Known as the gas exchange organ, the lung is also critical for responding to the aerosol environment in part through interaction with the nervous system. The diversity and specificity of lung innervating neurons remains poorly understood. Here, we interrogated the cell body location, molecular signature and projection pattern of lung innervating sensory neurons. Retrograde tracing from the lung coupled with whole tissue clearing highlighted neurons primarily in the vagal ganglia. Centrally, they project specifically to the nucleus of the solitary tract in the brainstem. Peripherally, they enter the lung alongside branching airways. Labeling of nociceptor Trpv1+ versus peptidergic Tac1+ vagal neurons showed shared and distinct terminal morphology and targeting to airway smooth muscles, vasculature including lymphatics, and alveoli. Notably, a small population of vagal neurons that are Calb1+ preferentially innervate pulmonary neuroendocrine cells, a demonstrated airway sensor population. This atlas of lung innervating neurons serves as a foundation for understanding their function in lung.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly rampaged worldwide, causing a pandemic of coronavirus disease (COVID -19), but the biology of SARS-CoV-2 is under investigation. We demonstrate that both SARS-CoV-2 spike protein and human coronavirus 229E (hCoV-229E) or its purified S protein, one of the main viruses responsible for the common cold, induce the transient opening of Pannexin-1 (Panx-1) channels in human lung epithelial cells. However, the Panx-1 channel opening induced by SARS-CoV-2 is greater and more prolonged than hCoV-229E/S protein, resulting in ATP, PGE2, and IL-1β release. Analysis of lung lavage and tissues indicate that Panx-1 mRNA expression is associated with increased ATP, PGE2, and IL-1β levels. Panx-1 channel opening induced by SARS-CoV-2 spike protein is angiotensin-converting enzyme 2 (ACE-2), endocytosis, and furin dependent. Overall, we demonstrated that Panx-1 is a critical contributor to SARS-CoV-2 infection and should be considered an alternative therapy.

RNA subcellular localization has recently emerged as a widespread phenomenon, which may apply to the majority of RNAs. The two main sources of data for characterization of RNA localization are sequence features and microscopy images, such as obtained from single-molecule fluorescent in situ hybridization-based techniques. Although such imaging data are ideal for characterization of RNA distribution, these techniques remain costly, time-consuming, and technically challenging. Given these limitations, imaging data exist only for a limited number of RNAs. We argue that the field of RNA localization would greatly benefit from complementary techniques able to characterize location of RNA. Here we discuss the importance of RNA localization and the current methodology in the field, followed by an introduction on prediction of location of molecules. We then suggest a machine learning approach based on the integration between imaging localization data and sequence-based data to assist in characterization of RNA localization on a transcriptome level.

The golden hamster model of SARS-CoV-2 infection recapitulates key characteristics of COVID-19. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in hamsters. We report that delivery of SARS-CoV-2 by a low versus high volume intranasal or intragastric route results in comparable viral titers in the lung and viral shedding. However, low-volume intranasal exposure results in milder weight loss while intragastric exposure leads to a diminished capacity to regain body weight. Male hamsters, and particularly older male hamsters, display an impaired capacity to recover from illness and delayed viral clearance. These factors were found to influence the nature of the host inflammatory cytokine response, but had a minimal effect on the quality and durability of the humoral immune response and susceptibility to re-infection. These data further elucidate key factors that impact pre-clinical challenge studies carried out in the hamster model of COVID-19.

CircRNAs belong to a family of RNA molecules which are conserved in evolution, have tissue-specific expression, and are abundant in neuronal cells. Here, we define several features of circ-Hdgfrp3 and describe interesting alterations occurring in motor neurons (MNs) carrying ALS-associated FUS mutations. Through a highly sensitive in situ approach we describe that circ-Hdgfrp3 traffics along neurites, while upon oxidative stress it is retained in the perinuclear region. While in wild-type stressed MNs, circ-Hdgfrp3 localizes in stress granules (SGs), in MNs carrying mutant FUS, a higher proportion of circ-Hdgfrp3 was trapped into cytoplasmic aggregates. Upon stress removal, circ-Hdgfrp3 was easily freed from SGs whereas it was less efficiently released from FUS-aggregates. We found that the human circ-Hdgfrp3 counterpart was also similarly associated to mutant FUS-aggregates in stressed neuronal cells. Overall, the alteration of circ-Hdgfrp3 trafficking adds a further layer of complexity to the role of FUS-aggregates in ALS disease.

The loss of uterine epithelial progesterone receptor (PGR) is crucial for successful embryo implantation in both humans and mice. The two major isoforms PGRA and PGRB have divergent functions under both physiological and pathological conditions. The present study compares phenotypes and gene signatures of PGRA and PGRB in uterine epithelium using uterine epithelial-specific constitutively expressed PGRA or PGRB mouse models. The cistrome and transcriptome analysis reveals substantial overlap between epithelial PGRA and PGRB, and both disrupt embryo implantation through FOXO1 pathways. Constitutive epithelial PGRA and PGRB expression impairs ESR1 occupancy at the promoter of Lif leading to reduced Lif transcription and further exaggerates SGK1 expression leading to enhanced PI3K-SGK1 activities, and both contribute to the decline of nuclear FOXO1 expression. Our study demonstrates that PGRA and PGRB in the uterine epithelium act on a similar set of target genes and commonly regulate the LIF-SGK1-FOXO1 signaling pathway for embryo implantation.

Absent in melanoma-2 (AIM2) is an inflammasome-forming innate immune sensor for dsDNA but also exhibits inflammasome-independent functions such as restricting cellular proliferation. AIM2 is expressed in the kidney, but its localization and function are not fully characterized. In normal human glomeruli, AIM2 localized to podocytes. In patients with glomerulonephritis, AIM2 expression increased in CD44+-activated parietal epithelial cells within glomerular crescents. To explore AIM2 effects in glomerular disease, studies in Aim2 -/- mice were performed. Aim2-/- glomeruli showed reduced expression of Wilm tumor gene-1 (WT1), WT1-driven podocyte genes, and increased proliferation in outgrowth assays. In a nephrotoxic serum (NTS)-induced glomerulonephritis model, Aim2-/- (B6) mice exhibited more severe glomerular crescent formation, tubular injury, inflammation, and proteinuria compared with wild-type controls. Inflammasome activation markers were absent in both Aim2 -/- and wild-type kidneys, despite an increased inflammatory transcriptomic signature in Aim2 -/- mice. Aim2 -/- mice also demonstrated dysregulated cellular proliferation and an increase in CD44+ parietal epithelial cells during glomerulonephritis. The augmented inflammation and epithelial cell proliferation in Aim2 -/- (B6) mice was not due to genetic background, as Aim2 -/- (B6.129) mice demonstrated a similar phenotype during NTS glomerulonephritis. The AIM2-like receptor (ALR) locus was necessary for the inflammatory glomerulonephritis phenotype observed in Aim2 -/- mice, as NTS-treated ALR -/- mice displayed equal levels of injury as wild-type controls. Podocyte outgrowth from ALR -/- glomeruli was still increased, however, confirming that the ALR locus is dispensable for AIM2 effects on epithelial cell proliferation. These results identify a noncanonical role for AIM2 in suppressing inflammation and epithelial cell proliferation during glomerulonephritis.

Neurogenic lower urinary tract dysfunction typically develops after spinal cord injury. We investigated the time course and the anatomical changes in the spinal cord that may be causing lower urinary tract symptoms following injury. Rats were implanted with a bladder catheter and external urethral sphincter electromyography electrodes. Animals underwent a large, incomplete spinal transection at the T8/9 spinal level. At 1, 2-3, and 4 weeks after injury, the animals underwent urodynamic investigations. Urodynamic investigations showed detrusor overactivity and detrusor-sphincter-dyssynergia appearing over time at 3-4 weeks after injury. Lower urinary tract dysfunction was accompanied by an increase in density of C-fiber afferents in the lumbosacral dorsal horn. CRF-positive Barrington's and 5-HT-positive bulbospinal projections drastically decreased after injury, with partial compensation for the CRF fibers at 3-4 weeks. Interestingly, a decrease over time was observed in the number of GABAergic neurons in the lumbosacral dorsal horn and lamina X, and a decrease of glutamatergic cells in the dorsal horn. Detrusor overactivity and detrusor-sphincter-dyssynergia might therefore arise from a discrepancy in inhibitory/excitatory interneuron activity in the lumbosacral cord as well as input changes which develop over time after injury. The processes point to spinal plastic changes leading to malfunction of the important physiological pathway of lower urinary tract control.

Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV-OPSCC) presents frequently as metastasis in a neck lymph node that may be cystic or necrotic. Fine-needle aspiration (FNA) biopsies are often first-line diagnostic procedures. p16 immunohistochemistry (IHC) is a surrogate marker for high-risk HPV (hrHPV) infection but can be challenging to interpret. This study evaluated the use of hrHPV in situ hybridization (ISH) in cytology cell blocks of cystic neck lesions.Twenty-four FNA cases with cell blocks and surgical correlates were evaluated. p16 IHC and hrHPV ISH were assessed on cell blocks (C-p16 and C-hrHPV ISH), and hrHPV ISH on surgical samples (S-hrHPV ISH). All results were classified as negative, positive, or equivocal.Two cases were excluded because of insufficient tissue on recut. On the basis of C-hrHPV ISH cases, 12 were positive, 5 were negative, and 5 were equivocal. All 12 positive C-hrHPV ISH cases had concordant S-hrHPV ISH with no false positives. Of the 5 negative C-hrHPV ISH cases, 4 had concordant S-hrHPV ISH, and 1 had a discordant S-hrHPV ISH. Of the 5 equivocal C-hrHPV ISH cases, S-hrHPV ISH were both positive and negative. Fourteen cases were equivocal by C-p16; 9 cases were reliably classified by C-hrHPV ISH (5 positive, 4 negative; 64%).C-hrHPV ISH can be reliably used, especially when positive. A negative or equivocal interpretation of C-hrHPV ISH may warrant repeat testing. Compared to C-p16, C-hrHPV ISH is more frequently diagnostic and could be helpful for HPV-OSCC diagnosis and management.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. Here, we sought to understand how S1P signaling affects neuronal excitability in the central amygdala (CeA), which is a brain region associated with fear learning, aversive memory, and the affective dimension of pain. Because the G-protein coupled S1P receptor 1 (S1PR1) has been shown to be the primary mediator of S1P signaling, we utilized S1PR1 agonist SEW2871 and S1PR1 antagonist NIBR to determine a potential role of S1PR1 in altering the cellular physiology of neurons in the lateral division of the CeA (CeL) that share the neuronal lineage marker somatostatin (Sst). CeL-Sst neurons play a critical role in expression of conditioned fear and pain modulation. Here we used transgenic breeding strategies to identify fluorescently labeled CeL-Sst neurons for electrophysiological recordings. Using principal component analysis, we identified two primary subtypes of Sst neurons within the CeL in both male and female mice. We denoted the two types regular-firing (type A) and late-firing (type B) CeL-Sst neurons. In response to SEW2871 application, Type A neurons exhibited increased input resistance, while type B neurons displayed a depolarized resting membrane potential and voltage threshold, increased current threshold, and decreased voltage height. NIBR application had no effect on CeL Sst neurons, indicating the absence of tonic S1P-induced S1PR1. Our findings reveal subtypes of Sst neurons within the CeL that are uniquely affected by S1PR1 activation, which may have implications for how S1P alters supraspinal circuits.

Human papillomavirus (HPV) infections are transmitted through sexual or other close contact and are etiologically associated with epithelial warts, papillomas, and intraepithelial lesions that may progress to cancer. Indeed, 4.8% of the global cancer burden is linked to HPV infection. Highly effective vaccines protect against two to nine of the most medically important HPV genotypes; yet vaccine uptake is inadequate and/or cost prohibitive in many settings. With HPV-related cancer incidence expected to rise over the coming decades, there is a need for effective HPV microbicides. Herein we demonstrate the strong inhibitory activity of the heparin-neutralizing drug protamine sulfate (PS) against HPV infection. Pretreatment of cells with PS greatly reduced infection regardless of HPV genotype or virus source. Vaginal application of PS prevented infection of the murine genital tract by HPV pseudovirions. Time-of-addition assays where PS was added to cells before infection, during infection, or after viral attachment demonstrated strong inhibitory activities on early infection steps. No effect on virus infection was found for cell lines deficient in heparan sulfate expression, suggesting that PS binds to heparan sulfate on the cell surface. Consistent with this, prophylactic PS exposure prevented viral attachment, including under low pH conditions akin to the human vaginal tract. Our findings suggest PS acts dually to prevent HPV infection: prophylactic treatment prevents HPV attachment to host cells and post-attachment administration alters viral entry. Clinical trials are warranted to determine whether protamine-based products are effective as topical microbicides against genital HPVs.

Central nervous system (CNS)-mediated symptoms, such as fatigue, depression, and hyperalgesia, are common complications among patients with rheumatoid arthritis (RA). However, it remains unclear how the peripheral pathology of RA spreads to the brain. Accumulated evidence showing an association between serum cytokine levels and aberrant CNS function suggests that humoral factors participate in this mechanism. In contrast to the well-known early responses of microglia (CNS-resident immune cells) in the area postrema [AP; a brain region lacking a blood-brain barrier (BBB)] to experimental inflammation, microglial alterations in the AP during chronic inflammation like RA remain unclear. Therefore, to determine whether microglia in the AP can react to persistent autoimmune-arthritis conditions, we analyzed these cells in a mouse model of collagen-induced arthritis (CIA).Microglial number and morphology were analyzed in the AP of CIA and control mice (administered Freund's adjuvant or saline). Immunostaining for ionized calcium-binding adaptor molecule-1 was performed at various disease phases: "pre-onset" [post-immunization day (PID) 21], "establishment" (PID 35), and "chronic" (PID 56 and 84). Quantitative analyses of microglial number and morphology were performed, with principal component analysis used to classify microglia. Interleukin-1β (IL-1β) mRNA expression was analyzed by multiple fluorescent in situ hybridization and real-time polymerase chain reaction. Behavioral changes were assessed by sucrose preference test.Microglia in the AP significantly increased in density and exhibited changes in morphology during the establishment and chronic phases, but not the pre-onset phase. Non-subjective clustering classification of cell morphology (CIA, 1,256 cells; saline, 852 cells) showed that the proportion of highly activated microglia increased in the CIA group during establishment and chronic phases. Moreover, the density of IL-1β-positive microglia, a hallmark of functional activation, was increased in the AP. Sucrose preferences in CIA mice negatively correlated with IL-1β expression in brain regions containing the AP.Our findings demonstrate that microglia in the AP can sustain their activated state during persistent autoimmune arthritis, which suggests that chronic inflammation, such as RA, may affect microglia in brain regions lacking a BBB and have various neural consequences.