tough hydrogel crosslinked both covalently and ionically. The hydrogel composites can achieve a fracture energy of over 30,000 J m(-2), a modulus of over 6 MPa, and can be stretched over 2.8 times even in the presence of large structural defects. The enhancement of toughness in the new hydrogel composites relies on multiple pairs of toughening mechanisms which span over multiple length scales. A theoretical model is further developed to predict the toughness and modulus of the hydrogel composites and guide the design of future materials.


Gene expression profiles characterize inflammation stages in the acute lung injury in mice.
Acute Lung Injury (ALI) carries about 50 percent mortality and is frequently associated with an infection (sepsis). Life-support treatment with mechanical ventilation rescues many patients, although superimposed infection or multiple organ failure can result in death. The outcome of a patient developing sepsis depends on two factors: the infection and the pre-existing inflammation. In this study, we described each stage of the inflammation process using a transcriptional approach and an animal model. Female C57BL6/J mice received an intravenous oleic acid injection to induce an acute lung injury (ALI). Lung expression patterns were analyzed using a 9900 cDNA mouse microarray (MUSV29K). Our gene-expression analysis revealed marked changes in the immune and inflammatory response metabolic pathways, notably lipid metabolism and transcription. The early stage (1 hour-1.5 hours) is characterized by a pro-inflammatory immune response. Later (3 hours-4 hours), the immune cells migrate into inflamed tissues through interaction with vascular endothelial cells. Finally, at late stages of lung inflammation (18 hours-24 hours), metabolism is deeply disturbed. Highly expressed pro-inflammatory cytokines activate transcription of many genes and lipid metabolism. In this study, we described a global overview of critical events occurring during lung inflammation which is essential to understand infectious pathologies such as sepsis where inflammation and infection are intertwined. Based on these data, it becomes possible to isolate the impact of a pathogen at the transcriptional level from the global gene expression modifications resulting from the infection associated with the inflammation.


Synchrotron X-ray scattering study of the mechanism of nanopore generation in nanoporous organosilicate thin films imprinted with a reactive six-armed porogen.
In situ grazing incidence small-angle X-ray scattering analysis was successfully performed during the thermal processing of film blends of polymethylsilsesquioxane (PMSSQ) precursor and triethoxysilyl-terminated six-arm poly(epsilon-caprolactone) (mPCL6) porogen. In addition, thermogravimetric analysis of the films was carried out in a nitrogen atmosphere. These measurements provide important information about the structures of the blend films and of the resulting porous films. In particular, they are used in this paper to establish the mechanism of the formation of imprinted pores within the blend films. During the heating run, the sacrificial thermal degradation of the porogen component commenced at 320 degrees C, generating pores in the resulting cured PMSSQ films. Only very limited porogen aggregation occurred during the blend film formation process (spin-coating and subsequent drying), and these porogen aggregates were of relatively small size and narrow size distribution. The observed restriction of the formation of such porogen aggregates was found to result from the favorable hybridization reaction of the porogen's reactive end groups with the reactive functional groups of the PMSSQ precursor, which competes with aggregation via reaction between the porogen molecules. The average radius (or half-size) of the porogen aggregates was in the range 2.45-3.98 nm, depending on the porogen loading (10-40 wt %). The porogen aggregates retained their size and size distribution until thermal degradation, which resulted in the imprinting of nanopores in the cured PMSSQ films with size and size distribution corresponding to those of the porogen aggregates. The porosities of the resulting nanoporous films were in the range 12.4-41.7%, depending on the initial porogen loading.


Peptide-based FeS4 complexes: the zinc ribbon fold is unsurpassed to stabilize both the FeII and FeIII states.
Whereas Zn(Cys)4 zinc fingers exist with different protein folds, only the zinc ribbon fold is found in rubredoxin Fe(Cys)4 sites. To assess the significance of this observation, we have investigated the binding and stability of Fe(2+) and Fe(3+) ions by a set of four peptides designed to model Zn(Cys)4 zinc fingers with various folds, i.e. zinc ribbon, treble clef and a loosened zinc ribbon fold. All peptides were shown by means of UV-Vis and CD spectroscopies to form stable 1 : 1 Fe(II)/peptide complexes with binding constants higher than 10(7) M(-1) at pH 7. Their oxidation into Fe(III) complexes and the stability of the latter were compared. The UV-Vis absorption and CD spectroscopic properties of the Fe(II) and Fe(III) complexes were analysed with respect to the structures of the zinc analogues in order to get insight into the local arrangement of the Fe(Cys)4 core around the metal ion. The chemical stability of these complexes was rationalized according to the shielding from the solvent provided by the various peptide folds to the FeS4 core. In addition, we showed that whereas UV-visible spectra inform only on the FeS4, the information derived from the corresponding CD spectra extend to the Cβ orientation and the peptide fold. The results presented here demonstrate that while the zinc ribbon fold is not strictly required to obtain a Fe(Cys)4 site, it affords a drastically superior protection of the site toward external redox agents. This finding brings new clues to engineer stable and redox-active Fe(Cys)4 sites in de novo proteins.


A novel deletion of IGF1 in a patient with idiopathic short stature provides insight Into IGF1 haploinsufficiency.
Short stature is a common reason for referral to pediatric endocrinology centers. Frequently, the underlying etiology of short stature is unknown, resulting in a diagnosis of idiopathic short stature. Rare genetic defects in the GH/IGF-1 axis have been found to cause short stature. The objective of this study was to identify the genetic etiology of short stature in a patient with Idiopathic Short Stature and to review the clinical presentation of patients with genetic defects in IGF1, and specifically IGF-1 haploinsufficiency. The index patient was evaluated at an academic medical center, and DNA was obtained from the proband and both parents. Genome-wide copy number analysis was performed in the proband with confirmatory quantitative PCR in the proband and his parents. We measured novel copy number variants (CNVs) thought to explain the patient's short stature. CNV analysis revealed that the proband carried a paternally inherited heterozygous IGF1 gene deletion. His phenotypic features are consistent with those found in previous case reports of IGF-1 deficiency. This study, as the first case of a complete heterozygous 1GF1 deletion, provides insight into the effects of true IGF-1 haploinsufficiency. Given the similarities in phenotype between the present proband and those previously described, it is highly likely that his IGF1 deletion is the cause for his short stature. Broadly, this study emphasizes how CNV analysis and other genetic sequencing techniques are evolving as an important tool to identify genetic causes underlying human disease, allowing for improved diagnosis and targeted treatment.


Lack of a relationship between serum ferritin levels and coronary atherosclerosis evaluated by coronary arteriography.
Many clinical and epidemiological studies have demonstrated the relationship between serum ferritin and ischemic heart disease. In the present study we evaluated the relationship between coronary heart disease (CHD) and serum ferritin levels in patients submitted to coronary arteriography. We evaluated 307 patients (210 (68.7%) males; median age: 60 years) who were submitted to coronary angiography, measurement of serum ferritin and identification of clinical events of ischemic heart disease. Serum ferritin is reported as quartiles. Ninety-six patients (31.27%) had normal coronary angiography (group 1) and 211 (68.73%) had coronary heart disease (group 2). Of the patients with CHD, 61 (28.9%) had serum ferritin levels higher than 194 ng/ml (4th quartile), as opposed to only 14 (14.58%) of those without CHD (P = 0.0067). In the 2nd quartile, 39 patients (18.48%) had CHD, while 35 patients (36.46%) had normal coronary arteries (P = 0.00064). Multivariate analysis of the data showed that the difference between groups was not statistically significant (P = 0.33). We conclude that there is no independent relationship between coronary heart disease and increased levels of serum ferritin.


Identifying modes of large whispering-gallery mode resonators from the spectrum and emission pattern.
Identifying the mode numbers in whispering-gallery mode resonators (WGMRs) is important for tailoring them to experimental needs. Here we report on a novel experimental mode analysis technique based on the combination of frequency analysis and far-field imaging for high mode numbers of large WGMRs. The radial mode numbers q and the angular mode numbers p = ℓ-m are identified and labeled via far-field imaging. The polar mode numbers ℓ are determined unambiguously by fitting the frequency differences between individual whispering gallery modes (WGMs). This allows for the accurate determination of the geometry and the refractive index at different temperatures of the WGMR. For future applications in classical and quantum optics, this mode analysis enables one to control the narrow-band phase-matching conditions in nonlinear processes such as second-harmonic generation or parametric down-conversion.


An online parenting intervention to prevent affective disorders in high-risk adolescents: the PIPA trial protocol.
Adolescent depression can place a young person at high risk of recurrence and a range of psychosocial and vocational impairments in adult life, highlighting the importance of early recognition and prevention. Parents/carers are well placed to notice changes in their child's emotional wellbeing which may indicate risk, and there is increasing evidence that modifiable factors exist within the family system that may help reduce the risk of depression and anxiety in an adolescent. A randomised controlled trial (RCT) of the online personalised 'Partners in Parenting' programme developed in Australia, focused on improving parenting skills, knowledge and awareness, showed that it helped reduce depressive symptoms in adolescents who had elevated symptom levels at baseline. We have adapted this programme and will conduct an RCT in a UK setting. In total, 433 family dyads (parents/carers and children aged 11-15) will be recruited through schools, social media and parenting/family groups in the UK. Following completion of screening measures of their adolescent's depressive symptoms, parents/carers of those with elevated scores will be randomised to receive either the online personalised parenting programme or a series of online factsheets about adolescent development and wellbeing. The primary objective will be to test whether the personalised parenting intervention reduces depressive symptoms in adolescents deemed at high risk, using the parent-reported Short Mood & Feelings Questionnaire. Follow-up assessments will be undertaken at 6 and 15 months and a process evaluation will examine context, implementation and impact of the intervention. An economic evaluation will also be incorporated with cost-effectiveness of the parenting intervention expressed in terms of incremental cost per quality-adjusted life year gained. Half of mental health problems emerge before mid-adolescence and approximately three-quarters by mid-20s, highlighting the need for effective preventative strategies. However, few early interventions are family focused and delivered online. We aim to conduct a National Institute for Health and Care Research (NIHR) funded RCT of the online personalised 'Partners in Parenting' programme, proven effective in Australia, targeting adolescents at risk of depression to evaluate its effectiveness, cost-effectiveness and usability in a UK setting. TRIAL REGISTRATION {2A}: ISRCTN63358736 . Registered 18 September 2019.


Sequence complexity for biological sequence analysis.
A new statistical model for DNA considers a sequence to be a mixture of regions with little structure and regions that are approximate repeats of other subsequences, i.e. instances of repeats do not need to match each other exactly. Both forward- and reverse-complementary repeats are allowed. The model has a small number of parameters which are fitted to the data. In general there are many explanations for a given sequence and how to compute the total probability of the data given the model is shown. Computer algorithms are described for these tasks. The model can be used to compute the information content of a sequence, either in total or base by base. This amounts to looking at sequences from a data-compression point of view and it is argued that this is a good way to tackle intelligent sequence analysis in general.


Norovirus recombination in ORF1/ORF2 overlap.
Norovirus (NoV) genogroups I and II (GI and GII) are now recognized as the predominant worldwide cause of outbreaks of acute gastroenteritis in humans. Three recombinant NoV GII isolates were identified and characterized, 2 of which are unrelated to any previously published recombinant NoV. Using data from the current study, published sequences, database searches, and molecular techniques, we identified 23 recombinant NoV GII and 1 recombinant NoV GI isolates. Analysis of the genetic relationships among the recombinant NoV GII isolates identified 9 independent recombinant sequences; the other 14 strains were close relatives. Two of the 9 independent recombinant NoV were closely related to other recombinants only in the polymerase region, and in a similar fashion 1 recombinant NoV was closely related to another only in the capsid region. Breakpoint analysis of recombinant NoV showed that recombination occurred in the open reading frame (ORF)1/ORF2 overlap. We provide evidence to support the theory of the role of subgenomic RNA promoters as recombination hotspots and describe a simple mechanism of how recombination might occur in NoV.


Dynamic visual information facilitates object recognition from novel viewpoints.
Normally, people have difficulties recognizing objects from novel as compared to learned views, resulting in increased reaction times and errors. Recent studies showed, however, that this "view-dependency" can be reduced or even completely eliminated when novel views result from observer's movements instead of object movements. This observer movement benefit was previously attributed to extra-retinal (physical motion) cues. In two experiments, we demonstrate that dynamic visual information (that would normally accompany observer's movements) can provide a similar benefit and thus a potential alternative explanation. Participants performed sequential matching tasks for Shepard-Metzler-like objects presented via head-mounted display. As predicted by the literature, object recognition performance improved when view changes (45° or 90°) resulted from active observer movements around the object instead of object movements. Unexpectedly, however, merely providing dynamic visual information depicting the viewpoint change showed an equal benefit, despite the lack of any extra-retinal/physical self-motion cues. Moreover, visually simulated rotations of the table and hidden target object (table movement condition) yielded similar performance benefits as simulated viewpoint changes (scene movement condition). These findings challenge the prevailing notion that extra-retinal (physical motion) cues are required for facilitating object recognition from novel viewpoints, and highlight the importance of dynamic visual cues, which have previously received little attention.


What drives the occurrence of the melioidosis bacterium Burkholderia pseudomallei in domestic gardens?
Melioidosis is an often fatal infectious disease affecting humans and animals in tropical regions and is caused by the saprophytic environmental bacterium Burkholderia pseudomallei. Domestic gardens are not only a common source of exposure to soil and thus to B. pseudomallei, but they also have been found to contain more B. pseudomallei than other environments. In this study we addressed whether anthropogenic manipulations common to gardens such as irrigation or fertilizers change the occurrence of B. pseudomallei. We conducted a soil microcosm experiment with a range of fertilizers and soil types as well as a longitudinal interventional study over three years on an experimental fertilized field site in an area naturally positive for B. pseudomallei. Irrigation was the only consistent treatment to increase B. pseudomallei occurrence over time. The effects of fertilizers upon these bacteria depended on soil texture, physicochemical soil properties and biotic factors. Nitrates and urea increased B. pseudomallei load in sand while phosphates had a positive effect in clay. The high buffering and cation exchange capacities of organic material found in a commercial potting mix led to a marked increase in soil salinity with no survival of B. pseudomallei after four weeks in the potting mix sampled. Imported grasses were also associated with B. pseudomallei occurrence in a multivariate model. With increasing population density in endemic areas these findings inform the identification of areas in the anthropogenic environment with increased risk of exposure to B. pseudomallei.


Effect of jaw muscle pain and soreness evoked by capsaicin before sleep on orofacial motor activity during sleep.
Sleep bruxism, which is a form of orofacial motor activity (OMA), and jaw muscle pain and soreness have for a long time been thought to be mutually linked. The aim of this study was to investigate the effect of clinical and experimental jaw muscle pain and soreness on sleep OMA. Twelve healthy subjects aged 21 to 31 years old participated in this study. All of them were aware of signs or symptoms of sleep OMA and were subdivided into a group with clinical pain complaints (n = 5) and a group without pain (n = 7). All subjects slept in the laboratory for 3 consecutive nights, including a habituation night, a baseline night, and an experimental night. Electroencephalographic (EEG) activity and electromyographic (EMG) activity from the masseter muscles were recorded during sleep. On the experimental night, before sleep, all subjects received an injection of capsaicin (0.1 mL, 100 micrograms/mL) into the masseter muscle that had demonstrated the most EMG activity during the previous recordings. The OMA events and episodes were quantified and were compared between the baseline night and the experimental night. Every evening and morning during the study period, pain intensity, unpleasantness, and soreness were scored by the subjects on a visual analog scale (VAS), and pain detection thresholds (PDTs) in the masseter muscles and maximal voluntary occlusal force (MVOF) were also measured. Pre-sleep injection of capsaicin did not cause significant differences between groups in peak pain intensity on the VAS. The PDT and MVOF did not show any significant differences between groups, injection and non-injection sides, or baseline and experimental nights and mornings. The number of EMG episodes/hour sleep, the number of bursts/hour sleep, and total area of all bursts and episodes during the baseline night were significantly higher in the subjects without pain than in the subjects with pain. However, the capsaicin injection did not cause any significant changes in these parameters. This study suggests that an acute pre-sleep painful stimulus does not have any effect on OMA during sleep, but the study extends previous findings that clinical jaw muscle pain and soreness are associated with less EMG activity in the masticatory muscles.


The effect of background babble on working memory in young and middle-aged adults.
Background noise has been found to negatively affect working memory. Numerous studies have also found that older adults perform more poorly on working memory tasks than young adults (YA). Hearing status has often been a confounding factor in older individuals. Therefore, it would be beneficial to investigate working memory functions in adverse listening conditions early in the aging process (i.e., middle-age), when hearing function is relatively unaffected. The focus of this study was to determine the influence of background babble on working memory in YA and middle-aged adults (MA) with normal hearing. Before testing was begun, we established that all participants could correctly identify words in a degraded experimental testing environment with 100% accuracy. Then, the participants listened to lists composed of five pairs of words in quiet and in 20-talker babble. After the final word pair, the participants were cued with the first word of one of the previous five word pairs. The participants were required to write down the second word of the pair. The percent correct scores for each of the five serial positions were analyzed comparing the two listening conditions for YA and MA. Ten YA and ten MA with normal hearing between 250-8000 Hz and a score of at least 26/30 on the Mini-Mental State Examination participated in the study. As different cognitive processes are used for initial, middle, and final serial positions, averaged scores were obtained for Positions 2 and 3 and for Positions 4 and 5. Subsequently, repeated-measures analyses of variance (ANOVAs) were conducted on mean scores of correctly recalled word pairs with serial positions (initial, middle, and final) and listening condition (quiet, babble) as the within-participant variables and age group (YA, MA) as the between-participant independent variable. This OMNIBUS repeated-measures ANOVA was then followed up with separate repeated-measures ANOVAS for the initial, middle, and final positions. Correct recall scores were lower for early positions compared with the latter positions, irrespective of listening condition. For Position 1, YA-but not MA-performed significantly better in babble than in quiet. For the middle positions (Positions 2 and 3), MA performed significantly more poorly than the YA irrespective of listening condition. For the final positions (Positions 4 and 5), no age differences or effects of listening condition were found. The results indicate that both YA and MA have trouble recalling earlier pieces of information in quiet and in babble. However, MA exhibited significantly poorer recall scores than YA in babble for Position 1, which suggest that cognitive processes related to memory encoding and retrieval are different in background babble for MA and YA.


Phagocytic activity and monocyte chemotactic protein expression by pulmonary macrophages in persistent pulmonary cryptococcosis.
The mechanisms by which Cryptococcus neoformans persists in an immunocompetent host are not well understood. Using a rat model of persistent infection, we investigated the ability of pulmonary macrophages (PuM) to phagocytize C. neoformans and produce monocyte chemotactic protein 1 (MCP-1) as a function of the length of time of infection and opsonin. The ability of macrophages to affect serum-mediated phagocytosis varied over the course of infection and was dependent on CD11b/c and CD18 expression. Infection resulted in increased MCP-1 levels within the lung, though the actual amounts varied over the course of infection. Immunohistochemical studies localized MCP-1 expression to macrophages and epithelioid cells. Enhanced production of MCP-1 by PuM from infected rats was confirmed by ex vivo studies. Induction of MCP-1 following serum-mediated phagocytosis was observed for PuM from both infected and noninfected rats and depended on the interaction of C. neoformans with CD11b/c and CD18. Specific antibody was more efficient than serum in promoting phagocytosis and consistently elicited more MCP-1. The relative amount of MCP-1 produced in association with phagocytosis was similar for PuM at all lengths of time of infection. Decreased MCP-1 production was observed for PuM obtained from older rats, including long-term (8 to 10 months)-infected and age-matched controls, suggesting that aging may affect the production of MCP-1 by PuM in response to cryptococcal infection. In summary, our results show that macrophages are an important source of MCP-1 during pulmonary cryptococcosis and that MCP-1 production is actively regulated during infection. Furthermore, we find that phagocytosis of C. neoformans can serve as an important stimulus for MCP-1 production by PuM, though the efficiency of this process is dependent on the opsonin type and may be affected by aging.


Myocardial infarction in the young.
An increasing number of patients under 40 years of age are being hospitalized with the diagnosis of acute myocardial infarction. This is partly due to the increased prevalance of risk factors for atherosclerosis in the younger age group; especially increased incidence of impaired fasting glucose, high triglyceride, low high-density lipoprotein levels and increased waist to hip ratio. However, non-atherosclerotic coronary artery disease or hypercoagulability should also be investigated or at least suspected in the younger patients. The pathophysiology of different clinical conditions and disease states which cause acute coronary syndromes in the young patients are reviewed, and the diagnostic modalities and therapatic options for these conditions are briefly discussed by searching for "premature atherosclerosis", "hypercoagulable states", "risk factors for atherosclerosis in youth", "novel risk factors for atherosclerosis", "non-atherosclerotic coronary artery diseases" in PubMed.


Honokiol Induces Autophagic Apoptosis in Neuroblastoma Cells through a P53-Dependent Pathway.
In children, neuroblastomas are the most common and deadly solid tumor. Our previous studies showed that honokiol can cross the blood-brain barrier and kill neuroblastoma cells. In this study, we further evaluated if exposure to honokiol for short periods could induce autophagy and subsequent apoptosis of neuroblastoma cells and possible mechanisms. Exposure of neuroblastoma neuro-2a cells to honokiol for 24 h induced morphological shrinkage and cell death. As to the mechanisms, honokiol consecutively induced cytochrome c release from mitochondria, caspase-3 activation, DNA fragmentation and cell apoptosis. Separately, honokiol time-dependently augmented the proportion of autophagic cells and the ratio of light chain 3 (LC3)-II/LC3-I. Pretreatment of neuro-2a cells with 3-methyladenine, an inhibitor of autophagy, attenuated honokiol-induced cell autophagy, caspase-3 activation, DNA damage and cell apoptosis. In contrast, stimulation of autophagy by rapamycin, an inducer of autophagy, significantly enhanced honokiol-induced cell apoptosis. Furthermore, honokiol-induced autophagic apoptosis was confirmed in neuroblastoma NB41A3 cells. Knocking down translation of p53 using RNA interference attenuated honokiol-induced autophagy and apoptosis in neuro-2a and NB41A3 cells. Taken together, this study showed that at early periods, honokiol can induce autophagic apoptosis of neuroblastoma cells through activating a p53-dependent mechanism. Consequently, honokiol has the potential to be a therapeutic option for neuroblastomas.


IV vs. IA TPA in acute ischemic stroke with CT angiographic evidence of major vessel occlusion: a feasibility study.
Studies suggest that stroke patients with thrombus in a major cerebral vessel respond less favorably to intravenous (IV) thrombolysis. The purpose of this study was to test the feasibility of a protocol comparing IV versus intra-arterial (IA) recombinant tissue plasminogen activator (TPA) in an acute ischemic stroke with major vessel occlusion. Consecutive ischemic stroke patients presenting <3 h from symptom onset with major vessel occlusion on CT angiogram (CTA) were randomly assigned to IV TPA (per NINDS protocol) or IA TPA (22 mg over 2 h). Demographics, times to presentation and thrombolysis, presenting NIH stroke scale (NIHSS) and 90-day NIHSS, Barthel Index, and modified Rankin Scale were recorded. CT-scans at 24-h were reviewed for presence of hemorrhage. Recanalization was determined by post-procedure MR angiograms, which are obtained the day after thrombolytic therapy. Seven patients (median NIHSS = 16) were randomized to IV (N = 4) or IA (N = 3) TPA. There were no significant differences in the presentation NIHSS, time to presentation, or time to treatment between the two groups. Hemorrhage was noted in one patient in the IA group (asymptomatic) and one patient in the IV group (symptomatic). Recanalization was seen in all patients treated with IA TPA and none treated with IV TPA (P = 0.03, Fisher's Exact test). We found that it is feasible to conduct a trial comparing IV vs. IA TPA in ischemic stroke patients with major vessel occlusion presenting <3 h from onset. Patients treated with IA TPA showed a trend toward higher rate of recanalization. A larger trial may be designed to test safety and effectiveness of IA TPA in this specific group of stroke patients.


Inhibitory effects of bupivacaine and lidocaine on adrenergic neuroeffector junctions in rat tail artery.
Various local anesthetic agents have been shown to cause relaxation of isolated vascular segments contracted by catecholamines and other constrictor drugs. This report describes the actions of the amide-linked local anesthetic, bupivacaine, on adrenergic responsiveness of isolated arterial smooth muscle, and compares bupivacaine effects with those of lidocaine. Helical strips of rat tail artery mounted in a muscle bath for measurement of isometric force generation were contracted in response to adrenergic nerve stimulation, increased potassium concentration, tyramine, or exogenous norepinephrine. Treatment with bupivacaine or lidocaine caused depression of contraction to all four stimuli. Contraction to adrenergic nerve stimulation was more sensitive to the inhibitory effects of local anesthetics than was contraction to elevated potassium, tyramine, or exogenous norepinephrine. Furthermore, bupivacaine was more effective in reducing contraction to adrenergic nerve stimulation than was lidocaine (EC50: bupivacaine = 4 x 10(-6) M; lidocaine = 61 x 10(-6) M). In arteries incubated in solutions containing [3H]-norepinephrine and mounted for superfusion and isometric force recording, both bupivacaine and lidocaine (10(-5) M) depressed the contractions and diminished the release of radioactivity evoked by nerve stimulation. At the concentration tested, bupivacaine was more effective than lidocaine in reducing both contraction and the efflux of radioactivity as indicated by the magnitude of depression compared with control activities. These findings suggest that lidocaine and bupivacaine depress adrenergic neurotransmission and inhibit smooth muscle contraction. Bupivacaine is a more potent inhibitor of adrenergic neurotransmission in the blood vessel wall than is lidocaine.


Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins.
Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or high- molecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28- to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.


A circuit supporting concentration-invariant odor perception in Drosophila.
Most odors are perceived to have the same quality over a large concentration range, but the neural mechanisms that permit concentration-invariant olfactory perception are unknown. In larvae of the vinegar fly Drosophila melanogaster, odors are sensed by an array of 25 odorant receptors expressed in 21 olfactory sensory neurons (OSNs). We investigated how subsets of larval OSNs with overlapping but distinct response properties cooperate to mediate perception of a given odorant across a range of concentrations. Using calcium imaging, we found that ethyl butyrate, an ester perceived by humans as fruity, activated three OSNs with response thresholds that varied across three orders of magnitude. Whereas wild-type larvae were strongly attracted by this odor across a 500-fold range of concentration, individuals with only a single functional OSN showed attraction across a narrower concentration range corresponding to the sensitivity of each ethyl butyrate-tuned OSN. To clarify how the information carried by different OSNs is integrated by the olfactory system, we characterized the response properties of local inhibitory interneurons and projection neurons in the antennal lobe. Local interneurons only responded to high ethyl butyrate concentrations upon summed activation of at least two OSNs. Projection neurons showed a reduced response to odors when summed input from two OSNs impinged on the circuit compared to when there was only a single functional OSN. Our results show that increasing odor concentrations induce progressive activation of concentration-tuned olfactory sensory neurons and concomitant recruitment of inhibitory local interneurons. We propose that the interplay of combinatorial OSN input and local interneuron activation allows animals to remain sensitive to odors across a large range of stimulus intensities.


Optical coherence tomography measurement of macular and nerve fiber layer thickness in normal and glaucomatous human eyes.
To evaluate the hypothesis that macular thickness correlates with the diagnosis of glaucoma. Cross-sectional study. We studied 367 subjects (534 eyes), including 166 eyes of 109 normal subjects, 83 eyes of 58 glaucoma suspects, 196 eyes of 132 early glaucoma patients, and 89 eyes of 68 advanced glaucoma patients. We used optical coherence tomography (OCT) to measure macular and nerve fiber layer (NFL) thickness and to analyze their correlation with each other and with glaucoma status. We used both the commercial and prototype OCT units and evaluated correspondence between measurements performed on the same eyes on the same days. Macular and NFL thickness as measured by OCT. All NFL parameters both in prototype and commercial OCT units were statistically significantly different comparing normal subjects and either early or advanced glaucoma (P < 0.001). Inner ring, outer ring, and mean macular thickness both in prototype and commercial OCT devices were found to be significantly different between normal subjects and advanced glaucomatous eyes (P < 0.001). The outer ring was the only macular parameter that could significantly differentiate between normal and early glaucoma with either the prototype or commercial OCT unit (P = 0.003, P = 0.008, respectively). The area under the receiver operator characteristic (AROC) curves comparing mean NFL thickness between normal and advanced glaucomatous eyes was 1.00 for both the prototype and commercial OCT devices for eyes scanned on both machines on the same day. The AROC comparing mean macular thickness in normal and advanced glaucomatous eyes scanned on both machines on the same day was 0.88 for the prototype OCT device and 0.80 for the commercial OCT. Both macular and NFL thickness as measured by OCT showed statistically significant correlations with glaucoma, although NFL thickness showed a stronger association than macular thickness. There was good correspondence between findings using both the prototype and commercial OCT units. Macular and NFL thickness measurements made with OCT may have usefulness in the clinical assessment of glaucoma.


Patient enablement requires physician empathy: a cross-sectional study of general practice consultations in areas of high and low socioeconomic deprivation in Scotland.
Patient 'enablement' is a term closely aligned with 'empowerment' and its measurement in a general practice consultation has been operationalised in the widely used patient enablement instrument (PEI), a patient-rated measure of consultation outcome. However, there is limited knowledge regarding the factors that influence enablement, particularly the effect of socio-economic deprivation. The aim of the study is to assess the factors influencing patient enablement in GP consultations in areas of high and low deprivation. A questionnaire study was carried out on 3,044 patients attending 26 GPs (16 in areas of high socio-economic deprivation and 10 in low deprivation areas, in the west of Scotland). Patient expectation (confidence that the doctor would be able to help) was recorded prior to the consultation. PEI, GP empathy (measured by the CARE Measure), and a range of other measures and variables were recorded after the consultation. Data analysis employed multi-level modelling and multivariate analyses with the PEI as the dependant variable. Although numerous variables showed a univariate association with patient enablement, only four factors were independently predictive after multilevel multivariate analysis; patients with multimorbidity of 3 or more long-term conditions (reflecting poor chronic general health), and those consulting about a long-standing problem had reduced enablement scores in both affluent and deprived areas. In deprived areas, emotional distress (GHQ-caseness) had an additional negative effect on enablement. Perceived GP empathy had a positive effect on enablement in both affluent and deprived areas. Maximal patient enablement was never found with low empathy. Although other factors influence patient enablement, the patients' perceptions of the doctors' empathy is of key importance in patient enablement in general practice consultations in both high and low deprivation settings.


Effect of chronic ethanol feeding on Kupffer cell-mediated antitumor cell activity.
We have previously reported that the Kupffer cell has antitumor activity through mitochondrial damage to tumor cells by nitric oxide production. In this study, the effect of chronic ethanol feeding on antihepatoma cell activity of the Kupffer cell was examined in rats. Male rats of the Wistar strain were fed ethanol chronically for 8 weeks by liquid diets. Kupffer cells were isolated from the control rat or the ethanol-fed rat, and cocultured with AH 70 cells, a rat hepatoma cell line. Fluorescence of rhodamine 123 or propidium iodide was observed as indicators of the mitochondrial damage or cell membrane injury, respectively, by a laser scanning confocal microscopy. Mitochondrial damage of AH 70 cells as indicated by reduction of rhodamine 123 fluorescence was smaller by the coculture with Kupffer cell from the ethanol rat than that from the control. Cell membrane barrier dysfunction of AH 70 cell was less frequently observed with the Kupffer cell from ethanol-fed rats. A metabolite of nitric oxide (nitrite and nitrate) was less in the cultured medium with the ethanol Kupffer cell than with the control Kupffer cell. Ca2+ mobilization, which induces inducible nitric oxide synthase and observed by the fluorescence of fluo-3, in Kupffer cells cocultured with AH 70 cells was suppressed in ethanol-fed rats. These result suggests that chronic ethanol feeding suppresses antitumor cell activity of Kupffer cell through the impairment of Ca2+ mobilization and nitric oxide production.