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Type 1 diabetes (T1D)-associated hyperglycemia develops, in part, from loss of insulin-secreting beta cells. The degree of glycemic dysregulation and the age at onset of disease can serve as indicators of the aggressiveness of the disease. Tracking blood glucose levels in prediabetic mice may demonstrate the onset of diabetes and, along with animal age, also presage disease severity. In this study, an analysis of blood glucose levels obtained from female NOD mice starting at 4 weeks until diabetes onset was undertaken. New onset diabetic mice were orally vaccinated with a Salmonella-based vaccine towards T1D-associated preproinsulin combined with TGFβ and IL10 along with anti-CD3 antibody. Blood glucose levels were obtained before and after development of disease and vaccination. Animals were classified as acute disease if hyperglycemia was confirmed at a young age, while other animals were classified as progressive disease. The effectiveness of the oral T1D vaccine was greater in mice with progressive disease that had less glucose excursion compared to acute disease mice. Overall, the Salmonella-based vaccine reversed disease in 60% of the diabetic mice due, in part, to lessening of islet inflammation, improving residual beta cell health, and promoting tolerance. In summary, the age of disease onset and severity of glucose dysregulation in NOD mice predicted response to vaccine therapy. This suggests a similar disease categorization in the clinic may predict therapeutic response.

Entomological sampling and storage conditions often prioritise efficiency, practicality and conservation of morphological characteristics, and may therefore be suboptimal for DNA preservation. This practice can impact downstream molecular applications, such as the generation of high-throughput genomic libraries, which often requires substantial DNA input amounts. Here, we use a practical Tn5 transposase tagmentation-based library preparation method optimised for 96-well plates and low yield DNA extracts from insect legs that were stored under sub-optimal conditions for DNA preservation. The samples were kept in field vehicles for extended periods of time, before long-term storage in ethanol in the freezer, or dry at room temperature. By reducing DNA input to 6ng, more samples with sub-optimal DNA yields could be processed. We matched this low DNA input with a 6-fold dilution of a commercially available tagmentation enzyme, significantly reducing library preparation costs. Costs and workload were further suppressed by direct post-amplification pooling of individual libraries. We generated medium coverage (>3-fold) genomes for 88 out of 90 specimens, with an average of approximately 10-fold coverage. While samples stored in ethanol yielded significantly less DNA compared to those which were stored dry, these samples had superior sequencing statistics, with longer sequencing reads and higher rates of endogenous DNA. Furthermore, we find that the efficiency of tagmentation-based library preparation can be improved by a thorough post-amplification bead clean-up which selects against both short and large DNA fragments. By opening opportunities for the use of sub-optimally preserved, low yield DNA extracts, we broaden the scope of whole genome studies of insect specimens. We therefore expect these results and this protocol to be valuable for a range of applications in the field of entomology.

IntroductionThe COVID-19 pandemic disproportionately affected older adults, particularly those with pre-existing chronic health conditions. To address the health disparity and challenges faced by under-resourced African American older adults in South Los Angeles during this period, we implemented a hybrid (virtual/in-person), pre-post, community-based participatory intervention research project utilizing a faith-based lay health advisor model (COVID-19 Health Ambassador Program (CHAP)). We recruited COVID-19 Health Ambassadors (CHAs) and African American older adults (participants) from faith-based organizations who partook in CHA-led meetings and follow-ups that educated and supported the participants. This paper seeks to evaluate this intervention's implementation using the Consolidated Framework for Implementation Research (CFIR) as a reporting tool with an emphasis on fidelity, challenges, and adaptations based on data collected via stakeholder interviews and surveys.ResultsCHAP was delivered to 152 participants by 19 CHAs from 17 faith-based organizations. CHAs assisted with chronic disease management, resolved medication-related challenges, encouraged COVID-19 vaccination, reduced psychological stress and addressed healthcare avoidance behaviors such as COVID-19 vaccine hesitancy among the participants. Challenges encountered include ensuring participant engagement and retention in the virtual format and addressing technological barriers for CHAs and participants. Adaptations made to better suit the needs of participants included providing communication tools and additional training to CHAs to improve their proficiency in using virtual platforms in addition to adapting scientific/educational materials to suit our participants' diverse cultural and linguistic needs.ConclusionThe community-centered hybrid approach in addition to our partnership with faith-based organizations and their respective COVID-19 health ambassadors proved to be essential in assisting underserved African American older adults manage chronic health conditions and address community-wide health disparities during the COVID-19 pandemic. Adaptability, cultural sensitivity, and teamwork are key to implementing health interventions especially in underserved populations.

Several neuroimaging studies have shown that listening to music activates brain regions that reside in the motor system, even when there is no overt movement. However, many of these studies report the activation of varying motor system areas that include the primary motor cortex, supplementary motor area, dorsal and ventral pre-motor areas and parietal regions. In order to examine what specific roles are played by various motor regions during music perception, we used activation likelihood estimation (ALE) to conduct a meta-analysis of neuroimaging literature on passive music listening. After extensive search of the literature, 42 studies were analyzed resulting in a total of 386 unique subjects contributing 694 activation foci in total. As suspected, auditory activations were found in the bilateral superior temporal gyrus, transverse temporal gyrus, insula, pyramis, bilateral precentral gyrus, and bilateral medial frontal gyrus. We also saw the widespread activation of motor networks including left and right lateral premotor cortex, right primary motor cortex, and the left cerebellum. These results suggest a central role of the motor system in music and rhythm perception. We discuss these findings in the context of the Action Simulation for Auditory Prediction (ASAP) model and other predictive coding accounts of brain function.

INTRODUCTION: Lupus nephritis (LN) is a serious organ manifestation of systemic lupus erythematosus. Histologic overlap is relatively common in the six pathologic classes (I to VI) of LN. For example, mixed proliferative LN (MPLN) often includes features of classes III & V or classes IV & V combined. We performed a comparative evaluation of renal outcomes in patients with MPLN to patients with pure proliferative LN (PPLN) against pre-specified renal outcomes, and we also identified predictor of clinical outcomes among those with PPLN and MPLN. HYPOTHESIS: Individuals with MPLN will have worse short-term renal outcomes compared to those with PPLN. METHODS: We retrospectively reviewed 278 adult LN patients (≥18 years old) identified from an Emory University Hospital registry of native renal biopsies performed between January 2000 and December 2011. The final analytic sample consisted of individuals with a diagnosis of PPLN (n = 60) and MPLN (n = 96). We analyzed differences in clinical and laboratory characteristics at baseline. We also assessed associations between LN category and renal outcomes (complete remission and time to ESRD) with logistic and Cox proportional hazards models within two years of baseline. RESULTS: The study population was predominantly female (83.97%) and African American (71.8%) with a mean age of 33.4 years at baseline. Over a median follow up of 1.02 years, we did not find any statistically significant associations between MPLN and the development of ESRD or remission when compared to patients with PPLN (adjusted HR = 0.30, 95% CI = 0.07, 1.26). CONCLUSION: There was no association between mixed or pure histopathologic features of LN at presentation and rate of complete or partial remission but higher baseline eGFR was associated with a lower probability of complete remission among patients with lupus nephritis.

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease, the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study, a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus, preventing it from infecting cells in an in vitro cell-based assay, including multiple viral variants that are currently circulating in the human population. In addition, we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease.

BackgroundBoth active and passive cigarette smoking have previously been associated with orofacial cleft aetiology. We aimed to analyse the impact of declining active smoking prevalence and the implementation of smoke-free legislation on the incidence of children born with a cleft lip and/or palate within the United Kingdom.Methods and findingsWe conducted regression analysis using national administrative data in the United Kingdom between 2000-2018. The main outcome measure was orofacial cleft incidence, reported annually for England, Wales and Northern Ireland and separately for Scotland. First, we conducted an ecological study with longitudinal time-series analysis using smoking prevalence data for females over 16 years of age. Second, we used a natural experiment design with interrupted time-series analysis to assess the impact of smoke-free legislation. Over the study period, the annual incidence of orofacial cleft per 10,000 live births ranged from 14.2-16.2 in England, Wales and Northern Ireland and 13.4-18.8 in Scotland. The proportion of active smokers amongst females in the United Kingdom declined by 37% during the study period. Adjusted regression analysis did not show a correlation between the proportion of active smokers and orofacial cleft incidence in either dataset, although we were unable to exclude a modest effect of the magnitude seen in individual-level observational studies. The data in England, Wales and Northern Ireland suggested an 8% reduction in orofacial cleft incidence (RR 0.92, 95%CI 0.85 to 0.99; P = 0.024) following the implementation of smoke-free legislation. In Scotland, there was weak evidence for an increase in orofacial cleft incidence following smoke-free legislation (RR 1.16, 95%CI 0.94 to 1.44; P = 0.173).ConclusionsThese two ecological studies offer a novel insight into the influence of smoking in orofacial cleft aetiology, adding to the evidence base from individual-level studies. Our results suggest that smoke-free legislation may have reduced orofacial cleft incidence in England, Wales and Northern Ireland.

Our study takes advantage of unique data to quantify deficits in the psychosocial and cognitive resources of an extremely vulnerable subpopulation-those experiencing housing vulnerability-in an advanced, high-income country (Australia). Groups such as these are often impossible to study using nationally representative data sources because they make up a small share of the overall population. We show that those experiencing housing vulnerability sleep less well, have more limited cognitive functioning, and less social capital than do those in the general population. They are also less emotionally stable, less conscientious, more external, and more risk tolerant. Collectively, these deficits in psychosocial and cognitive resources account for between 24-42% of their reduced life satisfaction and their increased mental distress and loneliness. These traits also account for a large proportion of the gap in mental wellbeing across different levels of housing vulnerability.

Several neuroimaging studies have shown that listening to music activates brain regions that reside in the motor system, even when there is no overt movement. However, many of these studies report the activation of varying motor system areas that include the primary motor cortex, supplementary motor area, dorsal and ventral pre-motor areas and parietal regions. In order to examine what specific roles are played by various motor regions during music perception, we used activation likelihood estimation (ALE) to conduct a meta-analysis of neuroimaging literature on passive music listening. After extensive search of the literature, 42 studies were analyzed resulting in a total of 386 unique subjects contributing 694 activation foci in total. As suspected, auditory activations were found in the bilateral superior temporal gyrus, transverse temporal gyrus, insula, pyramis, bilateral precentral gyrus, and bilateral medial frontal gyrus. We also saw the widespread activation of motor networks including left and right lateral premotor cortex, right primary motor cortex, and the left cerebellum. These results suggest a central role of the motor system in music and rhythm perception. We discuss these findings in the context of the Action Simulation for Auditory Prediction (ASAP) model and other predictive coding accounts of brain function.

The global health community is beginning to understand the burden of norovirus-associated disease, which has a significant impact in both developed and developing countries. Norovirus virus like particle (VLP)-based vaccines are currently under development and have been shown to elicit systemic and mucosal immune responses when delivered intranasally. In the present study, we describe the use of a dry powder formulation (GelVac™) with an in situ gelling polysaccharide (GelSite™) extracted from Aloe vera for nasal delivery of a bivalent vaccine formulation containing both GI and GII.4 norovirus VLPs. Dose-ranging studies were performed to identify the optimal antigen dosages based on systemic and mucosal immune responses in guinea pigs and determine any antigenic interference. A dose-dependent increase in systemic and mucosal immunogenicity against each of the VLPs were observed as well as a boosting effect for each VLP after the second dosing. A total antigen dose of ≥50 μg of each GI and GII.4 VLPs was determined to be the maximally immunogenic dose in guinea pigs. The immunogenicity results of this bivalent formulation, taken together with previous work on monovalent GelVac™ norovirus vaccine formulation, provides a basis for future development of this norovirus VLP vaccine.

Rhizobia-legume symbiosis is the most well researched biological nitrogen fixation system. Coating legume seeds with rhizobia is now a recognized practical measure for improving the production of legume corp. However, the efficacy of some commercial rhizobia inoculants cannot be guaranteed in China due to the low rate of live rhizobia in these products. A greenhouse experiment was conducted to assess the effects of different rhizobial inoculant formulations on alfalfa productivity and nitrogen fixation. Two rhizobia strains, (ACCC17631 and ACCC17676), that are effective partners with alfalfa variety Zhongmu No. 1 were assessed with different concentrations of ammonium molybdate in seed-coat formulations with two different coating adhesives. Our study showed that the growth, nodulation, and nitrogen fixation ability of the plants inoculated with the ACCC17631 rhizobial strain were greatest when the ammonium molybdate application was0.2% of the formulation. An ammonium molybdate concentration of 0.1% was most beneficial to the growth of the plants inoculated with the ACCC17676 rhizobial strain. The sodium carboxymethyl cellulose and sodium alginate, used as coating adhesives, did not have a significant effect on alfalfa biomass and nitrogen fixation. However, the addition of skimmed milk to the adhesive improved nitrogenase activity. These results demonstrate that a new rhizobial seed-coat formulation benefitted alfalfa nodulation and yield.

While Toll-like receptors (TLRs) represent one of the best characterized innate immune pathways, evidence suggests that TLRs are not restricted to innate leukocytes and some epithelial cells, but are also expressed in T cells. Specifically, published evidence focusing on FoxP3+ regulatory T cells demonstrate that they express functional TLR2, which is already known among the TLR family for its association with immune suppression; however, little is known about the relationship between T cell-intrinsic TLR2 binding and cytokine production, T cell differentiation, or T cell receptor (TCR) stimulation. Here, we demonstrate that TCR and TLR2 co-stimulation provides a T cell-intrinsic signal which generates a dramatic, synergistic cytokine response dominated by IL-10. Importantly, the response was not seen in either CD4+CD25+ or CD4+FoxP3+ Tregs, yet resulted in the expansion of a suppressive CD4+CD25+CD62L-CD44+CD45Rbhi effector/memory T cell subset not typically associated with immune inhibition. This study reveals the striking ability of a prototypical innate immune receptor to trigger a potent and suppressive IL-10 response in effector/memory T cells, supporting the notion that TLR2 is a co-regulatory receptor on T cells.

Social networks influence health behavior, including tobacco use and cessation. To date, little is known about whether and how the networks of online smokers and non-smokers may differ, or the potential implications of such differences with regards to intervention efforts. Understanding how social networks vary by smoking status could inform public health efforts to accelerate cessation or slow the adoption of tobacco use.These secondary analyses explore the structure of ego networks of both smokers and non-smokers collected as part of a randomized control trial conducted within Facebook.During the trial, a total of 14,010 individuals installed a Facebook smoking cessation app: 9,042 smokers who were randomized in the trial, an additional 2,881 smokers who did not meet full eligibility criteria, and 2,087 non-smokers. The ego network for all individuals was constructed out to second-degree connections. Four kinds of networks were constructed: friendship, family, photo, and group networks. From these networks we measured edges, isolates, density, mean betweenness, transitivity, and mean closeness. We also measured diameter, clustering, and modularity without ego and isolates. Logistic regressions were performed with smoking status as the response and network metrics as the primary independent variables and demographics and Facebook utilization metrics as covariates.The four networks had different characteristics, indicated by different multicollinearity issues and by logistic regression output. Among Friendship networks, the odds of smoking were higher in networks with lower betweenness (p = 0.00), lower transitivity (p = 0.00), and larger diameter (p = 0.00). Among Family networks, the odds of smoking were higher in networks with more vertices (p = .01), less transitivity (p = .04), and fewer isolates (p = .01). Among Photo networks, none of the network metrics were predictive of smoking status. Among Group networks, the odds of smoking were higher when diameter was smaller (p = .04). Together, these findings suggested that compared to non-smokers, smokers in this sample had less connected, more dispersed Facebook Friendship networks; larger but more fractured Family networks with fewer isolates; more compact Group networks; and Photo networks that were similar in network structure to those of non-smokers.This study illustrates the importance of examining structural differences in online social networks as a critical component for network-based interventions and lays the foundation for future research that examines the ways that social networks differ based on individual health behavior. Interventions that seek to target the behavior of individuals in the context of their social environment would be well served to understand social network structures of participants.

Lupus nephritis (LN) is a serious organ manifestation of systemic lupus erythematosus. Histologic overlap is relatively common in the six pathologic classes (I to VI) of LN. For example, mixed proliferative LN (MPLN) often includes features of classes III & V or classes IV & V combined. We performed a comparative evaluation of renal outcomes in patients with MPLN to patients with pure proliferative LN (PPLN) against pre-specified renal outcomes, and we also identified predictor of clinical outcomes among those with PPLN and MPLN.Individuals with MPLN will have worse short-term renal outcomes compared to those with PPLN.We retrospectively reviewed 278 adult LN patients (≥18 years old) identified from an Emory University Hospital registry of native renal biopsies performed between January 2000 and December 2011. The final analytic sample consisted of individuals with a diagnosis of PPLN (n = 60) and MPLN (n = 96). We analyzed differences in clinical and laboratory characteristics at baseline. We also assessed associations between LN category and renal outcomes (complete remission and time to ESRD) with logistic and Cox proportional hazards models within two years of baseline.The study population was predominantly female (83.97%) and African American (71.8%) with a mean age of 33.4 years at baseline. Over a median follow up of 1.02 years, we did not find any statistically significant associations between MPLN and the development of ESRD or remission when compared to patients with PPLN (adjusted HR = 0.30, 95% CI = 0.07, 1.26).There was no association between mixed or pure histopathologic features of LN at presentation and rate of complete or partial remission but higher baseline eGFR was associated with a lower probability of complete remission among patients with lupus nephritis.

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer's disease.

Liver enzyme levels and total serum bilirubin are under genetic control and in recent years genome-wide population-based association studies have identified different susceptibility loci for these traits. We conducted a genome-wide association study in European ancestry participants from the Electronic Medical Records and Genomics (eMERGE) Network dataset of patient medical records with available genotyping data in order to identify genetic contributors to variability in serum bilirubin levels and other liver function tests and to compare the effects between adult and pediatric populations.The process of whole genome imputation of eMERGE samples with standard quality control measures have been described previously. After removing missing data and outliers based on principal components (PC) analyses, 3294 samples from European ancestry were used for the GWAS study. The association between each single nucleotide polymorphism (SNP) and total serum bilirubin and other liver function tests was tested using linear regression, adjusting for age, gender, site, platform and ancestry principal components (PC).Consistent with previous results, a strong association signal has been detected for UGT1A gene cluster (best SNP rs887829, beta = 0.15, p = 1.30x10-118) for total serum bilirubin level. Indeed, in this region more than 176 SNPs (or indels) had p

Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others.

Rett syndrome (RTT) is a genetic disorder characterized by a range of features including cognitive impairment, gait abnormalities and a reduction in purposeful hand skills. Mice harbouring knockout mutations in the Mecp2 gene display many RTT-like characteristics and are central to efforts to find novel therapies for the disorder. As hand stereotypies and gait abnormalities constitute major diagnostic criteria in RTT, it is clear that motor and gait-related phenotypes will be of importance in assessing preclinical therapeutic outcomes. We therefore aimed to assess gait properties over the prodromal phase in a functional knockout mouse model of RTT. In male Mecp2 knockout mice, we observed alterations in stride, coordination and balance parameters at 4 weeks of age, before the onset of other overt phenotypic changes as revealed by observational scoring. These data suggest that gait measures may be used as a robust and early marker of MeCP2-dysfunction in future preclinical therapeutic studies.

UNLABELLED:To explore the potential influence of the polymorphic 8p23.1 inversion on known autoimmune susceptibility risk at or near BLK locus, we validated a new bioinformatics method that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion in a Caucasian population. METHODS:Principal components analysis (PCA) was performed using markers inside the inversion territory followed by k-means cluster analyses on 7416 European derived and 267 HapMaP CEU and TSI samples. A logistic regression conditional analysis was performed. RESULTS:Three subgroups have been identified; inversion homozygous, heterozygous and non-inversion homozygous. The status of inversion was further validated using HapMap samples that had previously undergone Fluorescence in situ hybridization (FISH) assays with a concordance rate of above 98%. Conditional analyses based on the status of inversion were performed. We found that overall association signals in the BLK region remain significant after controlling for inversion status. The proportion of lupus cases and controls (cases/controls) in each subgroup was determined to be 0.97 for the inverted homozygous group (1067 cases and 1095 controls), 1.12 for the inverted heterozygous group (1935 cases 1717 controls) and 1.36 for non-inverted subgroups (924 cases and 678 controls). After calculating the linkage disequilibrium between inversion status and lupus risk haplotype we found that the lupus risk haplotype tends to reside on non-inversion background. As a result, a new association effect between non-inversion status and lupus phenotype has been identified ((p = 8.18×10(-7), OR = 1.18, 95%CI = 1.10-1.26). CONCLUSION:Our results demonstrate that both known lupus risk haplotype and inversion status act additively in the pathogenesis of lupus. Since inversion regulates expression of many genes in its territory, altered expression of other genes might also be involved in the development of lupus.

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.

ObjectivesGenome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA.MethodsA cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data.ResultsSignificant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10(-9)).ConclusionsThis study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.

The four glycoforms of the cellular prion protein (PrP(C)) variably glycosylated at the two N-linked glycosylation sites are converted into their pathological forms (PrP(Sc)) in most cases of sporadic prion diseases. However, a prominent molecular characteristic of PrP(Sc) in the recently identified variably protease-sensitive prionopathy (VPSPr) is the absence of a diglycosylated form, also notable in familial Creutzfeldt-Jakob disease (fCJD), which is linked to mutations in PrP either from Val to Ile at residue 180 (fCJD(V180I)) or from Thr to Ala at residue 183 (fCJD(T183A)). Here we report that fCJD(V180I), but not fCJD(T183A), exhibits a proteinase K (PK)-resistant PrP (PrP(res)) that is markedly similar to that observed in VPSPr, which exhibits a five-step ladder-like electrophoretic profile, a molecular hallmark of VPSPr. Remarkably, the absence of the diglycosylated PrP(res) species in both fCJD(V180I) and VPSPr is likewise attributable to the absence of PrP(res) glycosylated at the first N-linked glycosylation site at residue 181, as in fCJD(T183A). In contrast to fCJD(T183A), both VPSPr and fCJD(V180I) exhibit glycosylation at residue 181 on di- and monoglycosylated (mono181) PrP prior to PK-treatment. Furthermore, PrP(V180I) with a typical glycoform profile from cultured cells generates detectable PrP(res) that also contains the diglycosylated PrP in addition to mono- and unglycosylated forms upon PK-treatment. Taken together, our current in vivo and in vitro studies indicate that sporadic VPSPr and familial CJD(V180I) share a unique glycoform-selective prion formation pathway in which the conversion of diglycosylated and mono181 PrP(C) to PrP(Sc) is inhibited, probably by a dominant-negative effect, or by other co-factors.

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl >> hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.

The Drosophila larva possesses just 21 unique and identifiable pairs of olfactory sensory neurons (OSNs), enabling investigation of the contribution of individual OSN classes to the peripheral olfactory code. We combined electrophysiological and computational modeling to explore the nature of the peripheral olfactory code in situ. We recorded firing responses of 19/21 OSNs to a panel of 19 odors. This was achieved by creating larvae expressing just one functioning class of odorant receptor, and hence OSN. Odor response profiles of each OSN class were highly specific and unique. However many OSN-odor pairs yielded variable responses, some of which were statistically indistinguishable from background activity. We used these electrophysiological data, incorporating both responses and spontaneous firing activity, to develop a bayesian decoding model of olfactory processing. The model was able to accurately predict odor identity from raw OSN responses; prediction accuracy ranged from 12%-77% (mean for all odors 45.2%) but was always significantly above chance (5.6%). However, there was no correlation between prediction accuracy for a given odor and the strength of responses of wild-type larvae to the same odor in a behavioral assay. We also used the model to predict the ability of the code to discriminate between pairs of odors. Some of these predictions were supported in a behavioral discrimination (masking) assay but others were not. We conclude that our model of the peripheral code represents basic features of odor detection and discrimination, yielding insights into the information available to higher processing structures in the brain.

Most living organisms use pheromones for inter-individual communication. In Drosophila melanogaster flies, several pheromones perceived either by contact/at a short distance (cuticular hydrocarbons, CHs), or at a longer distance (cis-vaccenyl acetate, cVA), affect courtship and mating behaviours. However, it has not previously been possible to precisely identify all potential pheromonal compounds and simultaneously monitor their variation on a time scale. To overcome this limitation, we combined Solid Phase Micro-Extraction with gas-chromatography coupled with mass-spectrometry. This allowed us (i) to identify 59 cuticular compounds, including 17 new CHs; (ii) to precisely quantify the amount of each compound that could be detected by another fly, and (iii) to measure the variation of these substances as a function of aging and mating. Sex-specific variation appeared with age, while mating affected cuticular compounds in both sexes with three possible patterns: variation was (i) reciprocal in the two sexes, suggesting a passive mechanical transfer during mating, (ii) parallel in both sexes, such as for cVA which strikingly appeared during mating, or (iii) unilateral, presumably as a result of sexual interaction. We provide a complete reassessment of all Drosophila CHs and suggest that the chemical conversation between male and female flies is far more complex than is generally accepted. We conclude that focusing on individual compounds will not provide a satisfactory understanding of the evolution and function of chemical communication in Drosophila.

Decline in insulin action is a metabolic feature of aging and is involved in the development of age-related diseases including Type 2 Diabetes Mellitus (T2DM) and Alzheimer's disease (AD). A novel mitochondria-associated peptide, Humanin (HN), has a neuroprotective role against AD-related neurotoxicity. Considering the association between insulin resistance and AD, we investigated if HN influences insulin sensitivity.Using state of the art clamp technology, we examined the role of central and peripheral HN on insulin action. Continuous infusion of HN intra-cerebro-ventricularly significantly improved overall insulin sensitivity. The central effects of HN on insulin action were associated with activation of hypothalamic STAT-3 signaling; effects that were negated by co-inhibition of hypothalamic STAT-3. Peripheral intravenous infusions of novel and potent HN derivatives reproduced the insulin-sensitizing effects of central HN. Inhibition of hypothalamic STAT-3 completely negated the effects of IV HN analog on liver, suggesting that the hepatic actions of HN are centrally mediated. This is consistent with the lack of a direct effect of HN on primary hepatocytes. Furthermore, single treatment with a highly-potent HN analog significantly lowered blood glucose in Zucker diabetic fatty rats. Based upon the link of HN with two age-related diseases, we examined if there were age associated changes in HN levels. Indeed, the amount of detectable HN in hypothalamus, skeletal muscle, and cortex was decreased with age in rodents, and circulating levels of HN were decreased with age in humans and mice.We conclude that the decline in HN with age could play a role in the pathogenesis of age-related diseases including AD and T2DM. HN represents a novel link between T2DM and neurodegeneration and along with its analogues offers a potential therapeutic tool to improve insulin action and treat T2DM.

BACKGROUND: The functional polymorphism that explains the established association of the androgen receptor (AR) with androgenetic alopecia (AGA) remains unidentified, but Copy Number Variation (CNV) might be relevant. CNV involves changes in copy number of large segments of DNA, leading to the altered dosage of gene regulators or genes themselves. Two recent reports indicate regions of CNV in and around AR, and these have not been studied in relation to AGA. The aim of this preliminary case-control study was to determine if AR CNV is associated with AGA, with the hypothesis that CNV is the functional AR variant contributing to this condition. METHODOLOGY/PRINCIPAL FINDINGS: Multiplex Ligation-dependent Probe Amplification was used to screen for CNV in five AR exons and a conserved, non-coding region upstream of AR in 85 men carefully selected as cases and controls for maximal phenotypic contrast. There was no evidence of CNV in AR in any of the cases or controls, and thus no evidence of significant association between AGA and AR CNV. CONCLUSIONS/SIGNIFICANCE: The results suggest this form of genomic variation at the AR locus is unlikely to predispose to AGA.

Diagnosis of acute infection in the critically ill remains a challenge. We hypothesized that circulating leukocyte transcriptional profiles can be used to monitor the host response to and recovery from infection complicating critical illness.A translational research approach was employed. Fifteen mice underwent intratracheal injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury, GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 27 mechanically ventilated patients every two days for up to three weeks. Significant heterogeneity of VAP microarray profiles was observed secondary to patient ethnicity, age, and gender, yet 85 genes were identified with consistent changes in abundance during the seven days bracketing the diagnosis of VAP. Principal components analysis of these 85 genes appeared to differentiate between the responses of subjects who did versus those who did not develop VAP, as defined by a general trajectory (riboleukogram) for the onset and resolution of VAP. As patients recovered from critical illness complicated by acute infection, the riboleukograms converged, consistent with an immune attractor.Here we present the culmination of a mouse pneumonia study, demonstrating for the first time that disease trajectories derived from microarray expression profiles can be used to quantitatively track the clinical course of acute disease and identify a state of immune recovery. These data suggest that the onset of an infection-specific transcriptional program may precede the clinical diagnosis of pneumonia in patients. Moreover, riboleukograms may help explain variance in the host response due to differences in ethnic background, gender, and pathogen. Prospective clinical trials are indicated to validate our results and test the clinical utility of riboleukograms.

Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila melanogaster, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, we show that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, we show that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. We conclude that the inhibitory pheromone tastes bitter to the fly.

We developed an oral Salmonella-based vaccine that prevents and reverses diabetes in non-obese diabetic (NOD) mice. Related to this, the gastrointestinal tract harbors a complex dynamic population of microorganisms, the gut microbiome, that influences host homeostasis and metabolism. Changes in the gut microbiome are associated with insulin dysfunction and type 1 diabetes (T1D). Oral administration of diabetic autoantigens as a vaccine can restore immune balance. However, it was not known if a Salmonella-based vaccine would impact the gut microbiome. We administered a Salmonella-based vaccine to prediabetic NOD mice. Changes in the gut microbiota and associated metabolome were assessed using next-generation sequencing and gas chromatography-mass spectrometry (GC-MS). The Salmonella-based vaccine did not cause significant changes in the gut microbiota composition immediately after vaccination although at 30 days post-vaccination changes were seen. Additionally, no changes were noted in the fecal mycobiome between vaccine- and control/vehicle-treated mice. Significant changes in metabolic pathways related to inflammation and proliferation were found after vaccine administration. The results from this study suggest that an oral Salmonella-based vaccine alters the gut microbiome and metabolome towards a more tolerant composition. These results support the use of orally administered Salmonella-based vaccines that induced tolerance after administration.

Expert consensus on the potential benefits of early cancer detection does not exist for most cancer types. We convened 10 practicing oncologists using a RAND/UCLA modified Delphi panel to evaluate which of 20 solid tumors, representing >40 American Joint Committee on Cancer (AJCC)-identified cancer types and 80% of total cancer incidence, would receive potential clinical benefits from early detection. Pre-meeting, experts estimated how long cancers take to progress and rated the current curability and benefit (improvement in curability) of an annual hypothetical multi-cancer screening blood test. Post-meeting, experts rerated all questions. Cancers had varying estimates of the potential benefit of early cancer detection depending on estimates of their curability and progression by stage. Cancers rated as progressing quickly and being curable in earlier stages (stomach, esophagus, lung, urothelial tract, melanoma, ovary, sarcoma, bladder, cervix, breast, colon/rectum, kidney, uterus, anus, head and neck) were estimated to be most likely to benefit from a hypothetical screening blood test. Cancer types rated as progressing quickly but having comparatively lower cure rates in earlier stages (liver/intrahepatic bile duct, gallbladder, pancreas) were estimated to have medium likelihood of benefit from a hypothetical screening blood test. Cancer types rated as progressing more slowly and having higher curability regardless of stage (prostate, thyroid) were estimated to have limited likelihood of benefit from a hypothetical screening blood test. The panel concluded most solid tumors have a likelihood of benefit from early detection. Even among difficult-to-treat cancers (e.g., pancreas, liver/intrahepatic bile duct, gallbladder), early-stage detection was believed to be beneficial. Based on the panel consensus, broad coverage of cancers by screening blood tests would deliver the greatest potential benefits to patients.

Peatlands account for 15 to 30% of the world's soil carbon (C) stock and are important controls over global nitrogen (N) cycles. However, C and N concentrations are known to vary among peatlands contributing to the uncertainty of global C inventories, but there are few global studies that relate peatland classification to peat chemistry. We analyzed 436 peat cores sampled in 24 countries across six continents and measured C, N, and organic matter (OM) content at three depths down to 70 cm. Sites were distinguished between northern (387) and tropical (49) peatlands and assigned to one of six distinct broadly recognized peatland categories that vary primarily along a pH gradient. Peat C and N concentrations, OM content, and C:N ratios differed significantly among peatland categories, but few differences in chemistry with depth were found within each category. Across all peatlands C and N concentrations in the 10-20 cm layer, were 440 ± 85.1 g kg-1 and 13.9 ± 7.4 g kg-1, with an average C:N ratio of 30.1 ± 20.8. Among peatland categories, median C concentrations were highest in bogs, poor fens and tropical swamps (446-532 g kg-1) and lowest in intermediate and extremely rich fens (375-414 g kg-1). The C:OM ratio in peat was similar across most peatland categories, except in deeper samples from ombrotrophic tropical peat swamps that were higher than other peatlands categories. Peat N concentrations and C:N ratios varied approximately two-fold among peatland categories and N concentrations tended to be higher (and C:N lower) in intermediate fens compared with other peatland types. This study reports on a unique data set and demonstrates that differences in peat C and OM concentrations among broadly classified peatland categories are predictable, which can aid future studies that use land cover assessments to refine global peatland C and N stocks.

Claws are involved in a number of behaviours including locomotion and prey capture, and as a result animals evolve claw morphologies that enable these functions. Past authors have found geometry of the keratinous sheath of the claw to correlate with mode of life for extant birds and squamates; this relationship has frequently been cited to infer lifestyles for Mesozoic theropods including Archaeopteryx. However, many fossil claws lack keratinous sheaths and thus cannot be analysed using current methods. As the ungual phalanx within the claw is more commonly preserved in the fossil record, geometry of this bone may provide a more useful metric for paleontological analysis. In this study, ungual bones of 108 birds and 5 squamates were imaged using X-ray techniques and a relationship was found between curvatures of the ungual bone within the claw of pedal digit III and four modes of life; ground-dwelling, perching, predatory, and scansorial; using linear discriminant analysis with weighted accuracy equal to 0.79. Our model predicts arboreal lifestyles for Archaeopteryx and Microraptor and a predatory ecology for Confuciusornis. These findings demonstrate the utility of our model in answering questions of palaeoecology, the theropod-bird transition, and the evolution of avian flight. Though the metric exhibits a strong correlation with lifestyle, morphospaces for PD-III curvatures overlap and so this metric should be considered alongside additional evidence.

Insect pollinator communities are thought to transition from bee-dominated communities at low elevations to fly-dominated communities at high elevations. We predicted that increased tree canopy cover and a subsequent decrease in meadows and flowering plants would limit bees but not flies at higher elevations. We tested and supported this prediction by examining changes in both abundance and species richness for 128 bee species and 96 fly species at key points along an elevational gradient in Northern Arizona represented by distinct vegetation life zones. In addition to an increase in fly species and abundance relative to bees with increasing elevation, there were changes in community structure). To better understand factors that might influence this transition we examined how tree canopy cover changed along the elevational gradient and how this influenced the change in insect pollinator communities. While bee communities were progressively divergent between forest and meadow habitats with increasing elevation and tree canopy cover, there was no significant pattern with flies between meadow and forest habitats. However, fly abundance did increase with increasing elevation relative to bees. Along a comparable elevational gradient on an adjacent mountain with no tree canopy cover (i.e., a fire burned mountain), the bee-to-fly transition did not occur; bees persisted as the dominant pollinator into the highest life zone. This suggests that tree canopy cover can in part explain the transition from bee-to fly-dominated communities. In conclusion, this is the first study in North America to document a bee-fly transition for both abundance and species richness and show that tree canopy cover may play a role in determining pollinator community composition, by restricting bees to open meadow habitats.

Despite strong interest over many years, the usage of quantitative trait loci in plant breeding has often failed to live up to expectations. A key weak point in the utilisation of QTLs is the "quality" of markers used during marker-assisted selection (MAS): unreliable markers result in variable outcomes, leading to a perception that MAS products fail to achieve reliable improvement. Most reports of markers used for MAS focus on markers derived from the mapping population. There are very few studies that examine the reliability of these markers in other genetic backgrounds, and critically, no metrics exist to describe and quantify this reliability. To improve the MAS process, this work proposes five core metrics that fully describe the reliability of a marker. These metrics give a comprehensive and quantitative measure of the ability of a marker to correctly classify germplasm as QTL[+]/[-], particularly against a background of high allelic diversity. Markers that score well on these metrics will have far higher reliability in breeding, and deficiencies in specific metrics give information on circumstances under which a marker may not be reliable. The metrics are applicable across different marker types and platforms, allowing an objective comparison of the performance of different markers irrespective of the platform. Evaluating markers using these metrics demonstrates that trait-specific markers consistently out-perform markers designed for other purposes. These metrics also provide a superb set of criteria for designing superior marker systems for a target QTL, enabling the selection of an optimal marker set before committing to design.

Osteomyelitis, or bone infection, is often induced by antibiotic resistant Staphylococcus aureus strains of bacteria. Although debridement and long-term administration of antibiotics are the gold standard for osteomyelitis treatment, the increase in prevalence of antibiotic resistant bacterial strains limits the ability of clinicians to effectively treat infection. Bacteriophages (phages), viruses that in a lytic state can effectively kill bacteria, have gained recent attention for their high specificity, abundance in nature, and minimal risk of host toxicity. Previously, we have shown that CRISPR-Cas9 genomic editing techniques could be utilized to expand temperate bacteriophage host range and enhance bactericidal activity through modification of the tail fiber protein. In a dermal infection study, these CRISPR-Cas9 phages reduced bacterial load relative to unmodified phage. Thus we hypothesized this temperate bacteriophage, equipped with the CRISPR-Cas9 bactericidal machinery, would be effective at mitigating infection from a biofilm forming S. aureus strain in vitro and in vivo. In vitro, qualitative fluorescent imaging demonstrated superiority of phage to conventional vancomycin and fosfomycin antibiotics against S. aureus biofilm. Quantitative antibiofilm effects increased over time, at least partially, for all fosfomycin, phage, and fosfomycin-phage (dual) therapeutics delivered via alginate hydrogel. We developed an in vivo rat model of osteomyelitis and soft tissue infection that was reproducible and challenging and enabled longitudinal monitoring of infection progression. Using this model, phage (with and without fosfomycin) delivered via alginate hydrogel were successful in reducing soft tissue infection but not bone infection, based on bacteriological, histological, and scanning electron microscopy analyses. Notably, the efficacy of phage at mitigating soft tissue infection was equal to that of high dose fosfomycin. Future research may utilize this model as a platform for evaluation of therapeutic type and dose, and alternate delivery vehicles for osteomyelitis mitigation.

PurposeSurgical education videos currently all use a single point of view (POV) with the trainee locked onto a fixed viewpoint, which may not deliver sufficient information for complex procedures. We developed a novel multiple POV video system and evaluated its training outcome compared with traditional single POV.MethodsWe filmed a hip resurfacing procedure performed by an expert attending using 8 cameras in theatre. 30 medical students were randomly and equally allocated to learn the procedure using the multiple POV (experiment group [EG]) versus single POV system (control group [CG]). Participants advanced a pin into the femoral head as demonstrated in the video. We measured the drilling trajectories and compared it with pre-operative plan to evaluate distance of the pin insertion and angular deviations. Two orthopedic attendings expertly evaluated the participants' performance using a modified global rating scale (GRS). There was a pre-video knowledge test that was repeated post-simulation alongside a Likert-scale questionnaire.ResultsThe angular deviation of the pin in EG was significantly less by 29% compared to CG (p = 0.037), with no significant difference in the entry point's distance between groups (p = 0.204). The GRS scores for EG were 3.5% higher than CG (p = 0.046). There was a 32% higher overall knowledge test score (pConclusionThe novel multiple POV provided significant objective and subjective advantages over single POV for acquisition of technical skills in hip surgery.

CD4+ effector/memory T cells (Tem) represent a leading edge of the adaptive immune system responsible for protecting the body from infection, cancer, and other damaging processes. However, a subset of Tem cells with low expression of CD45Rb (RbLoTem) has been shown to suppress inflammation despite their effector surface phenotype and the lack of FoxP3 expression, the canonical transcription factor found in most regulatory T cells. In this report, we show that RbLoTem cells can suppress inflammation by influencing Treg behavior. Co-culturing activated RbLoTem and Tregs induced high expression of IL-10 in vitro, and conditioned media from RbLoTem cells induced IL-10 expression in FoxP3+ Tregs in vitro and in vivo, indicating that RbLoTem cells communicate with Tregs in a cell-contact independent fashion. Transcriptomic and multi-analyte Luminex data identified both IL-2 and IL-4 as potential mediators of RbLoTem-Treg communication, and antibody-mediated neutralization of either IL-4 or CD124 (IL-4Rα) prevented IL-10 induction in Tregs. Moreover, isolated Tregs cultured with recombinant IL-2 and IL-4 strongly induced IL-10 production. Using house dust mite (HDM)-induced airway inflammation as a model, we confirmed that the in vivo suppressive activity of RbLoTem cells was lost in IL-4-ablated RbLoTem cells. These data support a model in which RbLoTem cells communicate with Tregs using a combination of IL-2 and IL-4 to induce robust expression of IL-10 and suppression of inflammation.

Hypertension is the leading risk factor for cardiovascular disease and leading cause of premature death globally. In 2008, approximately 40% of adults were diagnosed with hypertension, with more than 1.5 billion people estimated to be affected globally by 2025. Hypertension disproportionally affects low- and middle-income countries, where the prevalence is higher and where the health systems are more fragile. This qualitative study explored patients' experiences on the management and control of hypertension in rural Bangladesh, Sri Lanka and Pakistan. We conducted sixty semi-structured interviews, with 20 participants in each country. Hypertensive individuals were recruited based on age, gender and hypertensive status. Overall, patients' reported symptoms across the three countries were quite similar, although perceptions of hypertension were mixed. The majority of patients reported low knowledge on how to prevent or treat hypertension. The main barriers to accessing health services, as reported by participants, were inadequate services and poor quality of existing facilities, shortage of medicine supplies, busyness of doctors due to high patient load, long travel distance to facilities, and long waiting times once facilities were reached. Patients also mentioned that cost was a barrier to accessing services and adhering to medication. Many patients, when asked for areas of improvement, reported on the importance of the provider-patient relationship and mentioned valuing doctors who spent time with them, provided advice, and could be trusted. However, most patients reported that, especially at primary health care level and in government hospitals, the experience with their doctor did not meet their expectations. Patients in the three countries reported desire for good quality local medical services, the need for access to doctors, medicine and diagnostics and decreased cost for medication and medical services. Patients also described welcoming health care outreach activities near their homes. Areas of improvement could focus on reorienting community health workers' activities; involving family members in comprehensive counseling for medication adherence; providing appropriate training for health care staff to deliver effective information and services for controlling hypertension to patients; enhancing primary health care and specialist services; improving supplies of hypertensive medication in public facilities; taking into account patients' cultural and social background when providing services; and facilitating access and treatment to those who are most vulnerable.

During pregnancy, women experience numerous physiological changes but, to date, there is limited published data that characterize accompanying changes in gene expression over pregnancy. This study sought to characterize the complexity of the transcriptome over the course of pregnancy among women with healthy pregnancies. Subjects provided a venous blood sample during early (6-15 weeks) and late (22-33 weeks) pregnancy, which was used to isolate peripheral blood mononuclear cells prior to RNA extraction. Gene expression was examined for 63 women with uncomplicated, term deliveries. We evaluated the association between weeks gestation at sample collection and expression of each transcript. Of the 16,311 transcripts evaluated, 439 changed over pregnancy after a Bonferroni correction to account for multiple comparisons. Genes whose expression increased over pregnancy were associated with oxygen transport, the immune system, and host response to bacteria. Characterization of changes in the transcriptome over the course of healthy term pregnancies may enable the identification of genes whose expression predicts complications or adverse outcomes of pregnancy.

ObjectivesBone material properties are a major determinant of bone health in older age, both in terms of fracture risk and implant fixation, in orthopaedics and dentistry. Bone is an anisotropic and hierarchical material so its measured material properties depend upon the scale of metric used. The scale used should reflect the clinical problem, whether it is fracture risk, a whole bone problem, or implant stability, at the millimetre-scale. Indentation, an engineering technique involving pressing a hard-tipped material into another material with a known force, may be able to assess bone stiffness at the millimetre-scale (the apparent elastic modulus). We aimed to investigate whether spherical-tip indentation could reliably measure the apparent elastic modulus of human cortical bone.Materials and methodsCortical bone samples were retrieved from the femoral necks of nineteen patients undergoing total hip replacement surgery (10 females, 9 males, mean age: 69 years). The samples underwent indentation using a 1.5 mm diameter, ruby, spherical indenter tip, with sixty indentations per patient sample, across six locations on the bone surfaces, with ten repeated indentations at each of the six locations. The samples then underwent mechanical compression testing. The repeatability of indentation measurements of elastic modulus was assessed using the co-efficient of repeatability and the correlation between the bone elastic modulus measured by indentation and compression testing was analysed by least-squares regression.ResultsIn total, 1140 indentations in total were performed. Indentation was found to be repeatable for indentations performed at the same locations on the bone samples with a mean co-efficient of repeatability of 0.4 GigaPascals (GPa), confidence interval (C.I): 0.33-0.42 GPa. There was variation in the indentation modulus results between different locations on the bone samples (mean co-efficient of repeatability: 3.1 GPa, C.I: 2.2-3.90 GPa). No clear correlation was observed between indentation and compression values of bone elastic modulus (r = 0.33, p = 0.17). The mean apparent elastic modulus obtained by spherical indentation was 9.9 GPa, the standard deviation for each indent cycle was 0.11 GPa, and the standard deviation between locations on the same sample was 1.01 GPa. The mean compression apparent elastic modulus was 4.42 GPa, standard deviation 1.02 GPa.DiscussionSpherical-tip indentation was found to be a repeatable test for measuring the elastic modulus of human cortical bone, demonstrated by a low co-efficient of repeatability in this study. It could not, however, reliably predict cortical bone elastic modulus determined by platens compression testing in this study. This may be due to indentation only probing mechanical properties at the micro-scale while platens compression testing assesses millimetre length-scale properties. Improvements to the testing technique, including the use of a larger diameter spherical indenter tip, may improve the measurement of bone stiffness at the millimetre scale and should be investigated further.

ObjectiveCohort selection is challenging for large-scale electronic health record (EHR) analyses, as International Classification of Diseases 9th edition (ICD-9) diagnostic codes are notoriously unreliable disease predictors. Our objective was to develop, evaluate, and validate an automated algorithm for determining an Autism Spectrum Disorder (ASD) patient cohort from EHR. We demonstrate its utility via the largest investigation to date of the co-occurrence patterns of medical comorbidities in ASD.MethodsWe extracted ICD-9 codes and concepts derived from the clinical notes. A gold standard patient set was labeled by clinicians at Boston Children's Hospital (BCH) (N = 150) and Cincinnati Children's Hospital and Medical Center (CCHMC) (N = 152). Two algorithms were created: (1) rule-based implementing the ASD criteria from Diagnostic and Statistical Manual of Mental Diseases 4th edition, (2) predictive classifier. The positive predictive values (PPV) achieved by these algorithms were compared to an ICD-9 code baseline. We clustered the patients based on grouped ICD-9 codes and evaluated subgroups.ResultsThe rule-based algorithm produced the best PPV: (a) BCH: 0.885 vs. 0.273 (baseline); (b) CCHMC: 0.840 vs. 0.645 (baseline); (c) combined: 0.864 vs. 0.460 (baseline). A validation at Children's Hospital of Philadelphia yielded 0.848 (PPV). Clustering analyses of comorbidities on the three-site large cohort (N = 20,658 ASD patients) identified psychiatric, developmental, and seizure disorder clusters.ConclusionsIn a large cross-institutional cohort, co-occurrence patterns of comorbidities in ASDs provide further hypothetical evidence for distinct courses in ASD. The proposed automated algorithms for cohort selection open avenues for other large-scale EHR studies and individualized treatment of ASD.

Marine fungi are an understudied group of eukaryotic microorganisms characterized by unresolved genealogies and unstable classification. Whereas DNA barcoding via the nuclear ribosomal internal transcribed spacer (ITS) provides a robust and rapid tool for fungal species delineation, accurate classification of fungi is often arduous given the large number of partial or unknown barcodes and misidentified isolates deposited in public databases. This situation is perpetuated by a paucity of cultivable fungal strains available for phylogenetic research linked to these data sets. We analyze ITS barcodes produced from a subsample (290) of 1781 cultured isolates of marine-derived fungi in the Bioresources Library located at the Australian Institute of Marine Science (AIMS). Our analysis revealed high levels of under-explored fungal diversity. The majority of isolates were ascomycetes including representatives of the subclasses Eurotiomycetidae, Hypocreomycetidae, Sordariomycetidae, Pleosporomycetidae, Dothideomycetidae, Xylariomycetidae and Saccharomycetidae. The phylum Basidiomycota was represented by isolates affiliated with the genera Tritirachium and Tilletiopsis. BLAST searches revealed 26 unknown OTUs and 50 isolates corresponding to previously uncultured, unidentified fungal clones. This study makes a significant addition to the availability of barcoded, culturable marine-derived fungi for detailed future genomic and physiological studies. We also demonstrate the influence of commonly used alignment algorithms and genetic distance measures on the accuracy and comparability of estimating Operational Taxonomic Units (OTUs) by the automatic barcode gap finder (ABGD) method. Large scale biodiversity screening programs that combine datasets using algorithmic OTU delineation pipelines need to ensure compatible algorithms have been used because the algorithm matters.

Expert consensus on the potential benefits of early cancer detection does not exist for most cancer types. We convened 10 practicing oncologists using a RAND/UCLA modified Delphi panel to evaluate which of 20 solid tumors, representing >40 American Joint Committee on Cancer (AJCC)-identified cancer types and 80% of total cancer incidence, would receive potential clinical benefits from early detection. Pre-meeting, experts estimated how long cancers take to progress and rated the current curability and benefit (improvement in curability) of an annual hypothetical multi-cancer screening blood test. Post-meeting, experts rerated all questions. Cancers had varying estimates of the potential benefit of early cancer detection depending on estimates of their curability and progression by stage. Cancers rated as progressing quickly and being curable in earlier stages (stomach, esophagus, lung, urothelial tract, melanoma, ovary, sarcoma, bladder, cervix, breast, colon/rectum, kidney, uterus, anus, head and neck) were estimated to be most likely to benefit from a hypothetical screening blood test. Cancer types rated as progressing quickly but having comparatively lower cure rates in earlier stages (liver/intrahepatic bile duct, gallbladder, pancreas) were estimated to have medium likelihood of benefit from a hypothetical screening blood test. Cancer types rated as progressing more slowly and having higher curability regardless of stage (prostate, thyroid) were estimated to have limited likelihood of benefit from a hypothetical screening blood test. The panel concluded most solid tumors have a likelihood of benefit from early detection. Even among difficult-to-treat cancers (e.g., pancreas, liver/intrahepatic bile duct, gallbladder), early-stage detection was believed to be beneficial. Based on the panel consensus, broad coverage of cancers by screening blood tests would deliver the greatest potential benefits to patients., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LS, SA, HB, JB, GTB, LC, MC, PC, BJM, DJW reported consulting fees from Partnership for Health Analytic Research (PHAR), LLC and GRAIL, LLC, a subsidiary of Illumina, Inc., currently held separate from Illumina, Inc. under the terms of the Interim Measures Order of the European Commission dated 29 October 2021. ARK and AK are employees of GRAIL, LLC, a subsidiary of Illumina, Inc., currently held separate from Illumina, Inc. under the terms of the Interim Measures Order of the European Commission dated 29 October 2021, who supported this study, and reported stock or stock options in Illumina (parent company of GRAIL, LLC, a subsidiary of Illumina, Inc., currently held separate from Illumina, Inc. under the terms of the Interim Measures Order of the European Commission dated 29 October 2021). MSB, IY, and CC are employees of Partnership for Health Analytic Research (PHAR), LLC which was paid by the following to conduct research related to the work described in the manuscript: Abbvie, Amgen, AstraZeneca, Biomarin Pharmaceuticals, BMS, Celgene, Dompe, Eisai, Exact Sciences Corporation, Genentech, GRAIL, LLC, a subsidiary of Illumina, Inc., currently held separate from Illumina, Inc. under the terms of the Interim Measures Order of the European Commission dated 29 October 2021, Ionis, Jazz, Kite, Novartis, Otsuka, Recordati, Regeneron, Sanofi US Services, Takeda Pharmaceuticals USA. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2022 Schwartzberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)