Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.

Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIbeta inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.

Stratification of risk of progression to colectomy in ulcerative colitis via measured and predicted gene expression.

An important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of individuals with ulcerative colitis (UC) require colectomy within 5 years of diagnosis, but polygenic risk scores (PRSs) utilizing findings from genome-wide association studies (GWASs) are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a transcriptional risk score (TRS). Here, we demonstrate that a measured TRS based on bulk rectal gene expression in the PROTECT inception cohort study has a positive predictive value approaching 50% for colectomy. Single-cell profiling demonstrates that the genes are active in multiple diverse cell types from both the epithelial and immune compartments. Expression quantitative trait locus (QTL) analysis identifies genes with differential effects at baseline and week 52 follow-up, but for the most part, differential expression associated with colectomy risk is independent of local genetic regulation. Nevertheless, a predicted polygenic transcriptional risk score (PPTRS) derived by summation of transcriptome-wide association study (TWAS) effects identifies UC-affected individuals at 5-fold elevated risk of colectomy with data from the UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with TWASs for stratification of risk of disease complications.

NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures.

OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

Inflamed Ulcerative Colitis Regions Associated With MRGPRX2-Mediated Mast Cell Degranulation and Cell Activation Modules, Defining a New Therapeutic Target.

BACKGROUND & AIMS: Recent literature has implicated a key role for mast cells in murine models of colonic inflammation, but their role in human ulcerative colitis (UC) is not well established. A major advance has been the identification of mrgprb2 (human orthologue, MRGPX2) as mediating IgE-independent mast cell activation. We sought to define mechanisms of mast cell activation and MRGPRX2 in human UC. METHODS: Colon tissues were collected from patients with UC for bulk RNA sequencing and lamina propria cells were isolated for MRGPRX2 activation studies and single-cell RNA sequencing. Genetic association of all protein-altering G-protein coupled receptor single-nucleotide polymorphism was performed in an Ashkenazi Jewish UC case-control cohort. Variants of MRGPRX2 were transfected into Chinese hamster ovary (CHO) and human mast cell (HMC) 1.1 cells to detect genotype-dependent effects on ß-arrestin recruitment, IP-1 accumulation, and phosphorylated extracellular signal-regulated kinase. RESULTS: Mast cell-specific mediators and adrenomedullin (proteolytic precursor of PAMP-12, an MRGPRX2 agonist) are up-regulated in inflamed compared to uninflamed UC. MRGPRX2 stimulation induces carboxypeptidase secretion from inflamed UC. Of all protein-altering GPCR alleles, a unique variant of MRGPRX2, Asn62Ser, was most associated with and was bioinformatically predicted to alter arrestin recruitment. We validated that the UC protective serine allele enhances ß-arrestin recruitment, decreases IP-1, and increases phosphorylated extracellular signal-regulated kinase with MRGPRX2 agonists. Single-cell RNA sequencing defines that adrenomedullin is expressed by activated fibroblasts and epithelial cells and that interferon gamma is a key upstream regulator of mast cell gene expression. CONCLUSION: Inflamed UC regions are distinguished by MRGPRX2-mediated activation of mast cells, with decreased activation observed with a UC-protective genetic variant. These results define cell modules of UC activation and a new therapeutic target.

Common and Rare Variant Prediction and Penetrance of IBD in a Large, Multi-ethnic, Health System-based Biobank Cohort.

BACKGROUND AND AIMS: Polygenic risk scores (PRS) may soon be used to predict inflammatory bowel disease (IBD) risk in prevention efforts. We leveraged exome-sequence and single nucleotide polymorphism (SNP) array data from 29,358 individuals in the multiethnic, randomly ascertained health system-based BioMe biobank to define effects of common and rare IBD variants on disease prediction and pathophysiology. METHODS: PRS were calculated from European, African American, and Ashkenazi Jewish (AJ) reference case-control studies, and a meta-GWAS run using all three association datasets. PRS were then combined using regression to assess which combination of scores best predicted IBD status in European, AJ, Hispanic, and African American cohorts in BioMe. Additionally, rare variants were assessed in genes associated with very early-onset IBD (VEO-IBD), by estimating genetic penetrance in each BioMe population. RESULTS: Combining risk scores based on association data from distinct ancestral populations improved IBD prediction for every population in BioMe and significantly improved prediction among European ancestry UK Biobank individuals. Lower predictive power for non-Europeans was observed, reflecting in part substantially lower African IBD case-control reference sizes. We replicated associations for two VEO-IBD genes, ADAM17 and LRBA, with high dominant model penetrance in BioMe. Autosomal recessive LRBA risk alleles are associated with severe, early-onset autoimmunity; we show that heterozygous carriage of an African-predominant LRBA protein-altering allele is associated with significantly decreased LRBA and CTLA-4 expression with T-cell activation. CONCLUSIONS: Greater genetic diversity in African populations improves prediction across populations, and generalizes some VEO-IBD genes. Increasing African American IBD case-collections should be prioritized to reduce health disparities and enhance pathophysiological insight.

Prioritizing Crohn's disease genes by integrating association signals with gene expression implicates monocyte subsets.

Genome-wide association studies have identified ~170 loci associated with Crohn's disease (CD) and defining which genes drive these association signals is a major challenge. The primary aim of this study was to define which CD locus genes are most likely to be disease related. We developed a gene prioritization regression model (GPRM) by integrating complementary mRNA expression datasets, including bulk RNA-Seq from the terminal ileum of 302 newly diagnosed, untreated CD patients and controls, and in stimulated monocytes. Transcriptome-wide association and co-expression network analyses were performed on the ileal RNA-Seq datasets, identifying 40 genome-wide significant genes. Co-expression network analysis identified a single gene module, which was substantially enriched for CD locus genes and most highly expressed in monocytes. By including expression-based and epigenetic information, we refined likely CD genes to 2.5 prioritized genes per locus from an average of 7.8 total genes. We validated our model structure using cross-validation and our prioritization results by protein-association network analyses, which demonstrated significantly higher CD gene interactions for prioritized compared with non-prioritized genes. Although individual datasets cannot convey all of the information relevant to a disease, combining data from multiple relevant expression-based datasets improves prediction of disease genes and helps to further understanding of disease pathogenesis.

A Frameshift in CSF2RB Predominant Among Ashkenazi Jews Increases Risk for Crohn's Disease and Reduces Monocyte Signaling via GM-CSF.

BACKGROUND & AIMS: Crohn's disease (CD) has the highest prevalence in Ashkenazi Jewish populations. We sought to identify rare, CD-associated frameshift variants of high functional and statistical effects. METHODS: We performed exome sequencing and array-based genotype analyses of 1477 Ashkenazi Jewish individuals with CD and 2614 Ashkenazi Jewish individuals without CD (controls). To validate our findings, we performed genotype analyses of an additional 1515 CD cases and 7052 controls for frameshift mutations in the colony-stimulating factor 2-receptor ß common subunit gene (CSF2RB). Intestinal tissues and blood samples were collected from patients with CD; lamina propria leukocytes were isolated and expression of CSF2RB and granulocyte-macrophage colony-stimulating factor-responsive cells were defined by adenomatous polyposis coli (APC) time-of-flight mass cytometry (CyTOF analysis). Variants of CSF2RB were transfected into HEK293 cells and the expression and functions of gene products were compared. RESULTS: In the discovery cohort, we associated CD with a frameshift mutation in CSF2RB (P = 8.52 × 10(-4)); the finding was validated in the replication cohort (combined P = 3.42 × 10(-6)). Incubation of intestinal lamina propria leukocytes with granulocyte-macrophage colony-stimulating factor resulted in high levels of phosphorylation of signal transducer and activator of transcription (STAT5) and lesser increases in phosphorylation of extracellular signal-regulated kinase and AK straining transforming (AKT). Cells co-transfected with full-length and mutant forms of CSF2RB had reduced pSTAT5 after stimulation with granulocyte-macrophage colony-stimulating factor, compared with cells transfected with control CSF2RB, indicating a dominant-negative effect of the mutant gene. Monocytes from patients with CD who were heterozygous for the frameshift mutation (6% of CD cases analyzed) had reduced responses to granulocyte-macrophage colony-stimulating factor and markedly decreased activity of aldehyde dehydrogenase; activity of this enzyme has been associated with immune tolerance. CONCLUSIONS: In a genetic analysis of Ashkenazi Jewish individuals, we associated CD with a frameshift mutation in CSF2RB. Intestinal monocytes from carriers of this mutation had reduced responses to granulocyte-macrophage colony-stimulating factor, providing an additional mechanism for alterations to the innate immune response in individuals with CD.

Higher moisture content is associated with greater emissions of DEHP from PVC wallpaper.

Water damage and moisture in buildings may become more prevalent due to the increasing frequency of extreme precipitation and flooding events resulting from climate change. However, the effects of moisture levels on phthalate emissions from building materials are still underreported. This study aims to evaluate the effect of moisture content (MC) on the level of di-(2ethylhexyl) phthalate (DEHP) emitted from plastic wallpaper (0.22wt% DEHP) within 15 days in a closed chamber. A scenario of short-term exposure to DEHP in buildings suffering from water damage was simulated. Experiments, controlled at 100% relative humidity (RH) of air and 28°C, were conducted under the following three conditions: (I) without wallpaper (control chamber), (II) dry wallpaper (MC at 3.57%) and (III) damp wallpaper (MC at 52.31%). Air and dust samples were collected at the elapsed time of 2, 4, 6, 8, 10, 13 and 15 days, and the wipe sample was collected on the last day. Higher DEHP concentrations were found to be emitted into the air and adsorbed on the dust for wallpapers with higher MC%. DEHP levels in the air exhibited an increasing trend with the length of the experiment. Overall, it was found that approximately 35.31% more total DEHP mass was released into the air, dust and wipe samples from damp wallpapers compared to dry wallpapers. It is concluded that DEHP emissions from plastic materials are affected by the inner moisture percentage.

Disease-associated variants in different categories of disease located in distinct regulatory elements.

BACKGROUND: The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. RESULTS: In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that regulatory regions are located within over 50% coding exon regions. Transcription promoters, methylation regions, and transcription insulators have the highest density of disease variants, with 472, 239, and 72 disease variants per one million base pairs, respectively. CONCLUSIONS: Disease-associated variants in different disease categories are preferentially located in particular regulatory elements. These results will be useful for an overall understanding about the differences among the pathogenic mechanisms of various disease-associated variants.

Feeding bottles usage and the prevalence of childhood allergy and asthma.

This study aimed to examine the association between the length of use of feeding bottles or pacifiers during childhood and the prevalence of respiratory and allergic morbidities. A large-scale questionnaire survey was performed in day care centers and kindergartens (with children's ages ranging from 2 to 7 years) in southern Taiwan, and a total of 14,862 questionnaires completed by parents were finally recruited for data analysis. Effects of using feeding bottles on children's wheezing/asthma (adjusted OR: 1.05, 95% CI 1.00-1.09), allergic rhinitis (adjusted OR: 1.04, 95% CI 1.00-1.08), and eczema (adjusted OR: 1.07, 95% CI 1.01-1.2) were found. Moreover, significant dose-dependent relationships were further established after an adjustment for confounders was performed that included children's ages, gender, gestational age, birth weight, length of breastfeeding, the age when first given infant formula or complementary foods, family history, parental educational levels, and smoking status, as well as the problem of indoor water damage. This study was the first to reveal the potential risk of using plastic consumer products such as feeding bottles on the reported health status of preschool children in Asian countries.

Airborne fungi and bacteria in child daycare centers and the effectiveness of weak acid hypochlorous water on controlling microbes.

A three-week-long biological sampling scheme was conducted in two child daycare centers (CDCCs) in order to investigate interdiurnal and diurnal variations in indoor airborne microbes as well as the efficiency of weak acid hypochlorous water (WAHW) on disinfecting indoor microbes. During the second week of sampling, WAHW was sprayed using a fogger in the classroom after children had left and before they returned the next morning. An identical cycle of experiments was performed twice in the winter and spring. Without WAHW intervention, the respective mean of the indoor concentrations and I/O ratios were 8732-47581 CFU m(-3) and 0.96-2.53 for fungi, and 6706-28998 CFU m(-3) and 1.10-11.92 for bacteria, showing severe bio-contamination in the CDCCs. Moreover, a relatively high level of bacterial pollution was found at noon, whereas a greater fungal pollution could be detected in the morning and at noon. Among five school days, the fungal and bacterial pollution may be higher on Monday and on Wednesday, Thursday, and Friday, respectively. Furthermore, with WAHW intervention, the indoor microbial concentrations and I/O ratios were decreased significantly. The reduction of I/O ratios caused by WAHW disinfection was accomplished in the morning for bacteria and in the morning, at noon, and in the afternoon for fungi. In conclusion, this study clearly clarified the risky period during which children may be exposed to hazardous environments, and demonstrated the effectiveness of spraying WAHW the night before on decontaminating indoor airborne microbes on the following day, especially in the case of fungi.

Influence of Indoor Temperature Exposure on Emergency Department Visits Due to Infectious and Non-Infectious Respiratory Diseases for Older People.

Previous studies have demonstrated that outdoor temperature exposure was an important risk factor for respiratory diseases. However, no study investigates the effect of indoor temperature exposure on respiratory diseases and further assesses cumulative effect. The objective of this study is to study the cumulative effect of indoor temperature exposure on emergency department visits due to infectious (IRD) and non-infectious (NIRD) respiratory diseases among older adults. Subjects were collected from the Longitudinal Health Insurance Database in Taiwan. The cumulative degree hours (CDHs) was used to assess the cumulative effect of indoor temperature exposure. A distributed lag nonlinear model with quasi-Poisson function was used to analyze the association between CDHs and emergency department visits due to IRD and NIRD. For IRD, there was a significant risk at 27, 28, 29, 30, and 31 °C when the CDHs exceeded 69, 40, 14, 5, and 1 during the cooling season (May to October), respectively, and at 19, 20, 21, 22, and 23 °C when the CDHs exceeded 8, 1, 1, 35, and 62 during the heating season (November to April), respectively. For NIRD, there was a significant risk at 19, 20, 21, 22, and 23 °C when the CDHs exceeded 1, 1, 16, 36, and 52 during the heating season, respectively; the CDHs at 1 was only associated with the NIRD at 31 °C during the cooling season. Our data also indicated that the CDHs was lower among men than women. We conclude that the cumulative effects of indoor temperature exposure should be considered to reduce IRD risk in both cooling and heating seasons and NIRD risk in heating season and the cumulative effect on different gender.

Associations among phthalate exposure, DNA methylation of TSLP, and childhood allergy.

BACKGROUND: Dysregulation of thymic stromal lymphopoietin (TSLP) expressions is linked to asthma and allergic disease. Exposure to phthalate esters, a widely used plasticizer, is associated with respiratory and allergic morbidity. Dibutyl phthalate (DBP) causes TSLP upregulation in the skin. In addition, phthalate exposure is associated with changes in environmentally induced DNA methylation, which might cause phenotypic heterogeneity. This study examined the DNA methylation of the TSLP gene to determine the potential mechanism between phthalate exposure and allergic diseases. RESULTS: Among all evaluated, only benzyl butyl phthalate (BBzP) in the settled dusts were negatively correlated with the methylation levels of TSLP and positively associated with children's respiratory symptoms. The results revealed that every unit increase in BBzP concentration in the settled dust was associated with a 1.75% decrease in the methylation level on upstream 775 bp from the transcription start site (TSS) of TSLP (ß = - 1.75, p = 0.015) after adjustment for child's sex, age, BMI, parents' smoking status, allergic history, and education levels, PM2.5, formaldehyde, temperature; and relative humidity. Moreover, every percentage increase in the methylation level was associated with a 20% decrease in the risk of morning respiratory symptoms in the children (OR 0.80, 95% CI 0.65-0.99). CONCLUSIONS: Exposure to BBzP in settled dust might increase children's respiratory symptoms in the morning through decreasing TSLP methylation. Therefore, the exposure to BBzP should be reduced especially for the children already having allergic diseases.

Development of Hourly Indoor PM2.5 Concentration Prediction Model: The Role of Outdoor Air, Ventilation, Building Characteristic, and Human Activity.

Exposure to indoor particulate matter less than 2.5 µm in diameter (PM2.5) is a critical health risk factor. Therefore, measuring indoor PM2.5 concentrations is important for assessing their health risks and further investigating the sources and influential factors. However, installing monitoring instruments to collect indoor PM2.5 data is difficult and expensive. Therefore, several indoor PM2.5 concentration prediction models have been developed. However, these prediction models only assess the daily average PM2.5 concentrations in cold or temperate regions. The factors that influence PM2.5 concentration differ according to climatic conditions. In this study, we developed a prediction model for hourly indoor PM2.5 concentrations in Taiwan (tropical and subtropical region) by using a multiple linear regression model and investigated the impact factor. The sample comprised 93 study cases (1979 measurements) and 25 potential predictor variables. Cross-validation was performed to assess performance. The prediction model explained 74% of the variation, and outdoor PM2.5 concentrations, the difference between indoor and outdoor CO2 levels, building type, building floor level, bed sheet cleaning, bed sheet replacement, and mosquito coil burning were included in the prediction model. Cross-validation explained 75% of variation on average. The results also confirm that the prediction model can be used to estimate indoor PM2.5 concentrations across seasons and areas. In summary, we developed a prediction model of hourly indoor PM2.5 concentrations and suggested that outdoor PM2.5 concentrations, ventilation, building characteristics, and human activities should be considered. Moreover, it is important to consider outdoor air quality while occupants open or close windows or doors for regulating ventilation rate and human activities changing also can reduce indoor PM2.5 concentrations.

Cumulative effect of indoor temperature on cardiovascular disease-related emergency department visits among older adults in Taiwan.

Studies have demonstrated that exposure to extreme outdoor temperatures increases cardiovascular disease mortality and morbidity. However, people spend 80%-90% of their time indoors, and the cumulative effects of exposure to high or low temperature on the risk of cardiovascular diseases had not been considered. This study investigated the cumulative effects of high or low indoor temperature exposure on the risk of cardiovascular diseases. We estimated indoor temperatures by using a prediction model of indoor temperature from a previous study and further calculated the cumulative degree hours at different indoor temperature ranges. Samples of emergency department visits due to cardiovascular diseases were collected from the Longitudinal Health Insurance Database (LHID) from 2000 to 2014 in Taiwan. We used a distributed lag nonlinear model to analyze the data. Our data demonstrated a significant risk of emergency department visits due to cardiovascular diseases at 27, 28, 29, 30, and 31 °C when cooling cumulative degree hours exceeded 62, 43, 16, 1, and 1 during the hot season (May to October), respectively, and at 19, 20, 21, 22, and 23 °C when heating cumulative degree hours exceeded 1, 1, 1, 11, and 33 during the cold season (November to April), respectively. Cumulative degree hours were different according to gender and age groups. Policymakers should further consider the cumulative effects to prevent hot- or cold-related cardiovascular diseases for populations.

Zebrafish modeling of intestinal injury, bacterial exposures and medications defines epithelial in vivo responses relevant to human inflammatory bowel disease.

Genome-wide association studies have identified over 200 genomic loci associated with inflammatory bowel disease (IBD). High-effect risk alleles define key roles for genes involved in bacterial response and innate defense. More high-throughput in vivo systems are required to rapidly evaluate therapeutic agents. We visualize, in zebrafish, the effects on epithelial barrier function and intestinal autophagy of one-course and repetitive injury. Repetitive injury induces increased mortality, impaired recovery of intestinal barrier function, failure to contain bacteria within the intestine and impaired autophagy. Prostaglandin E2 (PGE2) administration protected against injury by enhancing epithelial barrier function and limiting systemic infection. Effects of IBD therapeutic agents were defined: mesalamine showed protective features during injury, whereas 6-mercaptopurine displayed marked induction of autophagy during recovery. Given the highly conserved nature of innate defense in zebrafish, it represents an ideal model system with which to test established and new IBD therapies targeted to the epithelial barrier.This article has an associated First Person interview with the first author of the paper.

Development of an efficient viral aerosol collector for higher sampling flow rate.

Viral aerosol infection through cough generates large amounts of viral aerosol and can result in many adverse health effects such as influenza flu and severe acute respiratory syndrome (SARS). To characterize the coughed viral aerosol, the sampler needs to sample at higher flow rate and possess high physical collection efficiency as well as high viral preservation. However, most current inertia-based high flow bioaerosol samplers are not suited for viral aerosol sampling since the viability will be lost doing the sampling process. Current condensation growth methods only have good physical collection efficiency and viral preservation at low flow rate (< 10 LPM). In this study, we developed a viral aerosol sampling system using a cooler and steam-jet aerosol collector (SJAC) for bioaerosol collection for the first time. The system is based on mixing condensation growth method and has high viral preservation at a higher flow rate (12.5 LPM). We control the inlet aerosol flow temperature and the SJAC mixing reservoir temperature to improve the physical collection efficiency and viability preservation of the viral aerosol. Results indicate that the physical collection efficiency is 70-99% for aerosol 30-100 nm when the aerosol flow and mixing reservoir temperature was 19 and 50 °C, respectively. In addition, the system was 7 and 22 times more efficient for viability preservation of MS2 bacteriophage than the commonly used All Glass Impinger 30 (AGI-30) and BioSampler®, respectively. Finally, the system can be applied to sample at a lower concentration (105 PFU/m3), and results shows the system was 4.7 times more efficient for viability preservation than using AGI-30 alone. The developed viral collection system will improve our understanding of the characteristics of coughed aerosol and can be used for future evaluation of respiratory protective equipment and environmental sampling.

Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease.

Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10-10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10-8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

Allostatic load model associated with indoor environmental quality and sick building syndrome among office workers.

This study investigates whether indoor environmental quality (IEQ) influences allostatic load (AL) and whether AL can be a predictor for sick building syndrome (SBS). We also assessed and compared the associations between AL and SBS versus 8-hydroxydeoxyguanosine (8-OHdG) and SBS. A total of 115 office workers from 21 offices completed self-reported SBS questionnaires, and provided 11 biomarkers for their AL. Multiple linear regressions and logistic regression analysis were applied to examine the correlations between IEQ and AL or 8-OHdG and between AL or 8-OHdG and SBS, respectively. Our data revealed that the neuroendocrine system was correlated with CO2, the difference between indoor and outdoor CO2 levels (dCO2), and the indoor-outdoor ratio of CO2 (CO2 I/O). Metabolic system effects were associated with illumination. The relationships between illumination, CO2, dCO2, CO2 I/O and 8-OHdG were consistent with those and AL in specific systems. Furthermore, we found that risks for SBS syndromes were related with neuroendocrine and metabolic system of the AL. 8-OHdG was associated with eye dryness or irritation, eye tiredness and vomiting. We conclude that IEQ significantly influences AL and that AL can be a predictor for reporting SBS with information on system-specific effects.

Enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication.

Cholesterol is a critical component of cellular membranes, regulating assembly and function of membrane-based protein/lipid complexes. Many RNA viruses, including enteroviruses, remodel host membranes to generate organelles with unique lipid blueprints on which they assemble replication complexes and synthesize viral RNA. Here we find that clathrin-mediated endocytosis (CME) is harnessed by enteroviruses to traffic cholesterol from the plasma membrane (PM) and extracellular medium to replication organelles, where cholesterol then regulates viral polyprotein processing and facilitates genome synthesis. When CME is disrupted, cellular cholesterol pools are instead stored in lipid droplets, cholesterol cannot be trafficked to replication organelles, and replication is inhibited. In contrast, replication is stimulated in cholesterol-elevated cells like those lacking caveolins or those from Niemann-Pick disease patients. Our findings indicate cholesterol as a critical determinant for enteroviral replication and outline roles for the endocytic machinery in both the enteroviral life cycle and host cell cholesterol homeostasis.