Probes for INS

The failure of remyelination in the human CNS contributes to axonal injury and disease progression in multiple sclerosis (MS). In contrast to regions of chronic demyelination in the human brain, remyelination in murine models is preceded by abundant oligodendrocyte progenitor cell (OPC) repopulation, such that OPC density within regions of demyelination far exceeds that of normal white matter (NWM). As such, we hypothesized that efficient OPC repopulation was a prerequisite of successful remyelination, and that increased lesion volume may contribute to the failure of OPC repopulation in human brain. In this study, we characterized the pattern of OPC activation and proliferation following induction of lysolecithin-induced chronic demyelination in adult rabbits. The density of OPCs never exceeded that of NWM and oligodendrocyte density did not recover even at 6 months post-injection. Rabbit OPC recruitment in large lesions was further characterized by chronic Sox2 expression in OPCs located in the lesion core and upregulation of quiescence-associated Prrx1 mRNA at the lesion border. Surprisingly, when small rabbit lesions of equivalent size to mouse were induced, they too exhibited reduced OPC repopulation. However, small lesions were distinct from large lesions as they displayed an almost complete lack of OPC proliferation following demyelination. These differences in the response to demyelination suggest that both volume dependent and species-specific mechanisms are critical in the regulation of OPC proliferation and lesion repopulation and suggest that alternate models will be necessary to fully understand the mechanisms that contribute to failed remyelination in MS.

Neuropathic pain is a chronic condition which can arise following damage to the somatosensory system and often involves both hyperalgesia and allodynia. The molecular mechanisms of neuropathic pain remain incompletely understood but require enduring alterations in specific gene program expression and protein synthesis affecting neuronal signaling and excitability. We investigate the roles of non-coding RNA regulatory pathways in impacting hyperalgesia and determining the mRNA complement recruited during the protein synthesis response in neuropathic pain. Nerve injury alters the expression of many miRNAs, including the highly conserved let-7 family miRNAs, which repress pro-growth mRNAs and are implicated in axon growth, neuronal plasticity, and brain circuit development. The Lin28 RNA binding protein can prevent maturation of let-7 precursor RNAs; consequently, increased Lin28 signaling promotes pro-growth gene expression. The regulation and potential roles role of Lin28/let-7 pathway in neuropathic pain remain largely unexplored. Using spared nerve injury (SNI) mouse models of neuropathic pain, we find in preliminary data that Lin28a loss of function in sensory neuron populations can result in a deficit in mechanical hypersensitivity post-surgery. In SNI and sciatic nerve transection (SNT) mouse models, we evaluate molecular mechanisms underlying pain using single molecule detection and genetic manipulation. A sensitive RNA imaging assays, RNAScope in situ hybridization (ISH), is used to amplify single RNA target signals in fixed tissues to allow mapping of the spatiotemporal patterns and cell type specificity of changes in non-coding RNA regulatory pathways. Digital PCR is used to provide sensitive and quantitative validation. We find lin28 mRNA level are elevated in injured dorsal root ganglion cells, injured sciatic nerves and their surrounding Schwann cells at 3 days post SNI and SNT surgery accordingly.

Antibodies have been commonly used to study protein phosphorylation since the first phospho-specific antibody was described in 1981. Antibodies can be developed so that they specifically recognize phosphorylated areas of particular proteins. In situ hybridization (ISH) is the technique where specific RNA or DNA molecules can be detected in a single cell without the need for antibodies. Using ACD's integrated Co-Detection Workflow (ICW), we have developed a protocol to use phospho-specific antibodies in combination with ISH to show co-localization of EGFR mRNA and EGFR proteins phosphorylated at different sites in tumor cells. Our protocol has been used for multiplexing Y1086 phosphorylated EGFR, Y1068 phosphorylated EGFR, and EGFR RNA in A431 human epidermoid carcinoma cells.

Background & Aims The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is increasing. Subjects with NASH often develop liver fibrosis and advanced liver fibrosis is the main determinant of mortality in NASH patients. We and others have reported that STAT3 contributes to liver fibrosis and hepatocellular carcinoma in mice. Methods Here, we explored whether STAT3 activation in hepatocytes and in non-hepatocytes areas, measured by phospho-STAT3 (pSTAT3), is associated with liver fibrosis progression in 133 patients with NAFLD. We further characterized the molecular and cellular determinants of STAT3 activation by integrating spatial distribution and transcriptomic changes in fibrotic NAFLD liver. Results pSTAT3 scores in non-hepatocytes areas progressively increased with fibrosis severity (r=0.53, p

The etiology of bladder cancer is known to be associated with behavioral and environmental factors. Moreover, several studies suggested a potential role of HPV infection in the pathogenesis with controversial results. A systematic review was conducted to assess the role of HPV. A total of 46 articles that reported the prevalence of HPV infection in squamous (SCC), urothelial (UC), and transitional cell carcinomas (TCC) were selected. A pooled prevalence of 19% was found, with a significant difference in SCC that was mainly driven by HPV-16. Moreover, infection prevalence in case-control studies showed a higher risk of bladder cancer in HPV-positive cases (OR: 7.84; p-value < 0.00001). The results may suggest an etiologic role of HPV in bladder cancer. HPV vaccine administration in both sexes could be key to prevent the infection caused by high-risk genotypes.

IL-38 is an IL-1 family receptor antagonist that restricts IL-17-driven inflammation by limiting cytokine production from macrophages and T cells. In the current study, we aimed to explore its role in experimental autoimmune encephalomyelitis in mice, which is, among others, driven by IL-17. Unexpectedly, IL-38-deficient mice showed strongly reduced clinical scores and histological markers of experimental autoimmune encephalomyelitis. This was accompanied by reduced inflammatory cell infiltrates, including macrophages and T cells, as well as reduced expression of inflammatory markers in the spinal cord. IL-38 was highly expressed by infiltrating macrophages in the spinal cord, and in vitro activated IL-38-deficient bone marrow-derived macrophages showed reduced expression of inflammatory markers, accompanied by altered cellular metabolism. These data suggest an alternative cell-intrinsic role of IL-38 to promote inflammation in the CNS.

In many human cancers, the expression of the prostaglandin receptor EP4 (EP4R) is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The objective of this study was to characterize the messenger RNA (mRNA) expression of EP4R in canine osteosarcoma (OSA). Gene expression of EP4R was evaluated using RNA in-situ hybridization (RNAscope). In all canine OSA samples evaluated, strong universal positive expression of EP4R was identified. Gene expression was significantly higher in OSA tissue samples than in normal nasal turbinate bone, possibly implicating EP4R in the pathogenesis of canine OSA.

Gastric cancer (GC) is one of the most common cancer types in the world with a high mortality rate. Hereditary predisposition for GC is not fully elucidated so far. The aim of this study was identification of possible new candidate genes, associated with the increased risk of gastric cancer development. Whole exome sequencing (WES) was performed on 18 DNA samples from adenocarcinoma specimens and non-tumor-bearing healthy stomach tissue from the same patient. Three pathogenic variants were identified: c.1320+1G>A in the CDH1 gene and c.27_28insCCCAGCCCCAGCTACCA (p.Ala9fs) of the VEGFA gene were found only in the tumor tissue, whereas c.G1874C (p.Cys625Ser) in the FANCA gene was found in both the tumor and normal tissue. These changes were found only in patients with diffuse gastric cancer and were absent in the DNA of healthy donors.

Imaging mass cytometry (IMC) is a new technology integrating mass spectrometry, high-resolution laser ablation and immunohistochemistry/cytochemistry. A unique high-dimensional perspective comprehensively and accurately depicts the complex interaction of phenotype, signalling pathway and tumour microenvironment and is widely used in solid tumours. However, the application scenarios of IMC in basic medicine and clinical research in solid tumours lack systematic introduction and classification. This paper reviews the application of IMC in depicting the panorama of the tumour microenvironment, revealing tumour spatial heterogeneity, clarifying tumour pharmacological mechanisms, assisting in new drug development, and dynamically evaluating the efficacy of immunotherapy in solid tumours.

Defining changes in gene expression during health and disease is critical for the understanding of human physiology. In recent years, single-cell/nuclei RNA sequencing (sc/snRNAseq) has revolutionized the definition and discovery of cell types and states as well as the interpretation of organ- and cell-type-specific signaling pathways. However, these advances require tissue dissociation to the level of the single cell or single nuclei level. Spatially resolved transcriptomics (SrT) now provides a platform to overcome this barrier in understanding the physiological contexts of gene expression and cellular microenvironment changes in development and disease. Some of these transcriptomic tools allow for high-resolution mapping of hundreds of genes simultaneously in cellular and subcellular compartments. Other tools offer genome depth mapping but at lower resolution. We review advances in SrT, considerations for using SrT in your own research, and applications for kidney biology.

Dysregulation of alternative splicing of hTERT gene to generate full-length Htert (hTERT-FL) that reactivate telomerase has been recognized as a major pathological alteration in pancreatic cancer (PrCa). Mechanism about the factors that regulate hTERT-FL splicing is lacking. Through bioinformatics approach, we focus on a candidate splicing factor RBM10, which leads to a switch in hTERT transcripts to generate a function-less isoform hTERT-s in PrCa, suppressed both telomerase activity and subsequent telomere shortening. RBM10 expression is negatively associated with PrCa progression. Gain or loss of RBM10 also significantly changed PrCa cell proliferation in vitro and in xenografts. RNA-IP and RNA pull-down assays reveal that RBM10 promotes the exclusion of exons7 and 8 which results in the production of TERT-s transcripts. This study may increase knowledge about potentially targetable cancer associated splicing factors and provide novel insights into therapeutic approach in PrCa. AJCR

Immunohistochemistry is a commonly used technique in research and pathology laboratories worldwide. However, in recent years, there has been a significant decrease in the number of Pubmed entries using the term immunohistochemistry. This decline can be attributed to two factors: increased awareness of the issue of unreliable research antibodies and the availability of novel RNA in situ hybridization techniques. Using the example of immunohistochemistry, this text discusses the factors that can affect good laboratory and publishing practices, or their lack thereof.