Probes for INS

Tenosynovial giant cell tumor (TSGCT) is a benign fibrohistiocytic tumor that affects the synovium of joints, bursa, and tendon sheaths and is categorized into localized TSGCT (LTSGCT) and diffuse TSGCT (DTSGCT). LTSGCT and DTSGCT are characterized by recurrent fusions involving the colony-stimulating factor 1 (CSF1) gene and its translocation partner collagen type VI alpha 3 chain. The fusion gene induces intratumoral overexpression of CSF1 mRNA and CSF1 protein. CSF1 expression is a characteristic finding of TSGCT and detection of CSF1 mRNA and CSF1 protein may be useful for the pathological diagnosis. Although there have been no effective anti-CSF1 antibodies to date, in situ hybridization (ISH) for CSF1 mRNA has been performed to detect CSF1 expression in TSGCT. We performed CSF1 immunohistochemistry (IHC) using anti-CSF1 antibody (clone 2D10) in cases of TSGCT, giant cell-rich tumor (GCRT), and GCRT-like lesion and verified its utility for the pathological diagnosis of TSGCT.We performed CSF1 IHC in 110 cases including 44 LTSGCTs, 20 DTSGCTs, 1 malignant TSGCT (MTSGCT), 10 giant cell tumors of bone, 2 giant cell reparative granulomas, 3 aneurysmal bone cysts, 10 undifferentiated pleomorphic sarcomas, 10 leiomyosarcomas, and 10 myxofibrosarcomas. We performed fluorescence ISH (FISH) for CSF1 rearrangement to confirm CSF1 expression on IHC in TSGCTs. We considered the specimens to have CSF1 rearrangement if a split signal was observed in greater than 2% of the tumor cells.Overall, 50 of 65 TSGCT cases, including 35 of the 44 LTSGCTs and 15 of the 20 DTSGCTs, showed distinct scattered expression of CSF1 in the majority of mononuclear tumor cells. MTSGCT showed no CSF1 expression. Non-TSGCT cases were negative for CSF1. FISH revealed CSF1 rearrangement in 6 of 7 CSF1-positive cases on IHC. On the other hand, FISH detected no CSF1 rearrangement in all CSF1-negative cases on IHC. Thus, the results of IHC corresponded to those of FISH.We revealed characteristic CSF1 expression on IHC in cases of TSGCT, whereas the cases of non-TSGCT exhibited no CSF1 expression. CSF1 IHC may be useful for differentiating TSGCTs from histologically mimicking GCRTs and GCRT-like lesions.

TREM2 is a transmembrane receptor expressed by myeloid cells and acts to regulate their immune response. TREM2 governs the response of microglia to amyloid and tau pathologies in the Alzheimer's disease (AD) brain. TREM2 is also present in a soluble form (sTREM2), and its CSF levels fluctuate as a function of AD progression. Analysis of stroke and AD mouse models revealed that sTREM2 proteins bind to neurons, which suggests sTREM2 may act in a non-cell autonomous manner to influence neuronal function. sTREM2 arises from the proteolytic cleavage of the membrane-associated receptor. However, alternatively spliced TREM2 species lacking a transmembrane domain have been postulated to contribute to the pool of sTREM2. Thus, both the source of sTREM2 species and its actions in the brain remain unclear.The expression of TREM2 isoforms in the AD brain was assessed through the analysis of the Accelerating Medicines Partnership for Alzheimer's Disease Consortium transcriptomics data, as well as qPCR analysis using post-mortem samples of AD patients and of the AD mouse model 5xFAD. TREM2 cleavage and secretion were studied in vitro using HEK-293T and HMC3 cell lines. Synaptic plasticity, as evaluated by induction of LTP in hippocampal brain slices, was employed as a measure of sTREM2 actions.Three distinct TREM2 transcripts, namely ENST00000373113 (TREM2230), which encodes the full-length transmembrane receptor, and the alternatively spliced isoforms ENST00000373122 (TREM2222) and ENST00000338469 (TREM2219), are moderately increased in specific brain regions of patients with AD. We provide experimental evidence that TREM2 alternatively spliced isoforms are translated and secreted as sTREM2. Furthermore, our functional analysis reveals that all sTREM2 species inhibit LTP induction, and this effect is abolished by the GABAA receptor antagonist picrotoxin.TREM2 transcripts can give rise to a heterogeneous pool of sTREM2 which acts to inhibit LTP. These results provide novel insight into the generation, regulation, and function of sTREM2 which fits into the complex biology of TREM2 and its role in human health and disease. Given that sTREM2 levels are linked to AD pathogenesis and progression, our finding that sTREM2 species interfere with LTP furthers our understanding about the role of TREM2 in AD.

Collagen VI (COLVI) is a critical myomatrix protein for skeletal muscle health and maintenance. There are 6 COL6 genes (COL6A1-COL6A6). Pathogenic variants in COL6A1, COL6A2, or COL6A3 cause COLVI-related dystrophies (COL6-RDs) with early-onset muscle weakness and loss of ambulation. Identifying novel therapeutic targets is critical for developing COL6-RDs therapies. Here, using in situ hybridization, we aim to identify, quantify, and locate all cell types that express the wild-type (WT) COL6 genes and a common, recurrent pseudoexon (PE)-inserting COL6A1 mutation in limb skeletal muscles and diaphragms of WT and COL6-RDs male mice at 10-day-old, and 6- and 20-month-old. We first analyzed published mouse skeletal muscle RNA-seq datasets to identify potential sensitive and specific mRNA cell markers (3/cell type). Then, we conducted a pilot marker validation experiment with RNAscope. We quantified and assessed the expression of the selected 33 markers’ transcripts in 3 non-overlapping fields of a quadriceps section from 2-month-old and 6-month-old mice (n=1). The specificity was reported as % of cells that had a marker for one cell type and lacked the markers of all other cell types. Our preliminary data indicated markers that were specific (Pdgfra: Fibro-adipogenic progenitors (FAPs) (98-100% specific), Myod1: satellite cells (SCs) (88-96%), and Pecam1: endothelial cells (endo) (92-100%)), non-specific (Esam: endo (57-77%), and Asb5: SCs (55-81%)), or specific in only one age group (Dpt: FAPs (2-month: 73%; 6-month: 89%), and Cdh5: endo (2-month: 80%; 6-month: 100%). We are currently validating each marker's specificity and sensitivity. We will identify and locate the cells that express the Col6 genes and the PE and quantify their transcripts expression levels in the various models and age groups. The findings of this project will provide additional insights into the roles of COLVI-producing cells in the pathogenesis of COL6-RDs and help direct therapeutic approaches.

Aryl hydrocarbon receptor (AhR) activation promotes intestinal barrier repair and enhances the gut mucosal barrier function in inflammatory bowel diseases (IBD). Spermidine is beneficial in several murine models of IBD and may affect AhR activity. However, the precise effects of spermidine on the intestinal barrier and AhR remain unclear. This study was designed to investigate whether spermidine affects AhR and gut barrier function in IBD models as well as, its underlying mechanism.We used dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mice, as well as, Caco2 cells incubated with TNF-α and IFN-γ to establish multiple IBD models, followed by spermidine intervention. Alcian blue/Periodic acid-Schiff (AB/PAS) staining, Fluorescein isothiocyanate (FITC)-dextran permeability assay, transepithelial electrical resistance (TER), tight junction protein (TJs) expression, and 16S rRNA scope in situ hybridization were performed to assess intestinal barrier function. AhR expression and the associated pathways were measured. AhR-targeted adeno-associated virus (AAV) and siRNA were used to explore the related molecular mechanisms.Spermidine significantly attenuated the increased intestinal permeability, decreased TER, abnormal distribution of TJs in colitis, and bacterial translocation from the gut tract. Additionally, it significantly increased AhR and Nrf2 expression and inhibited STAT3 phosphorylation. However, the protective effects of spermidine and the related alterations in pathway proteins were largely abolished by the specific inhibition of AhR.Our study demonstrated that spermidine rescues intestinal barrier defects in mice with colitis via the AhR-Nrf2 and AhR-STAT3 pathways, providing a potential therapeutic agent for IBD and other conditions associated with dysregulated gut barrier function.

Background Prostate cancer (PCa) is one of the main causes of death in men all over the world. To date, the emerging issue is the search for new diagnostic and prognostic biomarkers to distinguish patients with different risk types. Recent studies of PCa biomarkers have drawn attention to the remarkable heterogeneity of this tumor. Tumor heterogeneity (TH) is the main limitation of the ability to use biomarkers’ gene panels in clinics because of different cell subclones within one tumor. Methods Here we describe the relationship between tumor cells in a model of heterogeneous prostate cancer. Our model was based on direct and indirect co-cultivation of 2 prostate cell lines with aggressive (PC3-GFP) and indolent (22Rv1) phenotypes. To evaluate the tumor aggressiveness, we described the surface phenotype of adhesion molecules, measured the expression of genes related to metastasis in prostate cancer (Ai et al., 2017; Fan et al., 2018) and performed the motility tests. Results In experiments with direct co-cultivation of PC3 (aggressive) and 22Rv1 (indolent) cancer cells, we found a decrease of CD29 (integrin beta 1) on PC3 cells within 3 days, while the expression of other surface adhesion molecules (CD54, CD38, CD24 and CD44) was not altered, the surface phenotype of 22Rv1 did not change. Next, we assessed the expression of genes related to metastasis and showed that after 3 days of direct co-cultivation the expression of FLNC, AMACR, SNCG, HPN genes increases at least 2.5 times in 22Rv1, while HPN and FASN are upregulated in PC3 cells (for all measurements p

Progressive respiratory muscle weakness and ineffective cough contributes to morbidity and mortality in children with neuromuscular diseases (NMD). Inspiratory muscle training (IMT) aims to preserve or improve respiratory muscle strength and reduce respiratory morbidity. This study aimed to determine the safety and efficacy of IMT in children with NMD. A randomised cross-over study compared three-month intervention (IMT) with control periods. During the intervention, children with NMD (5-18 years) from two provinces in South Africa performed 30 breaths (at 30% of inspiratory muscle strength (Pimax)) with an electronic threshold device, twice daily. During the control period participants did not perform any IMT. Twenty-three participants (median (IQR) age of 12.33 (10.03-14.17) years), mostly male (n=20) and non-ambulant (n=14) were included. No adverse events related to IMT were reported. There was no evidence of a difference in median patient hospitalisation and respiratory tract infection rates between control and intervention periods (p=0.60; p=0.21). During IMT, Pimax and peak cough flow improved with a mean (SD) of 14.57 (±15.67) cmH2O and 32.27 (±36.60) L/min, compared to a change of 3.04(±11.93)cmH2O (p=0.01) and -16.59 (±48.29) L/min (p=0.0005) during the control period. There was no evidence of change in spirometry, functional ability and total health-related quality of life scores following intervention. Patient satisfaction with IMT was high (median 8/10 (IQR 5-10)) and adherence was good. A three-month IMT programme in children with NMD is well tolerated, appears to be safe and is associated with a significant improvement in respiratory muscle strength and cough efficacy.

Over the past decade, considerable efforts have been made to accelerate pathophysiological understanding of fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) with brain banks at the forefront. In addition to exploratory disease mechanisms, brain banks have aided our understanding with regard to clinical diagnosis, genetics and cell biology. Across neurodegenerative disorders, the impact of brain tissue in ALS research has yet to be quantified. This review aims to outline (i) how postmortem tissues from brain banks have influenced our understanding of ALS over the last 15 years, (ii) correlate the location of dedicated brain banks with the geographical prevalence of ALS, (iii) identify the frequency of features reported from postmortem studies and (iv) propose common reporting standards for materials obtained from dedicated brain banks. A systematic review was conducted using PubMed and Web of Science databases using key words. From a total of 1439 articles, 73 articles were included in the final review, following PRISMA guidelines. Following a thematic analysis, articles were categorised into five themes; clinico-pathological (13), genetic (20), transactive response DNA binding protein 43 (TDP-43) pathology (12), non-TDP-43 neuronal pathology (nine) and extraneuronal pathology (19). Research primarily focused on the genetics of ALS, followed by protein pathology. About 63% of the brain banks were in the United States of America and United Kingdom. The location of brain banks overall aligned with the incidence of ALS worldwide with 88% of brain banks situated in Europe and North America. An overwhelming lack of consistency in reporting and replicability was observed, strengthening the need for a standardised reporting system. Overall, postmortem material from brain banks generated substantial new knowledge in areas of genetics and proteomics and supports their ongoing role as an important research tool.

Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.

Stress management is necessary for vertebrate survival. Chronic stress drives depression by excitation of the lateral habenula (LHb), which silences dopaminergic neurons in the ventral tegmental area (VTA) via GABAergic neuronal projection from the rostromedial tegmental nucleus (RMTg). However, the effect of acute stress on this LHb-RMTg-VTA pathway is not clearly understood. Here, we used fluorescent in situ hybridisation and in vivo electrophysiology in mice to show that LHb aromatic L-amino acid decarboxylase-expressing neurons (D-neurons) are activated by acute stressors and suppress RMTg GABAergic neurons via trace aminergic signalling, thus activating VTA dopaminergic neurons. We show that the LHb regulates RMTg GABAergic neurons biphasically under acute stress. This study, carried out on male mice, has elucidated a molecular mechanism in the efferent LHb-RMTg-VTA pathway whereby trace aminergic signalling enables the brain to manage acute stress by preventing the hypoactivity of VTA dopaminergic neurons.

Type 2 diabetes (T2D) is a major health and economic burden worldwide. Despite the availability of multiple drugs for short-term management, sustained remission of T2D is currently not achievable pharmacologically. Intracerebroventricular administration of fibroblast growth factor 1 (icvFGF1) induces sustained remission in T2D rodents, propelling intense research efforts to understand its mechanism of action. Whether other FGFs possess similar therapeutic benefits is currently unknown. Here, we show that icvFGF4 also elicits a sustained antidiabetic effect in both male db/db mice and diet-induced obese mice by activating FGF receptor 1 (FGFR1) expressed in glucose-sensing neurons within the mediobasal hypothalamus. Specifically, FGF4 excites glucose-excited (GE) neurons while inhibiting glucose-inhibited (GI) neurons. Moreover, icvFGF4 restores the percentage of GI neurons in db/db mice. Importantly, intranasal delivery of FGF4 alleviates hyperglycemia in db/db mice, paving the way for non-invasive therapy. We conclude that icvFGF4 holds significant therapeutic potential for achieving sustained remission of T2D.

Chronic hepatitis B is usually treated with nucleos(t)ide analogues (NAs). However, a cure is rarely achieved even with years of treatment. Here, we investigated whether viral replication is completely halted and how long covalently closed circular DNA (cccDNA) persists in NAs successfully treated patients.A series of longitudinal serum samples and a collection of cross-sectional liver biopsies were obtained from NAs successfully treated patients. Viral variants in serum HBV RNA were enumerated by deep sequencing. Viral replication intermediates in hepatocytes were directly visualized by in situ hybridization. The apparent half-life of each cccDNA was estimated.Three out of six successfully treated patients demonstrated clear evidence of a small proportion of virus evolution, although the overwhelming variants were identical or possessed a similar degree of divergence through time. The apparent half-life of variants was estimated from approximately 7.42 weeks to infinite. Moreover, hepatocytes remained positive for cytoplasmic nucleocapsids-associated relaxed circular DNA (rcDNA) in four out of seven liver needle biopsies.We therefore conclude that even after prolonged treatment, a small proportion of the cccDNA reservoir is constantly replenished by continued low-level HBV replication, whereas the greatest amount of cccDNA reservoir persists over time.

Psoriasis and sarcoidosis are inflammatory skin and systemic diseases that may share a similar immunopathogenesis involving a Th1 and/or Th17 polarized immune response. Although the coexistence of sarcoidosis and psoriasis in the same individuals has been reported, the potential association between these diseases at a population-level in the United States has not been evaluated. To evaluate this association, we performed a matched cross-sectional study in the All of Us research program database. In the multivariable analysis of 4932 psoriasis cases and 19,728 controls, sarcoidosis was found to be significantly associated with psoriasis (OR 2.37 [95% CI 1.73-3.23], p < 0.001). The relative strength of this association between psoriasis and sarcoidosis may be, in part, explained by overlapping immunopathogenesis and common genetic susceptibility of these diseases. Taken together, these observations underscore the need for screening psoriasis patients for development of new cardiopulmonary symptoms. Further research into the mechanism of this relationship and its implications is warranted.