A CRISPR screen using BV2 cells identifies sialic acid synthesis genes as required for reovirus attachment and infection

Engagement of cell-surface receptors by viruses is a critical determinant of tropism and disease. The reovirus attachment protein, σ1, first binds sialylated and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are not entirely known. To identify host factors required for reovirus replication and subsequent cell killing, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus-induced cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of T3SA+, a strain known to engage sialic acid, and no change in the low-level binding of T3SA-, a strain that does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in CRISPR-modified cells. Following exogenous expression of Cmas and Slc35a1 into the respective null cells, sialic acid expression was restored. These results demonstrate that Cmas and Slc35a1, which are required for cell-surface expression of sialic acid, enhance reovirus attachment. Moreover, these findings provide additional evidence that sialic acid, which is expressed on most cells, serves as an attachment factor for reovirus. While reovirus is currently not a major public health concern, reoviruses are a highly tractable experimental model for the study of viral pathogenesis. These findings shed light on general principles of virus-receptor interactions which are important determinants of dissemination and tropism of many viruses affecting public health today

Expression of Ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of IFN-lambda against norovirus and reovirus

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1. We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity. IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo. Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1(−/−) mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections

Expression of SMARCD1 interacts with age in association with asthma control on inhaled corticosteroid therapy.

BackgroundGlobal gene expression levels are known to be highly dependent upon gross demographic features including age, yet identification of age-related genomic indicators has yet to be comprehensively undertaken in a disease and treatment-specific context.MethodsWe used gene expression data from CD4+ lymphocytes in the Asthma BioRepository for Integrative Genomic Exploration (Asthma BRIDGE), an open-access collection of subjects participating in genetic studies of asthma with available gene expression data. Replication population participants were Puerto Rico islanders recruited as part of the ongoing Genes environments & Admixture in Latino Americans (GALA II), who provided nasal brushings for transcript sequencing. The main outcome measure was chronic asthma control as derived by questionnaires. Genomic associations were performed using regression of chronic asthma control score on gene expression with age in years as a covariate, including a multiplicative interaction term for gene expression times age.ResultsThe SMARCD1 gene (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1) interacted with age to influence chronic asthma control on inhaled corticosteroids, with a doubling of expression leading to an increase of 1.3 units of chronic asthma control per year (95% CI [0.86, 1.74], p = 6 × 10- 9), suggesting worsening asthma control with increasing age. This result replicated in GALA II (p = 3.8 × 10- 8). Cellular assays confirmed the role of SMARCD1 in glucocorticoid response in airway epithelial cells.ConclusionFocusing on age-dependent factors may help identify novel indicators of asthma medication response. Age appears to modulate the effect of SMARCD1 on asthma control with inhaled corticosteroids

The Association Between Ankle-Brachial Index and Daily Patterns of Physical Activity: Results From the Hispanic Community Health Study/Study of Latinos.

BACKGROUND: Peripheral artery disease (PAD) is associated with lower physical activity but less is known about its association with daily patterns of activity. We examined the cross-sectional association between ankle-brachial index (ABI) and objectively measured patterns of physical activity among Hispanic/Latino adults. METHODS: We analyzed data from 7 688 participants (aged 45-74 years) in the Hispanic Community Health Study/Study of Latinos. ABI was categorized as low (≤0.90, indicating PAD), borderline low (0.91-0.99), normal (1.00-1.40), and high (>1.40, indicating incompressible ankle arteries). Daily physical activity metrics derived from accelerometer data included: log of total activity counts (LTAC), total log-transformed activity counts (TLAC), and active-to-sedentary transition probability (ASTP). Average differences between ABI categories in physical activity, overall and by 4-hour time-of-day intervals, were assessed using linear regression and mixed-effects models, respectively. RESULTS: In Hispanic/Latino adults, 5.3% and 2.6% had low and high ABIs, respectively. After adjustment, having a low compared to a normal ABI was associated with lower volume (LTAC = -0.13, p < .01; TLAC = -74.4, p = .04) and more fragmented physical activity (ASTP = 1.22%, p < .01). Having a low ABI was linked with more fragmented physical activity after 12 pm (p < .01). Having a high ABI was associated with lower volumes of activity (TLAC = -132.0, p = .03). CONCLUSIONS: Having a low or high ABI is associated with lower and more fragmented physical activity in Hispanic/Latino adults. In adults with low ABI, physical activity is more fragmented in the afternoon to evening. Longitudinal research is warranted to expand these findings to guide targeted interventions for PAD or incompressible ankle arteries

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells

Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors

Daily steps and all-cause mortality: a meta-analysis of 15 international cohorts

Background Although 10000 steps per day is widely promoted to have health benefits, there is little evidence to support this recommendation. We aimed to determine the association between number of steps per day and stepping rate with all-cause mortality. Methods In this meta-analysis, we identified studies investigating the effect of daily step count on all-cause mortality in adults (aged ≥18 years), via a previously published systematic review and expert knowledge of the field. We asked participating study investigators to process their participant-level data following a standardised protocol. The primary outcome was all-cause mortality collected from death certificates and country registries. We analysed the dose– response association of steps per day and stepping rate with all-cause mortality. We did Cox proportional hazards regression analyses using study-specific quartiles of steps per day and calculated hazard ratios (HRs) with inversevariance weighted random effects models. Findings We identified 15 studies, of which seven were published and eight were unpublished, with study start dates between 1999 and 2018. The total sample included 47 471 adults, among whom there were 3013 deaths (10·1 per 1000 participant-years) over a median follow-up of 7·1 years ([IQR 4·3–9·9]; total sum of follow-up across studies was 297 837 person-years). Quartile median steps per day were 3553 for quartile 1, 5801 for quartile 2, 7842 for quartile 3, and 10 901 for quartile 4. Compared with the lowest quartile, the adjusted HR for all-cause mortality was 0·60 (95% CI 0·51–0·71) for quartile 2, 0·55 (0·49–0·62) for quartile 3, and 0·47 (0·39–0·57) for quartile 4. Restricted cubic splines showed progressively decreasing risk of mortality among adults aged 60 years and older with increasing number of steps per day until 6000–8000 steps per day and among adults younger than 60 years until 8000–10000 steps per day. Adjusting for number of steps per day, comparing quartile 1 with quartile 4, the association between higher stepping rates and mortality was attenuated but remained significant for a peak of 30 min (HR 0·67 [95% CI 0·56–0·83]) and a peak of 60 min (0·67 [0·50–0·90]), but not significant for time (min per day) spent walking at 40 steps per min or faster (1·12 [0·96–1·32]) and 100 steps per min or faster (0·86 [0·58–1·28]). Interpretation Taking more steps per day was associated with a progressively lower risk of all-cause mortality, up to a level that varied by age. The findings from this meta-analysis can be used to inform step guidelines for public health promotion of physical activity

Daily steps and all-cause mortality: a meta-analysis of 15 international cohorts

Background Although 10 000 steps per day is widely promoted to have health benefits, there is little evidence to support this recommendation. We aimed to determine the association between number of steps per day and stepping rate with all-cause mortality. Methods In this meta-analysis, we identified studies investigating the effect of daily step count on all-cause mortality in adults (aged ≥18 years), via a previously published systematic review and expert knowledge of the field. We asked participating study investigators to process their participant-level data following a standardised protocol. The primary outcome was all-cause mortality collected from death certificates and country registries. We analysed the dose–response association of steps per day and stepping rate with all-cause mortality. We did Cox proportional hazards regression analyses using study-specific quartiles of steps per day and calculated hazard ratios (HRs) with inverse-variance weighted random effects models. Findings We identified 15 studies, of which seven were published and eight were unpublished, with study start dates between 1999 and 2018. The total sample included 47 471 adults, among whom there were 3013 deaths (10·1 per 1000 participant-years) over a median follow-up of 7·1 years ([IQR 4·3–9·9]; total sum of follow-up across studies was 297 837 person-years). Quartile median steps per day were 3553 for quartile 1, 5801 for quartile 2, 7842 for quartile 3, and 10 901 for quartile 4. Compared with the lowest quartile, the adjusted HR for all-cause mortality was 0·60 (95% CI 0·51–0·71) for quartile 2, 0·55 (0·49–0·62) for quartile 3, and 0·47 (0·39–0·57) for quartile 4. Restricted cubic splines showed progressively decreasing risk of mortality among adults aged 60 years and older with increasing number of steps per day until 6000–8000 steps per day and among adults younger than 60 years until 8000–10 000 steps per day. Adjusting for number of steps per day, comparing quartile 1 with quartile 4, the association between higher stepping rates and mortality was attenuated but remained significant for a peak of 30 min (HR 0·67 [95% CI 0·56–0·83]) and a peak of 60 min (0·67 [0·50–0·90]), but not significant for time (min per day) spent walking at 40 steps per min or faster (1·12 [0·96–1·32]) and 100 steps per min or faster (0·86 [0·58–1·28]). Interpretation Taking more steps per day was associated with a progressively lower risk of all-cause mortality, up to a level that varied by age. The findings from this meta-analysis can be used to inform step guidelines for public health promotion of physical activity. Funding US Centers for Disease Control and Prevention

Cardiovascular development: towards biomedical applicability: Epicardium-derived cells in cardiogenesis and cardiac regeneration

During cardiogenesis, the epicardium grows from the proepicardial organ to form the outermost layer of the early heart. Part of the epicardium undergoes epithelial-mesenchymal transformation, and migrates into the myocardium. These epicardium- derived cells differentiate into interstitial fibroblasts, coronary smooth muscle cells, and perivascular fibroblasts. Moreover, epicardium-derived cells are important regulators of formation of the compact myocardium, the coronary vasculature, and the Purkinje fiber network, thus being essential for proper cardiac development. The fibrous structures of the heart such as the fibrous heart skeleton and the semilunar and atrioventricular valves also depend on a contribution of these cells during development. We hypothesise that the essential properties of epicardium-derived cells can be recapitulated in adult diseased myocardium. These cells can therefore be considered as a novel source of adult stem cells useful in clinical cardiac regeneration therapy