Probes for INS

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated pain targets. Clinical block of Cav2.2 (e.g., with Prialt) or indirect modulation (e.g., with gabapentinoids) mitigates chronic pain but is constrained by side effects. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release and pain. Homology-guided mutagenesis of CBD3 revealed an antinociceptive core in the N-terminal A1RSR4. Here, we developed and applied a novel molecular dynamics approach to identify the Cav2.2 recognition motif of the core CBD3 peptide as the A1R2 dipeptide and used its presenting motif to design pharmacophore models to screen 27 million compounds in the open access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization‐evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Nine compounds were tested using electrophysiology and one compound (CBD3063) was evaluated biochemically, electrophysiologically, and behaviorally in models of experimental pain. CBD3063 reduced membrane Cav2.2 expression and currents, uncoupled the Cav2.2-CRMP2 interaction, inhibited neuronal excitability, decreased spinal cord transmission, induced analgesia in naïve rats and reversed mechanical allodynia in rats with spared nerve injury. These results identify CBD3063, as a selective, first-in-class, CRMP2-based peptidomimetic, which allosterically regulates Cav2.2 to achieve analgesia.

Ribonuclease inhibitor 1, also known as angiogenin inhibitor 1, encoded by RNH1, is a ubiquitously expressed leucine-rich repeat protein, which is highly conserved in mammalian species. Inactivation of rnh1 in mice causes an embryonically lethal anemia, but the exact biological function of RNH1 in humans remains unknown and no human genetic disease has so far been associated with RNH1. Here, we describe a family with two out of seven siblings affected by a disease characterized by congenital cataract, global developmental delay, myopathy and psychomotor deterioration, seizures and periodic anemia associated with upper respiratory tract infections. A homozygous splice-site variant (c.615-2A > C) in RNH1 segregated with the disease. Sequencing of RNA derived from patient fibroblasts and cDNA analysis of skeletal muscle mRNA showed aberrant splicing with skipping of exon 7. Western blot analysis revealed a total lack of the RNH1 protein. Functional analysis revealed that patient fibroblasts were more sensitive to RNase A exposure, and this phenotype was reversed by transduction with a lentivirus expressing RNH1 to complement patient cells. Our results demonstrate that loss-of-function of RNH1 in humans is associated with a multiorgan developmental disease with recessive inheritance. It may be speculated that the infection-induced deterioration resulted from an increased susceptibility toward extracellular RNases and/or other inflammatory responses normally kept in place by the RNase inhibitor RNH1.

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.

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.

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.

Nuclear depletion and cytoplasmic mislocalisation of the RNA-binding protein heterogeneous ribonucleoprotein K (hnRNP K) within pyramidal neurons of the frontal cortex have been shown to be a common neuropathological feature in frontotemporal lobar degeneration (FTLD) and elderly control brain. Here, we describe a second neuronal subtype vulnerable to mislocalisation within the dentate nucleus of the cerebellum. In contrast to neurons within the cerebellar cortex that typically exhibited normal, nuclear staining, many neurons of the dentate nucleus exhibited striking mislocalisation of hnRNP K to the cytoplasm within neurodegenerative disease brain. Mislocalisation frequency in this region was found to be significantly higher in both FTLD-TDP A and Alzheimer's disease (AD) brain than in age-matched controls. However, within control (but not disease) subjects, mislocalisation frequency was significantly associated with age-at-death with more elderly controls typically exhibiting greater levels of the pathology. This study provides further evidence for hnRNP K mislocalisation being a more anatomically diverse pathology than previously thought and suggests that potential dysfunction of the protein may be more broadly relevant to the fields of neurodegeneration and ageing.