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α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate receptors (NMDARs) are key mediators of central cholinergic and glutamatergic neurotransmission, respectively. In addition to numerous well-established functional interactions between α7 nAChRs and NMDARs, the two receptors have been proposed to form a multimeric complex, and in the present study we have investigated this putative α7 nAChR/NMDAR assembly in human and murine brain tissues. By α-bungarotoxin (BGT) affinity purification, α7 and NMDAR subunits were co-purified from human and murine cortical and hippocampal homogenates, substantiating the notion that the receptors are parts of a multimeric complex in the human and rodent brain. Interestingly, the ratios between GluN1 and α7 levels in BGT pull-downs from cortical homogenates from Alzheimer's disease (AD) brains were significantly lower than those in pull-downs from non-AD controls, indicating a reduced degree of α7 nAChR/NMDAR complex formation in the diseased tissue. A similar difference in GluN1/α7 ratios was observed between pull-downs from cortical homogenates from adult 3xTg-AD and age-matched wild type (WT) mice, whereas the GluN1/α7 ratios determined in pull-downs from young 3xTg-AD and age-matched WT mice did not differ significantly. The observation that pretreatment with oligomeric amyloid-β1-42 reduced GluN1/α7 ratios in BGT pull-downs from human cortical homogenate in a concentration-dependent manner provided a plausible molecular mechanism for this observed reduction. In conclusion, while it will be important to further challenge the existence of the putative α7 nAChR/NMDAR complex in future studies applying other methodologies than biochemical assays and to investigate the functional implications of this complex for cholinergic and glutamatergic neurotransmission, this work supports the formation of the complex and presents new insights into its regulation in healthy and diseased brain tissue.

BACKGROUND: Heart rate is routinely measured as part of the clinical examination but is rarely acted upon unless it is well outside a population-based normal range. With wearable sensor technologies, heart rate can now be continuously measured, making it possible to accurately identify an individuals normal heart rate and potentially important variations in it over time. Our objective is to describe inter- and intra-individual variability in resting heart rate (RHR) collected over the course of two years using a wearable device, studying the variations of resting heart rate as a function of time of year, as well as individuals characteristics like age, sex, average sleep duration, and body mass index (BMI). METHODS AND FINDINGS: Our retrospective, longitudinal cohort study includes 92,457 de-identified individuals from the United States (all 50 states), who consistently-over at least 35 weeks in the period from March 2016 to February 2018, for at least 2 days per week, and at least 20 hours per day-wore a heart rate wrist-worn tracker. In this study, we report daily RHR and its association with age, BMI, sex, and sleep duration, and its variation over time. Individual daily RHR was available for a median of 320 days, providing nearly 33 million daily RHR values. We also explored the range in daily RHR variability between individuals, and the long- and short-term changes in the trajectory of an individuals daily RHR. Mean daily RHR was 65 beats per minute (bpm), with a range of 40 to 109 bpm among all individuals. The mean RHR differed significantly by age, sex, BMI, and average sleep duration. Time of year variations were also noted, with a minimum in July and maximum in January. For most subjects, RHR remained relatively stable over the short term, but 20% experienced at least 1 week in which their RHR fluctuated by 10 bpm or more. CONCLUSIONS: Individuals have a daily RHR that is normal for them but can differ from another individuals normal by as much as 70 bpm. Within individuals, RHR was much more consistent over time, with a small but significant seasonal trend, and detectable discrete and infrequent episodes outside their norms.

Eastern equine encephalitis virus (EEEV) causes a highly pathogenic mosquito-borne zoonosis that is responsible for sporadic outbreaks of severe illness in humans and equines in the eastern USA. Culiseta (Cs.) melanura is the primary vector of EEEV in most geographic regions but its feeding patterns on specific avian and mammalian hosts are largely unknown in the mid-Atlantic region. The objectives of our study were to: 1) identify avian hosts of Cs. melanura and evaluate their potential role in enzootic amplification of EEEV, 2) assess spatial and temporal patterns of virus activity during a season of intense virus transmission, and 3) investigate the potential role of Cs. melanura in epidemic/epizootic transmission of EEEV to humans and equines. Accordingly, we collected mosquitoes at 55 sites in Suffolk, Virginia in 2013, and identified the source of blood meals in engorged mosquitoes by nucleotide sequencing PCR products of the mitochondrial cytochrome b gene. We also examined field-collected mosquitoes for evidence of infection with EEEV using Vector Test, cell culture, and PCR. Analysis of 188 engorged Cs. melanura sampled from April through October 2013 indicated that 95.2%, 4.3%, and 0.5% obtained blood meals from avian, mammalian, and reptilian hosts, respectively. American Robin was the most frequently identified host for Cs. melanura (42.6% of blood meals) followed by Northern Cardinal (16.0%), European Starling (11.2%), Carolina Wren (4.3%), and Common Grackle (4.3%). EEEV was detected in 106 mosquito pools of Cs. melanura, and the number of virus positive pools peaked in late July with 22 positive pools and a Maximum Likelihood Estimation (MLE) infection rate of 4.46 per 1,000 mosquitoes. Our findings highlight the importance of Cs. melanura as a regional EEEV vector based on frequent feeding on virus-competent bird species. A small proportion of blood meals acquired from mammalian hosts suggests the possibility that this species may occasionally contribute to epidemic/epizootic transmission of EEEV.

Frailty is an established risk factor for adverse outcomes following non-cardiac surgery. The Hospital Frailty Risk Score (HFRS) is a recently described frailty assessment tool that harnesses administrative data and is composed of 109 International Classification of Disease variables. We aimed to examine the incremental prognostic utility of the HFRS in a generalizable surgical population. Using linked administrative databases, a retrospective cohort of patients admitted for non-cardiac surgery between October 1st, 2008 and September 30th, 2019 in Alberta, Canada was created. Our primary outcome was a composite of death, myocardial infarction or cardiac arrest at 30-days. Multivariable logistic regression was undertaken to assess the impact of HFRS on outcomes after adjusting for age, sex, components of the Charlson Comorbidity Index (CCI), Revised Cardiac Risk Index (RCRI) and peri-operative biomarkers. The final cohort consisted of 712,808 non-cardiac surgeries, of which 55·1% were female and the average age was 53·4 +/- 22·4 years. Using the HFRS, 86.3% were considered low risk, 10·7% were considered intermediate risk and 3·1% were considered high risk for frailty. Intermediate and high HFRS scores were associated with increased risk of the primary outcome with an adjusted odds ratio of 1·61 (95% CI 1·50-1.74) and 1·55 (95% CI 1·38-1·73). Intermediate and high HFRS were also associated with increased adjusted odds of prolonged hospital stay, in-hospital mortality, and 1-year mortality. The HFRS is a minimally onerous frailty assessment tool that can complement perioperative risk stratification in identifying patients at high risk of short- and long-term adverse events.

Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians.

BackgroundThis systematic review described the association between electronic nicotine delivery systems and electronic non-nicotine delivery systems (ENDS/ENNDS) use among non-smoking children and adolescents aged MethodsWe searched five electronic databases and the grey literature up to end of September 2020. Prospective longitudinal studies that described the association between ENDS/ENNDS use, and subsequent tobacco use in those aged < 20 years who were non-smokers at baseline were included. The Joanna Briggs Institute Critical Appraisal Checklist was used to assess risk of bias. Data were extracted by two reviewers and pooled using a random-effects meta-analysis. We generated unadjusted and adjusted risk ratios (ARRs) describing associations between ENDS/ENNDS and tobacco use.FindingsA total of 36 publications met the eligibility criteria, of which 25 were included in the systematic review (23 in the meta-analysis) after exclusion of overlapping studies. Sixteen studies had high to moderate risk of bias. Ever users of ENDS/ENNDS had over three times the risk of ever cigarette use (ARR 3·01 (95% CI: 2·37, 3·82; pConclusionThere is an urgent need for policies that regulate the availability, accessibility, and marketing of ENDS/ENNDS to children and adolescents. Governments should also consider adopting policies to prevent ENDS/ENNDS uptake and use in children and adolescents, up to and including a ban for this group.

BackgroundHeart rate is routinely measured as part of the clinical examination but is rarely acted upon unless it is well outside a population-based normal range. With wearable sensor technologies, heart rate can now be continuously measured, making it possible to accurately identify an individual's "normal" heart rate and potentially important variations in it over time. Our objective is to describe inter- and intra-individual variability in resting heart rate (RHR) collected over the course of two years using a wearable device, studying the variations of resting heart rate as a function of time of year, as well as individuals characteristics like age, sex, average sleep duration, and body mass index (BMI).Methods and findingsOur retrospective, longitudinal cohort study includes 92,457 de-identified individuals from the United States (all 50 states), who consistently-over at least 35 weeks in the period from March 2016 to February 2018, for at least 2 days per week, and at least 20 hours per day-wore a heart rate wrist-worn tracker. In this study, we report daily RHR and its association with age, BMI, sex, and sleep duration, and its variation over time. Individual daily RHR was available for a median of 320 days, providing nearly 33 million daily RHR values. We also explored the range in daily RHR variability between individuals, and the long- and short-term changes in the trajectory of an individual's daily RHR. Mean daily RHR was 65 beats per minute (bpm), with a range of 40 to 109 bpm among all individuals. The mean RHR differed significantly by age, sex, BMI, and average sleep duration. Time of year variations were also noted, with a minimum in July and maximum in January. For most subjects, RHR remained relatively stable over the short term, but 20% experienced at least 1 week in which their RHR fluctuated by 10 bpm or more.ConclusionsIndividuals have a daily RHR that is normal for them but can differ from another individual's normal by as much as 70 bpm. Within individuals, RHR was much more consistent over time, with a small but significant seasonal trend, and detectable discrete and infrequent episodes outside their norms.

BackgroundRisk factors for cardiovascular disease (CVD) are negatively correlated with socio-economic status (SES) in high-income countries (HIC) but there has been little research on their distribution by household SES within low-and middle-income countries (LMICs). Considering the limited data from LMICs, this paper examines the association between behavioural and cardiovascular risk factors and household SES in Papua New Guinea (PNG).MethodsReported here are results of 671 participants from the 900 randomly selected adults aged 15-65 years. These adults were recruited from three socioeconomically and geographically diverse surveillance sites (peri-urban community, rural Highland and an Island community) in PNG in 2013-2014. We measured their CVD risk factors (behavioural and metabolic) using a modified WHO STEPS risk factor survey and analysis of blood samples. We assessed SES by education, occupation and creating a household wealth index based on household assets. We calculated risk ratios (RR) and their 95% confidence intervals (CI) using a generalized linear model to assess the associations between risks and SES.FindingsElevated CVD risk factors were common in all SES groups but the CVD metabolic risk factors were most prevalent among homemakers, peri-urban and rural highlands, and the highest (4th and 5th) wealth quintile population. Adults in the highest wealth quintile had high risks of obesity, elevated HbA1c and metabolic syndrome (MetS) that were greater than those in the lowest quintile although those in the highest wealth quintiles were less likely to smoke tobacco. Compared to people from the Island community, peri-urban residents had increased risks of increased waist circumference (WC) (RR: 1.67, 95%CI: 1.21-2.31), hypertension (RR: 2∙29, 95%CI: 1∙89-4.56), high cholesterol (RR: 2∙22, 95%CI: 1∙20-4∙10), high triglycerides (RR: 1∙49, 95%CI: 1∙17-1∙91), elevated HbA1c (RR: 5∙54, 95%CI: 1∙36-21∙56), and Metabolic syndrome (MetS) (RR: 2∙04, 95%CI: 1∙25-3∙32). Similarly, Rural Highland residents had increased risk of obesity (Waist Circumference RR: 1∙70, 95%CI: 1∙21-3∙38, Waist-Hip-Ratio RR:1∙48, 95%CI: 1∙28-1∙70), hypertension (RR: 2∙60, 95%CI: 1∙71-3∙95), high triglycerides (RR: 1∙34, 95%CI: 1∙06-1∙70) and MetS (RR: 1∙88, 95%CI: 1∙12-3∙16) compared to those in the rural Island site.InterpretationCVD risk factors are common in PNG adults but their association with SES varies markedly and by location. Our findings show that all community members are at risk of CVD weather they are part of high or low SES groups. These results support the notion that the association between CVD risk factors and SES differ greatly accordingly to the type of SES measure used, risk factors and the population studied. In addition, our findings contribute further to the limited literature in LMIC. Longitudinal studies are needed to monitor changes in rapidly changing societies such as PNG to inform public health policy for control and prevention of NCDs in the country.

More countries are using verbal autopsy as a part of routine mortality surveillance. The length of time required to complete a verbal autopsy interview is a key logistical consideration for planning large-scale surveillance.We use the PHMRC shortened questionnaire to conduct verbal autopsy interviews at three sites and collect data on the length of time required to complete the interview. This instrument uses a novel checklist of keywords to capture relevant information from the open response. The open response section is timed separately from the section consisting of closed questions.We found the median time to complete the entire interview was approximately 25 minutes and did not vary substantially by age-specific module. The median time for the open response section was approximately 4 minutes and 60% of interviewees mentioned at least one keyword within the open response section.The length of time required to complete the interview was short enough for large-scale routine use. The open-response section did not add a substantial amount of time and provided useful information which can be used to increase the accuracy of the predictions of the cause of death. The novel checklist approach further reduces the burden of transcribing and translating a large amount of free text. This makes the PHMRC instrument ideal for national mortality surveillance.

In recent years, the government of the Philippines embarked upon an ambitious Universal Health Care program, underpinned by the rapid scale-up of subsidized insurance coverage for poor and vulnerable populations. With a view of reducing the stubbornly high maternal mortality rates in the country, the program has a strong focus on maternal health services and is supported by a national policy of universal facility-based delivery (FBD). In this study, we examine the impact that recent reforms expanding health insurance coverage have had on FBD.Data from the most recent Philippines 2013 Demographic Health Survey was employed. This study applies quasi-experimental methods using propensity scores along with alternative matching techniques and weighted regression to control for self-selection and investigate the impact of health insurance on the utilization of FBD.Our findings reveal that the likelihood of FBD for women who are insured is between 5 to 10 percent higher than for those without insurance. The impact of health insurance is more pronounced amongst rural and poor women for whom insurance leads to a 9 to 11 per cent higher likelihood of FBD.We conclude that increasing health insurance coverage is likely to be an effective approach to increase women's access to FBD. Our findings suggest that when such coverage is subsidized, as it is the case in the Philippines, women from poor and rural populations are likely to benefit the most.

Human mate choice research often concerns sex differences in the importance of traits such as physical attractiveness and social status. A growing number of studies indicate that cues to social context, including other people who appear in stimulus photographs, can alter that individual's attractiveness. Fewer studies, however, consider judgements of traits other than physical attractiveness, such as wealth. Here we manipulate the presence/absence of other people in photographs of target models, and test the effects on judgments of both attractiveness and earnings (a proxy for status). Participants (N = 2044) rated either male or female models for either physical attractiveness or social/economic status when presented alone, with same sex others or with opposite sex others. We collectively refer to this manipulation as 'social context'. Male and female models received similar responses for physical attractiveness, but social context affected ratings of status differently for women and men. Males presented alongside other men received the highest status ratings while females presented alone were given the highest status ratings. Further, the status of females presented alongside a male was constrained by the rated status of that male. Our results suggests that high status may not directly lead to high attractiveness in men, but that status is more readily attributed to men than to women. This divide in status between the sexes is very clear when men and women are presented together, possibly reflecting one underlying mechanism of the modern day gender gap and sexist attitudes to women's economic participation. This adds complexity to our understanding of the relationship between attractiveness, status, and sex in the light of parental investment theory, sexual conflict and economic theory.

One of the greatest obstacles facing efforts to address quality of care in low and middle income countries is the absence of relevant and reliable data. This article proposes a methodology for creating a single "Quality Index" (QI) representing quality of maternal and neonatal health care based upon data collected as part of the Demographic and Health Survey (DHS) program.Using the 2012 Indonesian Demographic and Health Survey dataset, indicators of quality of care were identified based on the recommended guidelines outlined in the WHO Integrated Management of Pregnancy and Childbirth. Two sets of indicators were created; one set only including indicators available in the standard DHS questionnaire and the other including all indicators identified in the Indonesian dataset. For each indicator set composite indices were created using Principal Components Analysis and a modified form of Equal Weighting. These indices were tested for internal coherence and robustness, as well as their comparability with each other. Finally a single QI was chosen to explore the variation in index scores across a number of known equity markers in Indonesia including wealth, urban rural status and geographical region.The process of creating quality indexes from standard DHS data was proven to be feasible, and initial results from Indonesia indicate particular disparities in the quality of care received by the poor as well as those living in outlying regions.The QI represents an important step forward in efforts to understand, measure and improve quality of MNCH care in developing countries.

The rapid progress in rice genotyping must be matched by advances in phenotyping. A better understanding of genetic variation in rice for drought response, root traits, and practical methods for studying them are needed. In this study, the OryzaSNP set (20 diverse genotypes that have been genotyped for SNP markers) was phenotyped in a range of field and container studies to study the diversity of rice root growth and response to drought. Of the root traits measured across more than 20 root experiments, root dry weight showed the most stable genotypic performance across studies. The environment (E) component had the strongest effect on yield and root traits. We identified genomic regions correlated with root dry weight, percent deep roots, maximum root depth, and grain yield based on a correlation analysis with the phenotypes and aus, indica, or japonica introgression regions using the SNP data. Two genomic regions were identified as hot spots in which root traits and grain yield were co-located; on chromosome 1 (39.7-40.7 Mb) and on chromosome 8 (20.3-21.9 Mb). Across experiments, the soil type/ growth medium showed more correlations with plant growth than the container dimensions. Although the correlations among studies and genetic co-location of root traits from a range of study systems points to their potential utility to represent responses in field studies, the best correlations were observed when the two setups had some similar properties. Due to the co-location of the identified genomic regions (from introgression block analysis) with QTL for a number of previously reported root and drought traits, these regions are good candidates for detailed characterization to contribute to understanding rice improvement for response to drought. This study also highlights the utility of characterizing a small set of 20 genotypes for root growth, drought response, and related genomic regions.

Managed aquifer recharge (MAR) can provide irrigation water and overcome water scarcity in agriculture. Removal of potentially present plant pathogens during MAR is essential to prevent crop diseases. We studied the die-off of three plant pathogenic bacteria in water microcosms with natural or filtered tile drainage water (TDW) at 10 and 25°C and with natural anoxic aquifer water (AW) at 10°C from a MAR site. These bacteria were: Ralstonia solanacearum (bacterial wilt), and the soft rot Pectobacteriaceae (SRP) Dickeya solani and Pectobacterium carotovorum sp. carotovorum (soft rot, blackleg). They are present in surface waters and cause destructive crop diseases worldwide which have been linked to contaminated irrigation water. Nevertheless, little is known about the survival of the SRP in aqueous environments and no study has investigated the persistence of R. solanacearum under natural anoxic conditions. We found that all bacteria were undetectable in 0.1 mL samples within 19 days under oxic conditions in natural TDW at 10°C, using viable cell counting, corresponding to 3-log10 reduction by die-off. The SRP were no longer detected within 6 days at 25°C, whereas R. solanacearum was detectable for 25 days. Whereas in anoxic natural aquifer water at 10°C, the bacterial concentrations declined slower and the detection limit was reached within 56 days. Finally, we modelled the inactivation curves with a modified Weibull model that can simulate different curve shapes such as shoulder phenomena in the beginning and long tails reflecting persistent bacterial populations. The non-linear model was shown to be a reliable tool to predict the die-off of the analysed plant pathogenic bacteria, suggesting its further application to other pathogenic microorganisms in the context of microbial risk assessment., Competing Interests: The authors declare no competing interests in general and with any of the commercial affiliations. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Careful design of experiments using living organisms (e.g. mice) is of critical importance from both an ethical and a scientific standpoint. Randomization should, whenever possible, be an integral part of such experimental design to reduce bias thereby increasing its reliability and reproducibility. To keep the sample size as low as possible, one might take randomization one step further by controlling for baseline variations in the dependent variable(s) and/or certain known covariates. To give an example, in animal experiments aimed to study atherosclerosis development, one would want to control for baseline characteristics such as plasma triglyceride and total cholesterol levels and body weight. This can be done by first defining blocks to create balance among groups in terms of group size and baseline characteristics, followed by random assignment of the blocks to the various control and intervention groups. In the current study we developed a novel, user-friendly tool that allows users to easily randomize animals into blocks and identify random block divisions that are well-balanced based on given baseline characteristics, making randomization time-efficient and easy-to-use. Here, we present the resulting software tool that we have named RandoMice., Competing Interests: The authors have declared that no competing interests exist.

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes., Competing Interests: The authors have declared that no competing interests exist.

Bordetella pertussis circulates even in highly vaccinated countries affecting all age groups. Insight into the scale of concealed reinfections is important as they may contribute to transmission. We therefore investigated whether current single-point serodiagnostic methods are suitable to estimate the prevalence of pertussis reinfection. Two methods based on IgG-Ptx plasma levels alone were used to evaluate the proportion of renewed seroconversions in the past year in a cohort of retrospective pertussis cases ≥ 24 months after a proven earlier symptomatic infection. A Dutch population database was used as a baseline. Applying a classical 62.5 IU/ml IgG-Ptx cut-off, we calculated a seroprevalence of 15% in retrospective cases, higher than the 10% observed in the population baseline. However, this method could not discriminate between renewed seroconversion and waning of previously infection-enhanced IgG-Ptx levels. Two-component cluster analysis of the IgG-Ptx datasets of both pertussis cases and the general population revealed a continuum of intermediate IgG-Ptx levels, preventing the establishment of a positive population and the comparison of prevalence by this alternative method. Next, we investigated the complementary serodiagnostic value of IgA-Ptx levels. When modelling datasets including both convalescent and retrospective cases we obtained new cut-offs for both IgG-Ptx and IgA-Ptx that were optimized to evaluate renewed seroconversions in the ex-cases target population. Combining these cut-offs two-dimensionally, we calculated 8.0% reinfections in retrospective cases, being below the baseline seroprevalence. Our study for the first time revealed the shortcomings of using only IgG-Ptx data in conventional serodiagnostic methods to determine pertussis reinfections. Improved results can be obtained with two-dimensional serodiagnostic profiling. The proportion of reinfections thus established suggests a relatively increased period of protection to renewed infection after clinical pertussis.

Background: Multiple Sclerosis is more common in women than men and females have more relapses than men. In a large international cohort we have evaluated the effect of gender on disability accumulation and disease progression to determine if male MS patients have a worse clinical outcome than females., Methods: Using the MSBase Registry, data from 15,826 MS patients from 25 countries was analysed. Changes in the severity of MS (EDSS) were compared between sexes using a repeated measures analysis in generalised linear mixed models. Kaplan-Meier analysis was used to test for sex difference in the time to reach EDSS milestones 3 and 6 and the secondary progressive MS., Results: In relapse onset MS patients (n = 14,453), males progressed significantly faster in their EDSS than females (0.133 vs 0.112 per year, P<0.001,). Females had a reduced risk of secondary progressive MS (HR (95% CI) = 0.77 (0.67 to 0.90) P = 0.001). In primary progressive MS (n = 1,373), there was a significant increase in EDSS over time in males and females (P<0.001) but there was no significant sex effect on the annualized rate of EDSS change., Conclusion: Among registrants of MSBase, male relapse-onset patients accumulate disability faster than female patients. In contrast, the rate of disability accumulation between male and female patients with primary progressive MS is similar.

Background: Concerns are present on the limited value of renal function alone in defining the optimal moment to start dialysis. Disease-related symptoms and health-related quality of life (HRQOL) may have additional clinical value in defining this moment, but little is known about how these parameters change during pre-dialysis care. The aims of our study were to describe the course of symptoms and HRQOL during pre-dialysis care and to investigate their association with poor health outcomes., Methods: In the prospective PREPARE-2 cohort, incident patients starting specialized pre-dialysis care were included when referred to one of the 25 participating Dutch outpatient clinics (2004-2011). In the present analysis, 436 patients with data available on symptoms and HRQOL were included. Clinical data, symptoms (revised illness perception questionnaire), and HRQOL (short form-36 questionnaire; physical and mental summary score) were collected every 6-month interval. A time-dependent Cox proportional hazard model was used to associate symptoms and HRQOL with the combined poor health outcome (i.e. starting dialysis, receiving a kidney transplant, and death)., Results: All symptoms increased, especially fatigue and loss of strength, and both the physical and mental summary score decreased over time, with the most pronounced change during the last 6-12 months of follow-up. Furthermore, each additional symptom (adjusted HR 1.04 (95% CI, 1.00-1.09)) and each 3-point lower physical and mental summary score (adjusted HR 1.04 (1.02-1.06) and 1.04 (1.02-1.06) respectively) were associated with a higher risk of reaching the combined poor health outcome within the subsequent 6 months., Conclusions: The number of symptoms increased and both the physical and mental HRQOL score decreased during pre-dialysis care and these changes were associated with starting dialysis, receiving a kidney transplant, and death. These results may indicate that symptoms and HRQOL are good markers for the medical condition and disease stage of pre-dialysis patients.

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (Bmem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and Bmem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of Bmem cell populations; higher levels of Bmem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects' levels of specific Bmem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific Bmem cell and plasma IgG levels.

Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4(+) T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4(+) T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4(+) T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.

Background: There is an increasing demand for accurate biomarkers for early non-invasive colorectal cancer detection. We employed a genome-scale marker discovery method to identify and verify candidate DNA methylation biomarkers for blood-based detection of colorectal cancer., Methodology/principal Findings: We used DNA methylation data from 711 colorectal tumors, 53 matched adjacent-normal colonic tissue samples, 286 healthy blood samples and 4,201 tumor samples of 15 different cancer types. DNA methylation data were generated by the Illumina Infinium HumanMethylation27 and the HumanMethylation450 platforms, which determine the methylation status of 27,578 and 482,421 CpG sites respectively. We first performed a multistep marker selection to identify candidate markers with high methylation across all colorectal tumors while harboring low methylation in healthy samples and other cancer types. We then used pre-therapeutic plasma and serum samples from 107 colorectal cancer patients and 98 controls without colorectal cancer, confirmed by colonoscopy, to verify candidate markers. We selected two markers for further evaluation: methylated THBD (THBD-M) and methylated C9orf50 (C9orf50-M). When tested on clinical plasma and serum samples these markers outperformed carcinoembryonic antigen (CEA) serum measurement and resulted in a high sensitive and specific test performance for early colorectal cancer detection., Conclusions/significance: Our systematic marker discovery and verification study for blood-based DNA methylation markers resulted in two novel colorectal cancer biomarkers, THBD-M and C9orf50-M. THBD-M in particular showed promising performance in clinical samples, justifying its further optimization and clinical testing.