Where Are the Disease-Associated eQTLs?

Most disease-associated variants, although located in putatively regulatory regions, do not have detectable effects on gene expression. One explanation could be that we have not examined gene expression in the cell types or conditions that are most relevant for disease. Even large-scale efforts to study gene expression across tissues are limited to human samples obtained opportunistically or postmortem, mostly from adults. In this review we evaluate recent findings and suggest an alternative strategy, drawing on the dynamic and highly context-specific nature of gene regulation. We discuss new technologies that can extend the standard regulatory mapping framework to more diverse, disease-relevant cell types and states.

An Airway Protection Program Revealed by Sweeping Genetic Control of Vagal Afferents.

Sensory neurons initiate defensive reflexes that ensure airway integrity. Dysfunction of laryngeal neurons is life-threatening, causing pulmonary aspiration, dysphagia, and choking, yet relevant sensory pathways remain poorly understood. Here, we discover rare throat-innervating neurons (∼100 neurons/mouse) that guard the airways against assault. We used genetic tools that broadly cover a vagal/glossopharyngeal sensory neuron atlas to map, ablate, and control specific afferent populations. Optogenetic activation of vagal P2RY1 neurons evokes a coordinated airway defense program-apnea, vocal fold adduction, swallowing, and expiratory reflexes. Ablation of vagal P2RY1 neurons eliminates protective responses to laryngeal water and acid challenge. Anatomical mapping revealed numerous laryngeal terminal types, with P2RY1 neurons forming corpuscular endings that appose laryngeal taste buds. Epithelial cells are primary airway sentinels that communicate with second-order P2RY1 neurons through ATP. These findings provide mechanistic insights into airway defense and a general molecular/genetic roadmap for internal organ sensation by the vagus nerve.

Genetic Determinants of Penicillin Tolerance in Vibrio cholerae.

Many bacteria are resistant to killing (tolerant) by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A penicillin binding proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. Transposon insertion sequencing (TnSeq) analyses revealed that lipopolysaccharide (LPS) and cell wall biogenesis genes, as well as the sigma E cell envelope stress response, were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for postantibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

Neural Sensing of Organ Volume.

Many internal organs change volume periodically. For example, the stomach accommodates ingested food and drink, the bladder stores urine, the heart fills with blood, and the lungs expand with every breath. Specialized peripheral sensory neurons function as mechanoreceptors that detect tissue stretch to infer changes in organ volume and then relay this information to the brain. Central neural circuits process this information and evoke perceptions (satiety, nausea), control physiology (breathing, heart rate), and impact behavior (feeding, micturition). Yet, basic questions remain about how neurons sense organ distension and whether common sensory motifs are involved across organs. Here, we review candidate mechanosensory receptors, cell types, and neural circuits, focusing on the stomach, bladder, and airways. Understanding mechanisms of organ stretch sensation may provide new ways to treat autonomic dysfunction.

Author Correction: Nociceptor sensory neurons suppress neutrophil and γδ T cell responses in bacterial lung infections and lethal pneumonia.

In the version of this article initially published, the line graph showing TNF-α levels in Fig. 2d was inadvertently duplicated. A graph of IL-6 levels should be shown in place of the duplication.These results were also incorrectly described in the main text, which originally stated: "At an early time point of infection (6 h), RTX-treated mice showed higher induction of total inflammatory-protein levels in the bronchoalveolar lavage fluid (BALF) (Fig. 2c), as well as levels of the cytokines TNF-α and IL-6, and the chemokine CXCL-1 (Fig. 2d)". This should instead read: "At an early time point of infection (6 h), RTX-treated mice showed higher induction of total inflammatory-protein levels in the bronchoalveolar lavage fluid (BALF) (Fig. 2c), as well as levels of the cytokine TNF-α and the chemokine CXCL-1 (Fig. 2d)".In the supplementary information initially posted online, incorrect bar graphs were presented in Supplementary Fig. 1b (VG, TRPV1+ data, top panel) and Supplementary Fig. 4b (DRG, CGRP+ data, middle panel).

Nociceptor sensory neurons suppress neutrophil and γδ T cell responses in bacterial lung infections and lethal pneumonia.

Lung-innervating nociceptor sensory neurons detect noxious or harmful stimuli and consequently protect organisms by mediating coughing, pain, and bronchoconstriction. However, the role of sensory neurons in pulmonary host defense is unclear. Here, we found that TRPV1+ nociceptors suppressed protective immunity against lethal Staphylococcus aureus pneumonia. Targeted TRPV1+-neuron ablation increased survival, cytokine induction, and lung bacterial clearance. Nociceptors suppressed the recruitment and surveillance of neutrophils, and altered lung γδ T cell numbers, which are necessary for immunity. Vagal ganglia TRPV1+ afferents mediated immunosuppression through release of the neuropeptide calcitonin gene-related peptide (CGRP). Targeting neuroimmunological signaling may be an effective approach to treat lung infections and bacterial pneumonia.

Regions of very low H3K27me3 partition the Drosophila genome into topological domains.

It is now well established that eukaryote genomes have a common architectural organization into topologically associated domains (TADs) and evidence is accumulating that this organization plays an important role in gene regulation. However, the mechanisms that partition the genome into TADs and the nature of domain boundaries are still poorly understood. We have investigated boundary regions in the Drosophila genome and find that they can be identified as domains of very low H3K27me3. The genome-wide H3K27me3 profile partitions into two states; very low H3K27me3 identifies Depleted (D) domains that contain housekeeping genes and their regulators such as the histone acetyltransferase-containing NSL complex, whereas domains containing moderate-to-high levels of H3K27me3 (Enriched or E domains) are associated with regulated genes, irrespective of whether they are active or inactive. The D domains correlate with the boundaries of TADs and are enriched in a subset of architectural proteins, particularly Chromator, BEAF-32, and Z4/Putzig. However, rather than being clustered at the borders of these domains, these proteins bind throughout the H3K27me3-depleted regions and are much more strongly associated with the transcription start sites of housekeeping genes than with the H3K27me3 domain boundaries. While we have not demonstrated causality, we suggest that the D domain chromatin state, characterised by very low or absent H3K27me3 and established by housekeeping gene regulators, acts to separate topological domains thereby setting up the domain architecture of the genome.

A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics.

Gram-negative bacteria are notoriously resistant to a variety of high-molecular-weight antibiotics due to the limited permeability of their outer membrane (OM). The basis of OM barrier function and the genetic factors required for its maintenance remain incompletely understood. Here, we employed transposon insertion sequencing to identify genes required for Vibrio cholerae resistance to vancomycin and bacitracin, antibiotics that are thought to be too large to efficiently penetrate the OM. The screen yielded several genes whose protein products are predicted to participate in processes important for OM barrier functions and for biofilm formation. In addition, we identified a novel factor, designated vigA (for vancomycin inhibits growth), that has not previously been characterized or linked to outer membrane function. The vigA open reading frame (ORF) codes for an inner membrane protein, and in its absence, cells became highly sensitive to glycopeptide antibiotics (vancomycin and ramoplanin) and bacitracin but not to other large antibiotics or detergents. In contrast to wild-type (WT) cells, the vigA mutant was stained with fluorescent vancomycin. These observations suggest that VigA specifically prevents the periplasmic accumulation of certain large antibiotics without exerting a general role in the maintenance of OM integrity. We also observed marked interspecies variability in the susceptibilities of Gram-negative pathogens to glycopeptides and bacitracin. Collectively, our findings suggest that the OM barrier is not absolute but rather depends on specific OM-antibiotic interactions.

Sensory Neurons that Detect Stretch and Nutrients in the Digestive System.

Neural inputs from internal organs are essential for normal autonomic function. The vagus nerve is a key body-brain connection that monitors the digestive, cardiovascular, and respiratory systems. Within the gastrointestinal tract, vagal sensory neurons detect gut hormones and organ distension. Here, we investigate the molecular diversity of vagal sensory neurons and their roles in sensing gastrointestinal inputs. Genetic approaches allowed targeted investigation of gut-to-brain afferents involved in homeostatic responses to ingested nutrients (GPR65 neurons) and mechanical distension of the stomach and intestine (GLP1R neurons). Optogenetics, in vivo ganglion imaging, and genetically guided anatomical mapping provide direct links between neuron identity, peripheral anatomy, central anatomy, conduction velocity, response properties in vitro and in vivo, and physiological function. These studies clarify the roles of vagal afferents in mediating particular gut hormone responses. Moreover, genetic control over gut-to-brain neurons provides a molecular framework for understanding neural control of gastrointestinal physiology.

Predictive MRI correlates of lesser metatarsophalangeal joint plantar plate tear.

PURPOSE: To identify correlated signs on non-enhanced MRI that might improve diagnostic detection of plantar plate (PP) tear. MATERIALS AND METHODS: We performed an IRB-approved, HIPAA-compliant retrospective analysis of 100 non-contrast MRI (50 PP tear, 50 controls). All were anonymized, randomized, and reviewed; 20 were duplicated to assess consistency. One musculoskeletal radiologist evaluated qualitative variables. A trained non-physician performed measurements. Consistency and concordance were assessed. Pearson's Chi-square test was used to test the correlation between qualitative findings and PP tear status. Correlation between measurements and PP status was assessed using t tests and Wilcoxon's rank-sum test (p values < 0.05 considered significant). Classification and regression trees were utilized to identify attributes that, taken together, would consistently distinguish PP tear from controls. RESULTS: Quantitative measurements were highly reproducible (concordance 0.88-0.99). Elevated 2nd MT protrusion, lesser MT supination and rotational divergence of >45° between the 1st-2nd MT axis correlated with PP tear. Pericapsular soft tissue thickening correlated most strongly with PP tear, correctly classifying 95 % of cases and controls. Excluding pericapsular soft tissue thickening, sequential assessment of 2nd toe enthesitis, 2nd flexor tendon subluxation, and splaying of the second and third toes accurately classified PP status in 92 %. CONCLUSIONS: Pericapsular soft tissue thickening most strongly correlated with PP tear. For cases in which it might be difficult to distinguish pericapsular fibrosis from neuroma, sequential assessment of 2nd toe enthesitis, flexor tendon subluxation and splaying of the 2nd and 3rd toe is most helpful for optimizing accurate diagnosis of PP tear.

Fatty acids linked to cardiovascular mortality are associated with risk factors.

BACKGROUND: Although saturated fatty acids (FAs) have been linked to cardiovascular mortality, it is not clear whether this outcome is attributable solely to their effects on low-density lipoprotein cholesterol (LDL-C) or whether other risk factors are also associated with FAs. The Western Alaskan Native population, with its rapidly changing lifestyles, shift in diet from unsaturated to saturated fatty acids and dramatic increase in cardiovascular disease (CVD), presents an opportunity to elucidate any associations between specific FAs and known CVD risk factors. OBJECTIVE: We tested the hypothesis that the specific FAs previously identified as related to CVD mortality are also associated with individual CVD risk factors. METHODS: In this community-based, cross-sectional study, relative proportions of FAs in plasma and red blood cell membranes were compared with CVD risk factors in a sample of 758 men and women aged ≥35 years. Linear regression analyses were used to analyze relations between specific FAs and CVD risk factors (LDL-C, high-density lipoprotein cholesterol, triglycerides, C-reactive protein, systolic blood pressure, diastolic blood pressure, heart rate, body mass index, fasting glucose and fasting insulin, 2-hour glucose and 2-hour insulin). RESULTS: The specific saturated FAs previously identified as related to CVD mortality, the palmitic and myristic acids, were adversely associated with most CVD risk factors, whereas unsaturated linoleic acid (18:2n-6) and the marine n-3 FAs were not associated or were beneficially associated with CVD risk factors. CONCLUSIONS: The results suggest that CVD risk factors are more extensively affected by individual FAs than hitherto recognized, and that risk for CVD, MI and stroke can be reduced by reducing the intake of palmitate, myristic acid and simple carbohydrates and improved by greater intake of linoleic acid and marine n-3 FAs.

Vagal Sensory Neuron Subtypes that Differentially Control Breathing.

Breathing is essential for survival and under precise neural control. The vagus nerve is a major conduit between lung and brain required for normal respiration. Here, we identify two populations of mouse vagus nerve afferents (P2ry1, Npy2r), each a few hundred neurons, that exert powerful and opposing effects on breathing. Genetically guided anatomical mapping revealed that these neurons densely innervate the lung and send long-range projections to different brainstem targets. Npy2r neurons are largely slow-conducting C fibers, while P2ry1 neurons are largely fast-conducting A fibers that contact pulmonary endocrine cells (neuroepithelial bodies). Optogenetic stimulation of P2ry1 neurons acutely silences respiration, trapping animals in exhalation, while stimulating Npy2r neurons causes rapid, shallow breathing. Activating P2ry1 neurons did not impact heart rate or gastric pressure, other autonomic functions under vagal control. Thus, the vagus nerve contains intermingled sensory neurons constituting genetically definable labeled lines with different anatomical connections and physiological roles.