Probes for INS

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.

Background Diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Hyperglycemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. Purpose Investigate if SGLT1 is involved in a glucotoxicity injury during IRI and whether inhibiting SGLT1 with an SGLT1 inhibitor may reduce infarct size. Method RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22mmol) on myocardium at reperfusion. Canagliflozin (CANA) a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 receptor and SGLT2 and 5nmol/L to block only the SGLT2 receptor) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results We showed that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. we demonstrate that hyperglycemia at reperfusion is injurious to myocardium with an increase of myocardial infarction. Our data reveal that glucose exacerbation of injury appears to be mediated via SGLT1. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model is abrogated through the administration of a clinically available mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, but by the SGLT2-selective concentration. Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this incre Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): The government of saudi Arabia

Primary bladder tumors have a high degree of malignancy. To investigate the expression of human papillomavirus type 16 (HPV-16) in primary bladder tumors and the loss of cell differentiation and to explore the significance of HPV-16 detection, it is expected to be a disease. Treatment provides a theoretical basis.Fifty-seven patients with primary bladder tumors admitted to our hospital from January 2019 to January 2022 were selected as the research subjects, and they were divided into HPV-related groups according to the human papillomavirus (HPV) infection status (n = 28) and HPV unrelated group (n = 29). The general data of patients were collected, the expression of HPV-16 in bladder tissue samples was detected, and the correlation between pathological parameters and HPV-16 expression was analyzed.Among HPV subtypes, HPV 16 subtype accounted for the highest proportion, followed by HPV-18 and HPV-6 subtypes; there was no significant difference in tumor stage (stage 1, stage a, stage 2a) between the HPV-related group and the HPV-unrelated group (stage 1, stage a, and stage 2a). P > 0.05); there was no significant difference in postoperative pathological expression (high expression and low expression) of patients (P > 0.05); there was no statistical difference in age and gender between HPV-related and HPV-unrelated groups (P > 0.05), HPV-related group and HPV-unrelated group compared daily regular drinking and smoking status, the difference was statistically significant (P < 0.05); HPV-16 expression was not correlated with tumor differentiation degree and age of patients (P > 0.05); the area under the curve (AUC) of HPV-16 for judging primary bladder tumor expression and cellular molecular deletion was 0.891, with a sensitivity of 83.94% and a specificity of 88.57%.HPV-16 is an upper, expressed in primary bladder tumors and will participate in the differentiation and loss of cells, which can provide effective guidance and basis for the diagnosis of primary bladder tumors, which is an important factor for judging the pathological stage and prognosis of patients and can provide a theoretical reference for the formulation of therapeutic measures.

Background: Analysis of reporter gene expression by fluorescent microscopy is a routine way to determine gene transfer efficiency in tissues. Quantifying transduction efficiency within tissues can be complicated and laborious to perform manually. There is a need for development of new semi- and fully automatic processing tools for analysis of microscopy images. To quantify lentiviral vector transduction efficacy within the pulmonary epithelium, we have developed medium- to high-throughput, user-friendly segmentation and analysis tools for the open-source, Javabased, image-analysis platform ImageJ. Methods: Air-liquid interface (ALI) cultures (n = 6) and rat lungs (n = 5) were transduced with a pseudotyped lentiviral vector expressing an EGFP fluorescent reporter (SiV-EGFP.F/HN) at a multiplicity of infection of 250/ ALI or 3.9e8 transduction units/animal. ALI cultures were dissociated and cytospun onto glass slides for imaging. Lung tissues were formalin fixed, embedded in paraffin, and sectioned. EGFP fluorescence was visualized by fluorescent microscopy using an anti-EGFP antibody, and nuclei were visualized with DAPI staining. Transduction efficiency was measured by in situ hybridization (RNAscope) with vector-specific probes. Cells and individual airways were imaged at 20× magnification. Whole lung lobes were imaged at 10× using an automatic tiling method. Results: Two ImageJ macros were developed to quantify fluorescence reporter protein expression and RNAscope signal in cytospun cells or lung tissue sections. These methodologies involve segmentation of the cell or tissue using a nucleus-based seeding and Voronoi tessellation maximal cell boundary heuristic approach to identify individual cells within the sample. Fluorescence reporter expression or RNAscope signal can thus be reported on a per-cell basis within the sample. Resolution of DAPI fluorescence in 10× tiled images was too low for accurate segmentation of the airway on a cellular basis. A third ImageJ macro and plugin was developed to semiautomatically define and segment airway epithelia using a multithreaded cellular-density parsing algorithm. EGFP fluorescence was quantified within defined airway regions of interest, and data were expressed as percentage green fluorescent protein-positive area over total airway area (15.8 ± 0.42%). The methodology was validated by manually quantifying EGFP-positive cells per total nuclei on a subset of airways, which resulted in similar efficiencies (macro: 16.6 ± 2.8%, manual: 14.6 ± 2.4%) Conclusion: We have designed several image analysis tools for ImageJ to accelerate our pulmonary gene therapy research. Our workflow allows for quantitative analysis of fluorescent reporter expression or RNAscope signal in cell and tissue models for pulmonary gene therapy.

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Methods: Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, as well as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. One week later, the medium was changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Results: Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 ± 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 ± 9%, n = 3). Conclusions: In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.

Background: Early after primary infection, HIV reservoirs are established within multiple tissues, including the brain. As these viral reservoirs are not targeted by antiretroviral therapy (cART), we require robust methods of detection, quantification, and characterization of these viral reservoirs in human tissues. Our recent work developed a multi-component imaging methodology that characterizes and quantifies viral reservoirs within the brain. Methods: The imaging methodology demonstrated utilizes the simultaneous staining of brain tissue from HIV-infected donors using DNAscope, RNAscope, and antibodies for HIV-DNA, HIV-mRNA, and either viral or host proteins, respectively. The panel of patients included in these analyses varied in cART regimen, viral load, years living with HIV, and neurocognitive status, all contrasted to age-matched tissues from uninfected patients. Results: Our group demonstrated that cART is sufficient to reduce the size of the viral reservoirs within the brains of HIV patients. We also found that about half of the cells positive for HIV-DNA expressed HIV-mRNA, and only about one-third expressed viral proteins. HIV proteins varied in expression and bystander uptake by uninfected cells but could provide insight into bystander toxicity. Conclusions: The results found were present irrespective of cART regimen and systemic viral replication but suggested that these viral reservoirs are a major barrier to curing HIV and treating associated neurocognitive disorders.

Detection of phosphorylated proteins in tissue sections using immunohistochemistry (IHC) is a challenging task. The absence of tissue staining may be caused by either a lack of protein expression or a lack of protein activation via its phosphorylation. To address this problem, we employed Integrated Co-detection Workflow (ICW) protocol to analyze lung cancer tissue sections by combining in situ hybridization (ISH) with IHC. The target protein of interest was epidermal growth factor receptor (EGFR, also known as ErbB1 and HER1) which is the founding member of the ErbB family of receptor tyrosine kinases. Using phospho-specific antibodies specific for a phosphorylated site Y1173 of EGFR molecule allowed us to analyze IHC and ISH staining at a single cell level in lung cancer tissue. We have observed both a co-localization of IHC with ISH signals and ISH-positive cells lacking IHC labeling for phosphorylated EGFR. ICW appears to be a very powerful spatial biology technique for accurate localization of cancer cells with phosphorylated/activated and non-phosphorylated/nonactivated proteins.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.