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Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

Tue, 05/30/2017 - 12:12pm

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.

Statistical analysis of genetic interactions in Tn-Seq data

Tue, 05/30/2017 - 12:12pm

Tn-Seq is an experimental method for probing the functions of genes through construction of complex random transposon insertion libraries and quantification of each mutant's abundance using next-generation sequencing. An important emerging application of Tn-Seq is for identifying genetic interactions, which involves comparing Tn mutant libraries generated in different genetic backgrounds (e.g. wild-type strain versus knockout strain). Several analytical methods have been proposed for analyzing Tn-Seq data to identify genetic interactions, including estimating relative fitness ratios and fitting a generalized linear model. However, these have limitations which necessitate an improved approach. We present a hierarchical Bayesian method for identifying genetic interactions through quantifying the statistical significance of changes in enrichment. The analysis involves a four-way comparison of insertion counts across datasets to identify transposon mutants that differentially affect bacterial fitness depending on genetic background. Our approach was applied to Tn-Seq libraries made in isogenic strains of Mycobacterium tuberculosis lacking three different genes of unknown function previously shown to be necessary for optimal fitness during infection. By analyzing the libraries subjected to selection in mice, we were able to distinguish several distinct classes of genetic interactions for each target gene that shed light on their functions and roles during infection.

Circulating and Exosome-Packaged Hepatitis C Single-Stranded RNA Induce Monocyte Differentiation via TLR7/8 to Polarized Macrophages and Fibrocytes

Tue, 05/30/2017 - 12:12pm

Monocytes and macrophages (MPhis) play a central role in the pathogenesis of chronic hepatitis C virus (HCV) infection. The tissue microenvironment triggers monocyte differentiation into MPhis, with polarization ranging within the spectrum of M1 (classical) to M2 (alternative) activation. Recently, we demonstrated that HCV infection leads to monocyte differentiation into polarized MPhis that mediate stellate cell activation via TGF-beta. In this study, we aimed to identify the viral factor(s) that mediate monocyte-to-MPhi differentiation. We performed coculture experiments using healthy monocytes with exosome-packaged HCV, cell-free HCV, or HCV ssRNA. Coculture of monocytes with exosome-packaged HCV, cell-free HCV, or HCV ssRNA induced differentiation into MPhis with high M2 surface marker expression and production of pro- and anti-inflammatory cytokines. The HCV ssRNA-induced monocyte activation and differentiation into MPhis could be prevented by TLR7 or TLR8 knockdown. Furthermore, TLR7 or TLR8 stimulation, independent of HCV, caused monocyte differentiation and M2 MPhi polarization. In vivo, in chronic HCV-infected patients, we found increased expression of TLR7/8 in circulating monocytes that was associated with increased intracellular expression of procollagen. Furthermore, knockdown of TLR8 completely attenuated collagen expression in monocytes exposed to HCV, and knockdown of TLR7 partially attenuated this expression, suggesting roles for TLR7/8 in induction of fibrocytes in HCV infection. We identified TLR7/8 as mediators of monocyte differentiation and M2 MPhi polarization during HCV infection. Further, we demonstrated that HCV ssRNA and other TLR7/8 ligands promote MPhi polarization and generation of circulating fibrocytes.

The connection between BRG1, CTCF and topoisomerases at TAD boundaries

Tue, 05/30/2017 - 12:12pm

The eukaryotic genome is partitioned into topologically associating domains (TADs). Despite recent advances characterizing TADs and TAD boundaries, the organization of these structures is an important dimension of genome architecture and function that is not well understood. Recently, we demonstrated that knockdown of BRG1, an ATPase driving the chromatin remodeling activity of mammalian SWI/SNF enzymes, globally alters long-range genomic interactions and results in a reduction of TAD boundary strength. We provided evidence suggesting that this effect may be due to BRG1 affecting nucleosome occupancy around CTCF sites present at TAD boundaries. In this review, we elaborate on our findings and speculate that BRG1 may contribute to the regulation of the structural and functional properties of chromatin at TAD boundaries by affecting the function or the recruitment of CTCF and DNA topoisomerase complexes.

Metabolic Inputs into the Epigenome

Tue, 05/30/2017 - 12:12pm

A number of molecular pathways play key roles in transmitting information in addition to the genomic sequence-epigenetic information-from one generation to the next. However, so-called epigenetic marks also impact an enormous variety of physiological processes, even under circumstances that do not result in heritable consequences. Perhaps inevitably, the epigenetic regulatory machinery is highly responsive to metabolic cues, as, for example, central metabolites are the substrates for the enzymes that catalyze the deposition of covalent modifications on histones, DNA, and RNA. Interestingly, in addition to the effects that metabolites exert over biological regulation in somatic cells, over the past decade multiple studies have shown that ancestral nutrition can alter the metabolic phenotype of offspring, raising the question of how metabolism regulates the epigenome of germ cells. Here, we review the widespread links between metabolism and epigenetic modifications, both in somatic cells and in the germline.

Subclinical Atherosclerosis, Statin Eligibility, and Outcomes in African American Individuals: The Jackson Heart Study

Tue, 05/30/2017 - 12:12pm

Importance: Modern prevention guidelines substantially increase the number of individuals who are eligible for treatment with statins. Efforts to refine statin eligibility via coronary calcification have been studied in white populations but not, to our knowledge, in large African American populations.

Objective: To compare the relative accuracy of US Preventive Services Task Force (USPSTF) and American College of Cardiology/American Heart Association (ACC/AHA) recommendations in identifying African American individuals with subclinical and clinical atherosclerotic cardiovascular disease (ASCVD).

Design, Setting, and Participants: In this prospective, community-based study, 2812 African American individuals aged 40 to 75 years without prevalent ASCVD underwent assessment of ASCVD risk. Of these, 1743 participants completed computed tomography.

Main Outcomes and Measures: Nonzero coronary artery calcium (CAC) score, abdominal aortic calcium score, and incident ASCVD (ie, myocardial infarction, ischemic stroke, or fatal coronary heart disease).

Results: Of the 2812 included participants, the mean (SD) age at baseline was 55.4 (9.4) years, and 1837 (65.3%) were female. The USPSTF guidelines captured 404 of 732 African American individuals (55.2%) with a CAC score greater than 0; the ACC/AHA guidelines identified 507 individuals (69.3%) (risk difference, 14.1%; 95% CI, 11.2-17.0; P < .001). Statin recommendation under both guidelines was associated with a CAC score greater than 0 (odds ratio, 5.1; 95% CI, 4.1-6.3; P < .001). While individuals indicated for statins under both guidelines experienced 9.6 cardiovascular events per 1000 patient-years, those indicated under only ACC/AHA guidelines were at low to intermediate risk (4.1 events per 1000 patient-years). Among individuals who were statin eligible by ACC/AHA guidelines, the 10-year ASCVD incidence per 1000 person-years was 8.1 (95% CI, 5.9-11.1) in the presence of CAC and 3.1 (95% CI, 1.6-5.9) without CAC (P = .02). While statin-eligible individuals by USPSTF guidelines did not have a significantly higher 10-year ASCVD event rate in the presence of CAC, African American individuals not eligible for statins by USPSTF guidelines had a higher ASCVD event rate in the presence of CAC (2.8 per 1000 person-years; 95% CI, 1.5-5.4) relative to without CAC (0.8 per 1000 person-years; 95%, CI 0.3-1.7) (P = .03).

Conclusions and Relevance: The USPSTF guidelines focus treatment recommendations on 38% of high-risk African American individuals at the expense of not recommending treatment in nearly 25% of African American individuals eligible for statins by ACC/AHA guidelines with vascular calcification and at low to intermediate ASCVD risk.

Interleukin-17: Why the Worms Squirm

Tue, 05/30/2017 - 12:12pm

IL-17 is a cytokine known primarily for its role in inflammation. In a recent issue of Nature, Chen et al. (2017) demonstrate that IL-17 plays a neuromodulatory role in Caenorhabditis elegans by acting directly on neurons to amplify neuronal responses to stimuli and produce changes in animal behavior.

Stroke and Circulating Extracellular RNAs

Tue, 05/30/2017 - 12:11pm

BACKGROUND AND PURPOSE: There is increasing interest in extracellular RNAs (ex-RNAs), with numerous reports of associations between selected microRNAs (miRNAs) and a variety of cardiovascular disease phenotypes. Previous studies of ex-RNAs in relation to risk for cardiovascular disease have investigated small numbers of patients and assayed only candidate miRNAs. No human studies have investigated links between novel ex-RNAs and stroke.

METHODS: We conducted unbiased next-generation sequencing using plasma from 40 participants of the FHS (Framingham Heart Study; Offspring Cohort Exam 8) followed by high-throughput polymerase chain reaction of 471 ex-RNAs. The reverse transcription quantitative polymerase chain reaction included 331 of the most abundant miRNAs, 43 small nucleolar RNAs, and 97 piwi-interacting RNAs in 2763 additional FHS participants and explored the relations of ex-RNAs and prevalent (n=63) and incident (n=51) stroke and coronary heart disease (prevalent=286, incident=69).

RESULTS: After adjustment for multiple cardiovascular disease risk factors, 7 ex-RNAs were associated with stroke prevalence or incidence; there were no ex-RNA associated with prevalent or incident coronary heart disease. Statistically significant ex-RNA associations with stroke were specific, with no overlap between prevalent and incident events.

CONCLUSIONS: This is the largest study of ex-RNAs in relation to stroke using an unbiased approach in an observational cohort and the first large study to examine human small noncoding RNAs beyond miRNAs. These results demonstrate that when studied in a large observational cohort, extracellular miRNAs are associated with stroke risk.

CXCR3 Depleting Antibodies Prevent and Reverse Vitiligo in Mice

Tue, 05/30/2017 - 12:11pm

Therefore, we sought to determine if targeting CXCR3 could serve as a new treatment for vitiligo.

Extracellular RNAs Are Associated With Insulin Resistance and Metabolic Phenotypes

Tue, 05/30/2017 - 12:11pm

OBJECTIVE: Insulin resistance (IR) is a hallmark of obesity and metabolic disease. Circulating extracellular RNAs (ex-RNAs), stable RNA molecules in plasma, may play a role in IR, though most studies on ex-RNAs in IR are small. We sought to characterize the relationship between ex-RNAs and metabolic phenotypes in a large community-based human cohort.

RESEARCH DESIGN AND METHODS: We measured circulating plasma ex-RNAs in 2,317 participants without diabetes in the Framingham Heart Study (FHS) Offspring Cohort at cycle 8 and defined associations between ex-RNAs and IR (measured by circulating insulin level). We measured association between candidate ex-RNAs and markers of adiposity. Sensitivity analyses included individuals with diabetes. In a separate cohort of 90 overweight/obese youth, we measured selected ex-RNAs and metabolites. Biology of candidate microRNAs was investigated in silico.

RESULTS: The mean age of FHS participants was 65.8 years (56% female), with average BMI 27.7 kg/m2; participants in the youth cohort had a mean age of 15.5 years (60% female), with mean BMI 33.8 kg/m2. In age-, sex-, and BMI-adjusted models across 391 ex-RNAs in FHS, 18 ex-RNAs were associated with IR (of which 16 were microRNAs). miR-122 was associated with IR and regional adiposity in adults and IR in children (independent of metabolites). Pathway analysis revealed metabolic regulatory roles for miR-122, including regulation of IR pathways (AMPK, target of rapamycin signaling, and mitogen-activated protein kinase).

CONCLUSIONS: These results provide translational evidence in support of an important role of ex-RNAs as novel circulating factors implicated in IR.

Developmental Role of Macrophage Cannabinoid-1 Receptor Signaling in Type 2 Diabetes

Tue, 05/30/2017 - 12:11pm

Islet inflammation promotes beta-cell loss and type 2 diabetes (T2D), a process replicated in Zucker Diabetic Fatty (ZDF) rats in which beta-cell loss has been linked to cannabinoid-1 receptor (CB1R)-induced proinflammatory signaling in macrophages infiltrating pancreatic islets. Here, we analyzed CB1R signaling in macrophages and its developmental role in T2D. ZDF rats with global deletion of CB1R are protected from beta-cell loss, hyperglycemia, and nephropathy that are present in ZDF littermates. Adoptive transfer of CB1R-/- bone marrow to ZDF rats also prevents beta-cell loss and hyperglycemia but not nephropathy. ZDF islets contain elevated levels of CB1R, interleukin-1beta, tumor necrosis factor-alpha, the chemokine CCL2, and interferon regulatory factor-5 (IRF5), a marker of inflammatory macrophage polarization. In primary cultured rodent and human macrophages, CB1R activation increased Irf5 expression, whereas knockdown of Irf5 blunted CB1R-induced secretion of inflammatory cytokines without affecting CCL2 expression, which was p38MAPKalpha dependent. Macrophage-specific in vivo knockdown of Irf5 protected ZDF rats from beta-cell loss and hyperglycemia. Thus, IRF5 is a crucial downstream mediator of diabetogenic CB1R signaling in macrophages and a potential therapeutic target.

Chemical-Induced Vitiligo

Tue, 05/30/2017 - 12:11pm

Chemical-induced depigmentation of the skin has been recognized for more than 75 years, first as an occupational hazard but then extending to those using household commercial products as common as hair dyes. Since their discovery, these chemicals have been used therapeutically in patients with severe vitiligo to depigment their remaining skin and improve their appearance. Because chemical-induced depigmentation is clinically and histologically indistinguishable from nonchemically induced vitiligo, and because these chemicals appear to induce melanocyte autoimmunity, this phenomenon should be known as "chemical-induced vitiligo," rather than less accurate terms that have been previously used.

Vitiligo Pathogenesis and Emerging Treatments

Tue, 05/30/2017 - 12:11pm

The pathogenesis of vitiligo involves interplay between intrinsic and extrinsic melanocyte defects, innate immune inflammation, and T-cell-mediated melanocyte destruction. The goal of treatment is to not only halt disease progression but also promote repigmentation through melanocyte regeneration, proliferation, and migration. Treatment strategies that address all aspects of disease pathogenesis and repigmentation are likely to have greatest efficacy, a strategy that may require combination therapies. Current treatments generally involve nontargeted suppression of autoimmunity, whereas emerging treatments are likely to use a more targeted approach based on in-depth understanding of disease pathogenesis, which may provide higher efficacy with a good safety profile.

Mammalian SWI/SNF Enzymes and the Epigenetics of Tumor Cell Metabolic Reprogramming

Tue, 05/30/2017 - 12:11pm

Tumor cells reprogram their metabolism to survive and grow in a challenging microenvironment. Some of this reprogramming is performed by epigenetic mechanisms. Epigenetics is in turn affected by metabolism; chromatin modifying enzymes are dependent on substrates that are also key metabolic intermediates. We have shown that the chromatin remodeling enzyme Brahma-related gene 1 (BRG1), an epigenetic regulator, is necessary for rapid breast cancer cell proliferation. The mechanism for this requirement is the BRG1-dependent transcription of key lipogenic enzymes and regulators. Reduction in lipid synthesis lowers proliferation rates, which can be restored by palmitate supplementation. This work has established BRG1 as an attractive target for breast cancer therapy. Unlike genetic alterations, epigenetic mechanisms are reversible, promising gentler therapies without permanent off-target effects at distant sites.

Dynamic Role of trans Regulation of Gene Expression in Relation to Complex Traits

Tue, 05/30/2017 - 12:10pm

Identifying causal genetic variants and understanding their mechanisms of effect on traits remains a challenge in genome-wide association studies (GWASs). In particular, how genetic variants (i.e., trans-eQTLs) affect expression of remote genes (i.e., trans-eGenes) remains unknown. We hypothesized that some trans-eQTLs regulate expression of distant genes by altering the expression of nearby genes (cis-eGenes). Using published GWAS datasets with 39,165 single-nucleotide polymorphisms (SNPs) associated with 1,960 traits, we explored whole blood gene expression associations of trait-associated SNPs in 5,257 individuals from the Framingham Heart Study. We identified 2,350 trans-eQTLs (at p < 10-7); more than 80% of them were found to have cis-associated eGenes. Mediation testing suggested that for 35% of trans-eQTL-trans-eGene pairs in different chromosomes and 90% pairs in the same chromosome, the disease-associated SNP may alter expression of the trans-eGene via cis-eGene expression. In addition, we identified 13 trans-eQTL hotspots, affecting from ten to hundreds of genes, suggesting the existence of master genetic regulators. Using causal inference testing, we searched causal variants across eight cardiometabolic traits (BMI, systolic and diastolic blood pressure, LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides, and fasting blood glucose) and identified several cis-eGenes (ALDH2 for systolic and diastolic blood pressure, MCM6 and DARS for total cholesterol, and TRIB1 for triglycerides) that were causal mediators for the corresponding traits, as well as examples of trans-mediators (TAGAP for LDL cholesterol). The finding of extensive evidence of genome-wide mediation effects suggests a critical role of cryptic gene regulation underlying many disease traits.

Cleaning House: Selective Autophagy of Organelles

Tue, 05/30/2017 - 12:10pm

The selective clearance of organelles by autophagy is critical for the regulation of cellular homeostasis in organisms from yeast to humans. Removal of damaged organelles clears the cell of potentially toxic byproducts and enables reuse of organelle components for bioenergetics. Thus, defects in organelle clearance may be detrimental to the health of the cells, contributing to cancer, neurodegeneration, and inflammatory diseases. Organelle-specific autophagy can clear mitochondria, peroxisomes, lysosomes, ER, chloroplasts, and the nucleus. Here, we review our understanding of the mechanisms that regulate the clearance of organelles by autophagy and highlight gaps in our knowledge of these processes.

Standardized Screening for Mental Health Needs of Detained Youths from Various Ethnic Origins: The Dutch Massachusetts Youth Screening Instrument-Second Version (MAYSI-2)

Sun, 05/28/2017 - 6:10pm

In the U.S., the Massachusetts Youth Screening Instrument-Second Version (MAYSI-2) has been shown to be a reliable and valid tool to identify youth with mental health needs upon entry in detention facilities. The present study examined the factor structure, internal consistency, and convergent validity of the Dutch MAYSI-2 administered as part of routine clinical assessments in up to 955 detained male adolescents. Standardized mental health screening questionnaires (Youth Self-Report and Strengths and Difficulties Questionnaire) were used to test the convergent validity of the Dutch MAYSI-2. Confirmatory factor analyses showed that the factor structure of the original MAYSI-2 could be replicated with the Dutch MAYSI-2. Internal consistency indices showed that the Dutch MAYSI-2 provides a reliable screening of mental health needs. In addition, the Dutch MAYSI-2 scales were related with conceptually parallel measures of the same targeted mental health needs in the total group. With a few exceptions, the internal consistency and convergent validity was supported across ethnic groups as well. Overall, these results suggest the psychometric properties of the Dutch MAYSI-2 to be promising. Implications and limitations of the current study's findings and directions for future research are discussed.

Has screening mammography become obsolete

Sun, 05/28/2017 - 6:10pm

With so much debate about the value of screening mammography and with the emergence of newer technologies, it seems reasonable to ask whether screening mammography, as we know it, has become obsolete.

Predictors of Gestational Weight Gain among White and Latina Women and Associations with Birth Weight

Sun, 05/28/2017 - 6:10pm

This study examined racial/ethnic differences in gestational weight gain (GWG) predictors and association of first-trimester GWG to overall GWG among 271 White women and 300 Latina women. Rates of within-guideline GWG were higher among Latinas than among Whites (28.7% versus 24.4%, p < 0.016). Adjusted odds of above-guideline GWG were higher among prepregnancy overweight (OR = 3.4, CI = 1.8-6.5) and obese (OR = 4.5, CI = 2.3-9.0) women than among healthy weight women and among women with above-guideline first-trimester GWG than among those with within-guideline first-trimester GWG (OR = 4.9, CI = 2.8-8.8). GWG was positively associated with neonate birth size (p < 0.001). Interventions targeting prepregnancy overweight or obese women and those with excessive first-trimester GWG are needed.