Single-Cell Transcriptomes Reveal a Complex Cellular Landscape in the Middle Ear and Differential Capacities for Acute Response to Infection.

Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens

Association of Systemic Inflammation With Retinal Vascular Caliber in Patients With AIDS.

PurposeTo evaluate relationships among retinal vascular caliber and biomarkers of systemic inflammation in patients with AIDS.MethodsA total of 454 participants with AIDS had retinal vascular caliber (central retinal artery equivalent and central retinal vein equivalent) determined from enrollment retinal photographs by reading center graders masked to clinical and biomarker information. Cryopreserved plasma specimens were assayed for inflammatory biomarkers, including C-reactive protein (CRP), IL-6, interferon-γ inducible protein (IP)-10, kynurenine/tryptophan (KT) ratio, and intestinal fatty acid binding protein (I-FABP).ResultsIn the simple linear regression of retinal vascular caliber on plasma biomarkers, elevated CRP, IL-6, and IP-10 were associated with retinal venular dilation, and elevated KT ratio with retinal arteriolar narrowing. In the multiple linear regression, including baseline characteristics and plasma biomarkers, AMD was associated with dilation of retinal arterioles (mean difference: 9.1 μm; 95% confidence interval [CI] 5.2, 12.9; P < 0.001) and venules (mean difference, 10.9 μm; 95% CI, 5.3, 16.6; P < 0.001), as was black race (P < 0.001). Hyperlipidemia was associated with retinal venular narrowing (mean difference, -7.5 μm; 95% CI, -13.7, -1.2; P = 0.02); cardiovascular disease with arteriolar narrowing (mean difference, -5.2 μm; 95% CI, -10.3, -0.1; P = 0.05); age with arteriolar narrowing (slope, -0.26 μm/year; 95% CI, -0.46, -0.06; P = 0.009); and IL-6 with venular dilation (slope, 5.3 μm/standard deviation log10[plasma IL-6 concentration]; 95% CI, 2.7, 8.0; P < 0.001).ConclusionsThese data suggest that retinal vascular caliber is associated with age, race, AMD, hyperlipidemia, cardiovascular disease, and selected biomarkers of systemic inflammation

Hand problems among endourologists.

BACKGROUND AND PURPOSE: Endourology has evolved rapidly for the management of both benign and malignant disease of the upper urinary tract. Limited data exist, however, on the occupational hazards posed by complex endourologic procedures. The aim of this study was to determine the prevalence and possible causes of hand problems among endourologists who routinely perform flexible ureteroscopy compared with controls. MATERIALS AND METHODS: An online computer survey targeted members of the Endourological Society and psychiatrists in academic and community settings. A total of 600 endourologists and 578 psychiatrists were contacted by e-mail. Invited physicians were queried regarding their practice settings and symptoms of hand pain, neuropathy, and/or discomfort. RESULTS: Survey responses were obtained from 122 (20.3%) endourologists and 74 (12.8%) psychiatrists. Of endourologists, 61% were in an academic setting and 70% devoted their practice to endourology. Endourologists were in practice for a mean 13 years, performing 4.5 ureteroscopic cases per week with a mean operative time of 50 minutes. Hand/wrist problems were reported by 39 (32%) endourologists compared with 14 (19%) psychiatrists (P=0.0486, relative risk [RR]=1.69). Surgeons who preferred counterintuitive ureteroscope deflection were significantly more likely to have problems (56%) compared with intuitive users (27%) (RR 2.07, P=0.0139) or those with no preference (26%) (RR 2.15, P=0.0451). Overall, most respondents (85%) with hand/wrist problems needed either medical or surgical intervention. CONCLUSIONS: Hand and wrist problems are very common among endourologists. Future studies are needed to develop more ergonomic platforms and thereby reduce the endourologist\u27s exposure to these occupational hazards

P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea.

Type I and type II spiral ganglion neurons (SGN) innervate the inner and outer hair cells of the cochlea, respectively. This neural system is established by reorganization of promiscuous innervation of the hair cells, immediately before hearing is established. The mechanism for this synaptic reorganization is unresolved but probably includes regulation of trophic support between the hair cells and the neurons. We provide evidence that P2X receptors (ATP-gated ion channels) contribute such a mechanism in the neonatal rat cochlea. Single-cell quantitative RT-PCR identified the differential expression of two P2X receptor subunits, splice variant P2X(2)(-3) and P2X(3), in a 1:2 transcript ratio. Downregulation of this P2X(2-3/3) receptor coincided with maturation of the SGN innervation of the hair cells. When the P2X(2-3) and P2X(3) subunits were co-expressed in Xenopus oocytes, the resultant P2X receptor properties corresponded to the SGN phenotype. This included enhanced sensitivity to ATP and extended agonist action. In P4 spiral ganglion explants, activation of the P2X receptor signaling pathway by ATPgammaS or alpha,betaMeATP inhibited BDNF-induced neurite outgrowth and branching. These findings indicate that P2X receptor signaling provides a mechanism for inhibiting neurotrophin support of SGN neurites when synaptic reorganization is occurring in the cochlea

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.National Human Genome Research Institute (U.S.) (Grant P50HG006193

Pan-viral serology implicates enteroviruses in acute flaccid myelitis.

Since 2012, the United States of America has experienced a biennial spike in pediatric acute flaccid myelitis (AFM)1-6. Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF)2. CSF from children with AFM (n = 42) and other pediatric neurologic disease controls (n = 58) were investigated for intrathecal antiviral antibodies, using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). Metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n = 20 cases) was also performed, both unbiased sequencing and with targeted enrichment for EVs. Using VirScan, the viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n = 29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n = 22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology frequently identified high levels of CSF EV-specific antibodies in AFM compared with controls, providing further evidence for a causal role of non-polio EVs in AFM

Perceptions of Teachers’ Interpersonal Styles and Well-Being and Ill-Being in Secondary School Physical Education Students: The Role of Need Satisfaction and Need Frustration

This study examined the associations among physical education students’ perceptions of their teachers’ autonomy-supportive and controlling interpersonal styles, need satisfaction and need frustration, and indices of psychological well-being (subjective vitality) and ill-being (negative affect). The results from 591 Chinese secondary school students in Hong Kong indicated that the relationship between students’ perceptions of autonomy-supportive teaching behaviors and subjective vitality was primarily mediated by need satisfaction, whereas the relationship between perceived controlling teaching behaviors and negative affect was primarily mediated by need frustration. The results obtained from the multi-group structural equation model also suggested that these relationships were invariant across sex

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.Grants supporting this project include the National Multiple Sclerosis Society and the American Academy of Neurology award FAN-1608-25607 (R.D.S.), Clinical Research Training Scholarship P0534134 (P.S.R.), Sandler and William K. Bowes Jr Foundations (M.R.W., J.L.D., L.M.K., H.A.S., K.C.Z.), Rachleff Family Foundation (M.R.W.), and NINDS of the NIH under award K08NS096117 (M.R.W.) and F31NS113432 (K.E.L.). This study was partially supported by a grant from the Spanish National Health Institute [grant number PI15CIII-00020] and the European Regional Development Fund (FEDER funds). UCSF Biomedical Sciences Program (I.A.H., K.E.L.), UCSF Medical Scientist Training Program (K.E.L.), and the Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., J.L.D., E.D.C.) also supported this project.S

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Peer reviewedPublisher PD