Probes for INS

The skin epidermis is a highly compartmentalized tissue consisting of a cornifying epithelium called the interfollicular epidermis (IFE) and associated hair follicles (HFs). Several stem cell populations have been described that mark specific compartments in the skin but none of them is specific to the IFE. Here, we identify Troy as a marker of IFE and HF infundibulum basal layer cells in developing and adult human and mouse epidermis. Genetic lineage-tracing experiments demonstrate that Troy-expressing basal cells contribute to long-term renewal of all layers of the cornifying epithelium. Single-cell transcriptomics and organoid assays of Troy-expressing cells, as well as their progeny, confirmed stem cell identity as well as the ability to generate differentiating daughter cells. In conclusion, we define Troy as a marker of epidermal basal cells that govern interfollicular epidermal renewal and cornification.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

Neurotransmitter release is a highly controlled process by which synapses can critically regulate information transfer within neural circuits. While presynaptic receptors - typically activated by neurotransmitters and modulated by neuromodulators - provide a powerful way of fine-tuning synaptic function, their contribution to activity-dependent changes in transmitter release remains poorly understood. Here, we report that presynaptic NMDA receptors (preNMDARs) at mossy fiber boutons in the rodent hippocampus can be activated by physiologically relevant patterns of activity and selectively enhance short-term synaptic plasticity at mossy fiber inputs onto CA3 pyramidal cells and mossy cells, but not onto inhibitory interneurons. Moreover, preNMDARs facilitate brain-derived neurotrophic factor release and contribute to presynaptic calcium rise. Taken together, our results indicate that by increasing presynaptic calcium, preNMDARs fine-tune mossy fiber neurotransmission and can control information transfer during dentate granule cell burst activity that normally occur in vivo.

Coccidioidomycosis, otherwise known as Valley Fever, is caused by the dimorphic fungi Coccidioides immitis and C. posadasii. While most clinical cases present with self-limiting pulmonary infection, dissemination of Coccidioides spp. results in prolonged treatment and portends higher mortality rates. While the structure, genome, and niches for Coccidioides have provided some insight into the pathogenesis of disease, the underlying immunological mechanisms of clearance or inability to contain the infection in the lung are poorly understood. This review focuses on the known innate and adaptive immune responses to Coccidioides and highlights three important areas of uncertainty and potential approaches to address them. Closing these gaps in knowledge may enable new preventative and therapeutic strategies to be pursued.

Proprioception is essential for behavior and provides a sense of our body movements in physical space. Proprioceptor organs are thought to be only in the periphery. Whether the central nervous system can intrinsically sense its own movement remains unclear. Here we identify a segmental organ of proprioception in the adult zebrafish spinal cord, which is embedded by intraspinal mechanosensory neurons expressing Piezo2 channels. These cells are late-born, inhibitory, commissural neurons with unique molecular and physiological profiles reflecting a dual sensory and motor function. The central proprioceptive organ locally detects lateral body movements during locomotion and provides direct inhibitory feedback onto rhythm-generating interneurons responsible for the central motor program. This dynamically aligns central pattern generation with movement outcome for efficient locomotion. Our results demonstrate that a central proprioceptive organ monitors self-movement using hybrid neurons that merge sensory and motor entities into a unified network.

Coronavirus disease-19 (COVID-19) is caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although SARS-CoV-2 is visualized on electron microscopy, there is an increasing demand for widely applicable techniques to visualize viral components within tissue specimens. Viral protein and RNA can be detected on formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. Herein, we evaluate the staining performance of ISH for SARS-CoV-2 and an IHC directed at the SARS-CoV nucleocapsid protein and compare these results to a gold standard, tissue quantitative real-time polymerase chain reaction (qRT-PCR). We evaluated FFPE sections from 8 COVID-19 autopsies, including 19 pulmonary and 39 extrapulmonary samples including the heart, liver, kidney, small intestine, skin, adipose tissue, and bone marrow. We performed RNA-ISH for SARS-CoV-2 on all cases with IHC for SARS-CoV and SARS-CoV-2 qRT-PCR performed on selected cases. Lungs from 37 autopsies performed before the COVID-19 pandemic served as negative controls. The ISH and IHC slides were reviewed by 4 observers to record a consensus opinion. Selected ISH and IHC slides were also reviewed by 4 independent observers. Evidence of SARS-CoV-2 was identified on both the IHC and ISH platforms. Within the postmortem lung, detected viral protein and RNA were often extracellular, predominantly within hyaline membranes in patients with diffuse alveolar damage. Among individual cases, there was regional variation in the amount of detectable virus in lung samples. Intracellular viral RNA and protein was localized to pneumocytes and immune cells. Viral RNA was detected on RNA-ISH in 13 of 19 (68%) pulmonary FFPE blocks from patients with COVID-19. Viral protein was detected on IHC in 8 of 9 (88%) pulmonary FFPE blocks from patients with COVID-19, although in 5 cases the stain was interpreted as equivocal. From the control cohort, FFPE blocks from all 37 patients were negative for SARS-CoV-2 RNA-ISH, whereas 5 of 13 cases were positive on IHC. Collectively, when compared with qRT-PCR on individual tissue blocks, the sensitivity and specificity for ISH was 86.7% and 100%, respectively, while those for IHC were 85.7% and 53.3%, respectively. The interobserver variability for ISH ranged from moderate to almost perfect, whereas that for IHC ranged from slight to moderate. All extrapulmonary samples from COVID-19-positive cases were negative for SARS-CoV-2 by ISH, IHC, and qRT-PCR. SARS-CoV-2 is detectable on both RNA-ISH and nucleocapsid IHC. In the lung, viral RNA and nucleocapsid protein is predominantly extracellular and within hyaline membranes in some cases, while intracellular locations are more prominent in others. The intracellular virus is detected within pneumocytes, bronchial epithelial cells, and possibly immune cells. The ISH platform is more specific, easier to analyze and the interpretation is associated with the improved interobserver agreement. ISH, IHC, and qRT-PCR failed to detect the virus in the heart, liver, and kidney.

Background: Mucociliary clearance (MCC) is a vital innate defense mechanism that is impaired in people with cystic fibrosis (CF) and animal CF models. Dysfunctional MCC contributes to airway inflammation and infection, which hasten lung function decline. Most people with CF benefit from highly effective CF transmembrane conductance regulator (CFTR) modulators, but some mutations are unresponsive to currently available modulators, and even people with CF who benefit from modulator therapy may be unable to clear chronic pulmonary infections. Accordingly, CFTR-independent methods to increase MCC are needed. We previously discovered that the combination of low-dose cholinergic with βadrenergic agonists synergistically increased MCC velocity (MCCV) in ex vivo tracheal preparations from ferrets and newborn piglets. MCC was also significantly greater in tracheas from CF ferrets to a value of approximately 55% of that in wild-type animals. The MCCV increases were produced without inducing airway narrowing [1]. To further our preclinical work, we tested three hypotheses. We hypothesized that synergistic increases in MCCV by the combined agonists involve epithelial sodium channel (ENaC) inhibition, greater secretion of bicarbonate, and additivity with CFTR modulators. Methods: To test these hypotheses, we measured MCCV in excised newborn piglet tracheas with 10 µM formoterol (beta-adrenergic agonist) plus 0.3 µM methacholine (cholinergic agonist) with and without 10 µM benzamil (ENaC inhibitor) using particle tracking. Bicarbonate secretion rates were measured in tracheal mucosa of Yucatan minipigs mounted in Ussing chambers using a pH-stat method with pH electrodes and automated titrators (Metrohm Titrando 902). To assess whether the synergy agonists improved CF tissues exposed to CFTR modulators, we used high-speed digital microscopy to measure the effective diffusivity (Deff in µm2 /msec) of approximately 2-µm fluorescent polystyrene spheres (0.1%, ThermoFisher) added to the apical surface fluid layer of human CF primary nasal cell cultures (F508del homozygote) grown under air-liquid interface conditions with and without elexacaftor/ tezacaftor/ivacaftor (ELX/TEZ/IVA) (3 μM ELX, 3 μM TEZ, 10 μM IVA). Results: Baseline MCCV was 6 times as high with benzamil inhibition of ENaC (0.5 ± 0.7 mm/min to 3.0 ± 0.7 mm/min; p = 0.02, 4 piglets), but when benzamil was present during synergistically stimulated MCCV, no further increase was seen, consistent with the hypothesis that ENaC was already inhibited by the synergy agonists (MCCV: synergy agonists, 13.9 ± 1.6 mm/ min vs. synergy agonists + benzamil, 14.0 ± 1.6 mm/min; p = 0.97, n = 4 piglets, each condition). The synergy agonists increased bicarbonate secretion rates by about 83% (0.6 ± 0.2 µmol/cm2 per hour at baseline vs. 1.1 ± 0.3 µmol/cm2 per hour with synergy agonists, 5 experiments with 3 pig tracheas). Particle diffusivity in CF primary nasal cell cultures showed synergy agonists plus ELX/TEZ/IVA > synergy agonists > ELX/TEZ/IVA > no treatment. Conclusions: Results were consistent with our hypotheses. The combination of beta adrenergic plus low-dose cholinergic agonists produces synergistic increases in MCCV by inhibiting ENaC and increasing bicarbonate secretion and appears to be at least additive to the effects induced by ELX/TEZ/IVA modulator therapy.

Background: SARS-CoV-2 (SARS2) continues to place an unprecedented burden on global health. SARS2 is a respiratory virus that, in a minority of patients, causes severe pneumonia, which portends a poor prognosis. There is emerging evidence of long-term respiratory sequelae secondary to SARS2, including impaired lung function and persistent lung imaging abnormalities. CF patients often face prolonged morbidity and exacerbation of lung disease as a consequence of respiratory virus infection. Although small observational studies indicate that outcomes of SARS2 infection in people with CF are similar to those of the general population, the impact of SARS2 infection on CF lung disease is not known. Accordingly, we investigated the clinical, pathological, and molecular impact of SARS2 infection in mice with CF-like lung disease. Methods: β-epithelial sodium channel (Scnn1b) transgenic mice (βENaCTg) and wild-type (WT) littermates were inoculated intranasally with a mouse-adapted SARS2 virus (maSARS2). Clinical characteristics, including body weight, were recorded daily. At 2, 15, and 30 days postinoculation (dpi), lungs were harvested and left lobes prepared as histological sections for analysis by light microscopy (hematoxylin and eosin, AB-PAS), immunohistochemistry (IHC), and RNA in situ hybridization (ISH, RNAScope). The remainder of the lungs were used to determine virus titers. Results: βENaC-Tg mice lost less weight than WT mice (5% vs 11% weight loss at 4 dpi, P < 0.05). SARS2 nucleocapsid protein was less abundant in βENaC-Tg mice than in WT mice as measured by IHC (2.2% total lung area infected vs 5.2%, P < 0.05). βENaC-Tg mice had significantly less SARS2 mRNA in the epithelial cells of the airways, as measured by RNA ISH (1341 µm2 /mm vs 7018 µm2 /mm of basement membrane, P < 0.001)—a measurement reproduced with IHC for SARS2 nucleocapsid. Airway epithelium of βENaC-Tg negative for SARS2 infection was overlaid by greater accumulations of mucus secretions as measured by AB-PAS staining. Analysis of chronic outcomes of infection at 15 and 30 dpi revealed that lungs of βENaC-Tg mice but not WT mice had accumulations of alternatively activated macrophages (Ym1) and eosinophils (major basic protein). In addition, at these later time points, βENaC-Tg mice had evidence of more airway goblet cells and basal cell proliferation (p63). Conclusion: In the early phase of infection, βENaC-Tg mice were less severely infected by SARS2 than WT mice. After this early phase, βENaC-Tg mice developed a Th2-type immune response with persistent accumulation of alternatively activated macrophages, eosinophils, and goblet cell metaplasia. Our findings suggest that airway mucus accumulation, as is seen in CF patients, may offer protection against initial SARS2 infection of the airway epithelium, although infection of the distal lung in CF patients may be associated with a more severe chronic course of disease

Here, we describe a protocol to visualize RNA oligos and proteins independently or together using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence in human colonoids, expanding on previously published research. Whole-mount staining is used to preserve the colonoid structure and fix onto glass slides. We describe procedures for efficient plating, fixation, and preservation of the colonoids. This workflow can be adapted to 3D organoid models from other tissues or organisms. For complete details on the use and execution of this protocol, please refer to In et al. (2020).

Genetic markers used to define discrete cell populations are seldom expressed exclusively in the population of interest and are, thus, unsuitable when evaluated individually, especially in the absence of spatial and morphological information. Here, we present fluorescence _in situ_ hybridization for flow cytometry to allow simultaneous analysis of multiple marker genes at the single whole-cell level, exemplified by application to the embryonic epicardium. The protocol facilitates multiplexed quantification of gene and protein expression and temporal changes across specific cell populations.

Background: People with CF are at risk for malnutrition and fat-soluble vitamin deficiencies due to pancreatic insufficiency and fat malabsorption. Highly effective CFTR modulators, ivacaftor and elexacaftor/tezacaftor/ ivacaftor, substantially improve CFTR activity, lung function and nutritional status (weight and body mass index) in people with CF with certain genetic mutations [1, 2]. Fat-soluble vitamin levels (vitamins A, D, and E) are assessed annually in children with CF. We sought to determine changes in fat-soluble vitamin levels following treatment with ivacaftor or elexacaftor/ tezacaftor/ivacaftor. Methods: We performed a retrospective study of children with CF who had at least 3 annual evaluations including vitamin A, D, and E prior to ivacaftor or elexacaftor/tezacaftor/ivacaftor start date and at least 1 evaluation ≥ 3 months post-modulator start date. Data collected included demographics, CF diagnostic data, pancreatic status, nutritional status, and lung function. Summary statistics were calculated and vitamin levels were compared pre to post modulator within group via signed rank tests. Results: There were 24 children with CF prescribed highly effective CFTR modulators who met annual evaluation criteria (15 on elexacaftor/ tezacaftor/ivacaftor [62.5%] and 9 on ivacaftor [37.5%]). Individuals had a median age 13.8 years (range 12-15) for elexacaftor/tezacaftor/ivacaftor and 9.7 years (range 6.6-13.2) for ivacaftor. All individuals treated with elexacaftor/tezacaftor/ivacaftor were pancreatic insufficient, whereas 7/9 (78%) of those treated with ivacaftor were pancreatic sufficient. Individuals had a median of 6 annual evaluations over a median 5.2 years (timing of each annual measurement ranged 0.04-11.9 years) prior to modulator and 1 evaluation post modulator over 3.2 years. For children treated with ivacaftor, vitamin levels were not significantly different following treatment with mean (SD) levels before and after modulator treatment: vitamin A 41 (9.8) mcg/dL vs 51 (21.4) mcg/dL, P = 0.13; vitamin D 40 (5.0) ng/mL vs 47 (20.3) ng/mL, P = 0.57; and vitamin E (alpha tocopherol) 14 (4.4) mcg/ mL vs 18 (0.6) mcg/mL, P = 0.5. Average change within individuals was also not significant. For children treated with elexacaftor/tezacaftor/ivacaftor, mean vitamin levels increased following modulator treatment: vitamin A 36 (6.2) mcg/dL vs 47 (9.8) mcg/dL, p < 0.01; vitamin D 34 (6.0) ng/mL vs 44 (15.5) ng/mL, P = 0.01. Change in vitamin levels within individuals was also significantly improved (vitamin D increased on average 9.9 ng/mL, p < 0.01; vitamin A increased on average 10.3 mcg/dL, p < 0.01). Conclusion: Children treated with elexacaftor/tezacaftor/ivacaftor had improvement in fat-soluble vitamins A and D following at least 3 months of treatment. Vitamin levels did not change in those treated with ivacaftor, possibly due to small numbers, fewer pancreatic-insufficient patients, or less impact on fat absorption compared to elexacaftor/tezacaftor/ivacaftor. Evaluation of additional children started on elexacaftor/tezacaftor/ivacaftor and longer follow-up are needed to determine if significant changes in vitamin levels persist.

Background In the large intestine, the multipotent stem cells are located at the base of the crypt and differentiate into three main cell types: enterocytes, goblet cells, and enteroendocrine cells. Goblet cells’ main function is the synthesis and secretion of mucins. Genetic and epigenetic changes that provide survival advantages for stem or progenitor cells resulting in the deregulation of cellular differentiation are major causes of all carcinomas. Methods Our laboratory has a large collection of colorectal cancer (CRC) cell lines, well characterised in terms of gene expression and mutations. We analysed the presence of goblet cells in CRC cell lines using the genes Mucin 2 (MUC2) and Trefoil factor 3 (TFF3). The genes both at the mRNA level and at the protein level were investigated. The effects of various transcription factors were assessed by knockdown and overexpression techniques. Results We found that most of the cell lines are unable to produce goblet cells and that the number of MUC2 and TFF3-positive cells among the goblet cell positive cell lines was quite variable. While in the normal colon, MUC2 and TFF3 are always co-expressed, but that is not always the case in the CRC cell lines. MUC2-negative and TFF3-positive cell lines appear to reflect a novel interesting subset. The investigation of several transcription factors on goblet cell differentiation showed that downregulation of Atonal homologue 1 (ATOH1) had a dramatic effect on goblet cell production, while knocking down of SAM pointed domain ETS transcription factor (SPDEF), Caudal type homeobox 1 (CDX1), and 2 (CDX2) had a modest effect. Individually, none of these factors are sufficient to trigger the goblet cell differentiation. Conclusions As a conclusion, the percentage of goblet cells differs substantially between cell lines. Classification of the cell lines reveals an interesting major subset that has TFF3 expression without expressing MUC2. ATOH1, SPDEF, CDX1, and CDX2 had a significant effect on goblet cell differentiation, but on their own, they are not sufficient to induce the goblet cell differentiation. Understanding the mechanisms of goblet cell differentiation is important for advances in the prevention and treatment of CRC.