Probes for INS

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.

Lactate is a circulating metabolite and a signalling molecule with pleiotropic physiological effects. Studies suggest that lactate modulates energy balance by lowering food intake, inducing adipose browning and increasing whole-body thermogenesis. Yet, like many other metabolites, lactate is often commercially produced as a counterion-bound salt and typically administered in vivo through hypertonic aqueous solutions of sodium L-lactate. Most studies have not controlled for injection osmolarity and the co-injected sodium ions. Here, we show that the anorectic and thermogenic effects of exogenous sodium L-lactate in male mice are confounded by the hypertonicity of the injected solutions. Our data reveal that this is in contrast to the antiobesity effect of orally administered disodium succinate, which is uncoupled from these confounders. Further, our studies with other counterions indicate that counterions can have confounding effects beyond lactate pharmacology. Together, these findings underscore the importance of controlling for osmotic load and counterions in metabolite research.

Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine.

Nociceptive input to the spinal cord is transmitted by primary afferent neurons in the dorsal root ganglia (DRG). A subset of DRG neurons has the ability to attenuate nociceptive transmission through expression of the µ-opioid receptor. The relationship between algesic and analgesic properties of individual DRG neurons has not yet been evaluated in the human. By using a combination of six different 4-Plex in-situ hybridization experiments, we were able to identify three different main nociceptive populations. First, we detected a population poly-responsive, poly-modulated small-diameter nociceptors that co-express TRPV1, OPRM1, and SCN11A, and in most cases additionally SCN10A and P2RX3 and in subpopulations additionally TAC1, TRPA1, TRPM8, OPRD1, PIEZO2, and/or OPRL1. Second, we detected a medium-sized set of multimodal putative nociceptors with a very similar molecular profile, apart from a lack of expression of TRPA1. Both nociceptor types do express the µ-opioid receptor (encoded by OPRM1) and their activity and input into the spinal cord likely can be attenuated by clinically used µ-receptor agonists. The third population consists of small-diameter neurons that have a molecular expression profile characteristic for nociceptive neurons, such that they express TRPV1 together with SCN11A, SCN10A, P2RX3, PIEZO2, OPRD1, and partially TRPA1. The most distinguishable characteristic of this population is the lack of expression of the µ-opioid receptor and thus are non-susceptible to clinically used opioids. Further molecular characterization of these nociceptive populations might reveal attractive molecular candidates for targeting different types of pain indications.

The expanding use of chemotherapy in curative cancer treatment has simultaneously resulted in a substantial and growing cohort of cancer survivors with prolonged disability from chemotherapy-induced peripheral neuropathy (CIPN). CIPN is associated with several commonly prescribed chemotherapeutics, including taxanes, platinum-based drugs, vinca alkaloids, bortezomib and thalidomide. These distinct classes of chemotherapeutics, with their varied neurotoxic mechanisms, often cause patients to suffer from a broad profile of neuropathic symptoms including chronic numbness, paraesthesia, loss of proprioception or vibration sensation and neuropathic pain. Decades of investigation by numerous research groups have provided substantial insights describing this disease. Despite these advances, there is currently no effective curative or preventative treatment option for CIPN and only the dual serotonin-norepinephrine reuptake inhibitor Duloxetine is recommended by clinical guidelines for the symptomatic treatment of painful CIPN.In this review, we examine current preclinical models, with our analysis focused on translational relevance and value.Animal models have been pivotal in achieving a better understanding of the pathogenesis of CIPN. However, it has been challenging for researchers to develop appropriate preclinical models that are effective vehicles for the discovery of translatable treatment options.Further development of preclinical models targeting translational relevance will promote value for preclinical outcomes in CIPN studies.