Probes for INS

The associative network of hippocampal CA3 is thought to contribute to rapid formation of contextual memory from one-trial learning, but the network mechanisms underlying decorrelation of neuronal ensembles in CA3 is largely unknown. Kv1.2 expressions in rodent CA3 pyramidal cells (CA3-PCs) are polarized to distal apical dendrites, and its downregulation specifically enhances dendritic responses to perforant pathway (PP) synaptic inputs. We found that haploinsufficiency of Kv1.2 (Kcna2+/-) in CA3-PCs, but not Kv1.1 (Kcna1+/-), lowers the threshold for long-term potentiation (LTP) at PP-CA3 synapses, and that the Kcna2+/- mice are normal in discrimination of distinct contexts but impaired in discrimination of similar but slightly distinct contexts. We further examined the neuronal ensembles in CA3 and dentate gyrus (DG), which represent the two similar contexts using in situ hybridization of immediate early genes, Homer1a and Arc. The size and overlap of CA3 ensembles activated by the first visit to the similar contexts were not different between wild type and Kcna2+/- mice, but these ensemble parameters diverged over training days between genotypes, suggesting that abnormal plastic changes at PP-CA3 synapses of Kcna2+/- mice is responsible for the impaired pattern separation. Unlike CA3, DG ensembles were not different between two genotype mice. The DG ensembles were already separated on the first day, and their overlap did not further evolve. Eventually, the Kcna2+/- mice exhibited larger CA3 ensemble size and overlap upon retrieval of two contexts, compared to wild type or Kcna1+/- mice. These results suggest that sparse LTP at PP-CA3 synapse probably supervised by mossy fiber inputs is essential for gradual decorrelation of CA3 ensembles.

We herein report the treatment outcome of oropharyngeal squamous cell carcinoma (OPSCC) at Kyushu University Hospital, the total number of OPSCC cases, and changes in the proportion of human papilloma virus (HPV)-related carcinomas over time.We performed a retrospective analysis of 237 cases treated for OPSCC at Kyushu University Hospital between 2013 and 2019. We performed HPV-mRNA in situ hybridization and p16 immunohistochemistry.This study included 197 males (82.1%) and 40 females (17.9%). The disease-specific, progression-free and overall survival (OS) were 69%, 62% and 61%, respectively, over the decade-long study period. p16-Immunohistochemistory and highrisk HPV mRNA in situ hybridization were positive in 114 (48.1%) and 105 (44.3%) cases, respectively. The number of HPV-related OPSCC cases increased according to an annual analysis. HPV+ cases had a significantly better prognosis than HPV- cases. In addition, p16+/HPV- cases had a significantly worse prognosis than p16+/HPV+ cases (OS: p = 0.0484). HPV+ OPSCC cases were associated with a younger age (< 60 years old) (p = 0.0429), non-smoker (p = 0.0001), lateral tumor site (< 0.00001), lymphoid metastasis (< 0.0001) and low clinical stage (< 0.0001).The frequency of HPV-related OPSCC cases is increasing in Japan as well as worldwide, and such cases are characterized by no smoking habit, a young age, and a good prognosis. Even in p16+ OPSCC, HPV- cases had a poor prognosis, suggesting the importance of accurate HPV determination. To determine the intensity of treatment for HPV-related and non-related OPSCC, it is necessary to accumulate cases for the accurate HPV determination and comparison of treatment effects.

Diversity is a key feature in the glioma ecosystem. Adaptation to a changing tumor microenvironment is achieved through cellular and metabolic plasticity. Here we show that slow-cycling, astrocyte-like glioma cell subpopulations activate distinct metabolic programs, rendering them susceptible to novel treatments. We performed multi-omics analysis on transgenic murine glioma models to characterize cellular heterogeneity. Bulk RNAseq on targeted time-dependent biopsies combined with scRNAseq uncovered distinct tumor cell populations, including a quiescent, astrocyte-like population relatively insensitive to conventional chemotherapy targeting proliferating cells. Using scRNAseq, we identified a persistently conserved astrocytic population in human IDH1-mt/wt high-grade gliomas. This astrocytic tumor population was more abundant in mouse models with constitutive Notch activation, however it was associated with alterations in several other transcriptional programs, suggesting that targeted therapies would likely be ineffective at eradicating it. Gene ontology analysis revealed enrichment in mitochondrial genes specifically regulating oxidative phosphorylation and tricarboxylic acid cycle. Energetic, lipidomic and metabolomic analyses revealed significant mitochondrial β-fatty acid oxidation and lipid catabolism, with less effective oxygen consumption rate and higher basal oxidative stress. Furthermore, this astrocytic tumor population had depleted levels of basal GSH and was more sensitive to reactive oxygen species. Leveraging this metabolic vulnerability, we performed drug screens and found that therapeutic inhibition of complex I or GPX4 was highly effective and synergistic. GPX4 inhibition induced ferroptosis, a newly-discovered form of programmed non-necroptotic cell death mediated by iron-driven lipid peroxidation. Using scRNAseq and RNAscope on ex vivo slice cultures from murine and human gliomas, we found that GPX4 inhibition and ferroptosis induction in the glioma microenvironment selectively eradicated the quiescent astrocytic subpopulation whereas proliferating glioma were less sensitive. Our data therefore supports a novel treatment paradigm, employing metabolic strategies, such as ferroptosis, in conjunction with chemotherapy and RT to target distinct tumor cell populations with different therapeutic vulnerabilities.

Background: Although recently developed CFTR modulator drugs can alleviate cystic fibrosis (CF) in the majority of patients, CF patients with mutations that form premature termination codons (PTCs) cannot benefit from modulator therapies. PTCs terminate translation before a full-length CF transmembrane regulator (CFTR) protein can be generated. In addition, a PTC can trigger nonsense-mediated mRNA decay of the CFTR transcript, further reducing expression of CFTR protein. Together, these 2 PTCmediated events result in negligible CFTR protein, abrogating the usefulness of CFTR modulators, whose action requires CFTR expression. Nonsense suppression therapy uses small molecules to suppress translation termination at in-frame PTCs (nonsense mutations) to restore partial levels of full-length, functional CFTR protein. Methods: Although some compounds have been identified that can suppress translation termination at PTCs (also called readthrough), poor efficacy of current readthrough agents prompted us to search for more effective compounds. To this end, we developed a NanoLuc reporter system in Fischer rat thyroid cells that responds to readthrough and nonsensemediated mRNA decay. This assay was used to screen 771 345 lowmolecular-weight compounds. Results: Of the 180 compounds identified with readthrough activity, SRI37240 and its more potent derivative SRI-41315 suppressed multiple CFassociated PTCs in immortalized and primary human bronchial epithelial cells, restoring partial CFTR expression and function. Mechanistically, we found that these compounds induce a prolonged pause at stop codons and suppress PTCs by targeting the termination factor eRF1 to the proteasome and thus reducing its abundance. Moreover, SRI-41315 enhances aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Conclusion: SRI-37240 and SRI-41315 are the first pharmacological agents known to alter eRF1 levels and thus represent a new class of readthrough compounds that could potentially be used as part of a nonsense suppression therapy to treat genetic diseases in patients who carry PTCs

The myotendinous junction (MTJ) is a specialised interface for transmitting high forces between muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses and the tissue was separated through a sequential cryo-sectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-SNE plots were used to select the muscle, MTJ and tendon samples from 5 horses for RNA-sequencing. The expression of previously known and unknown genes identified through RNA-sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA-sequencing identified expression of a panel of 61 genes enriched at the MTJ. 48 of these genes were novel for the MTJ, and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known (COL22A1, NCAM, POSTN, NES, OSTN) and previously undescribed (MNS1 and LCT) MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.

Esophageal verrucous carcinoma (VSCC) is a rare and morphologically distinct type of esophageal squamous cell carcinoma (SCC). Diagnosing VSCC on biopsy material is challenging given the lack of significant atypia and the presence of keratinizing epithelium and exophytic growth. The molecular pathogenesis of VSCC remains unclear. The aim of this study was to characterize the genomic landscape of VSCC in comparison to conventional esophageal SCC. Three cases of VSCC from the Brigham and Women's Hospital pathology archive were identified. Formalin-fixed, paraffin-embedded (FFPE) tumor tissue was used for p16 immunohistochemistry (IHC), high-risk HPV in situ mRNA hybridization (ISH), and DNA isolation. Tumor DNA was sequenced using a targeted massively parallel sequencing assay enriched for cancer-associated genes. Three additional cases of VSCC were identified by image review of The Cancer Genome Atlas (TCGA) esophageal SCC cohort. VSCC cases were negative for p16 IHC and high-risk HPV ISH. TP53 mutations (p<0.001) and copy number variants (CNVs) for CDKN2A (p<0.001), CDKN2B (p<0.01) and CCND1 (p<0.01) were absent in VSCC and significantly less frequent in comparison to conventional SCC. Five VSCC cases featured SMARCA4 missense mutations or inframe deletions compared to only 4/88 conventional SCC cases (p<0.001). VSCC featured driver mutations in PIK3CA, HRAS, and GNAS. Recurrent CNVs were rare in VSCC. VSCC is not only morphologically but also genetically distinct from conventional esophageal SCC, featuring frequent SMARCA4 mutations and infrequent TP53 mutations or CDKN2A/B CNVs. Molecular findings may aid in establishing the challenging diagnosis of VSCC. This article is protected by

Background: The amygdala is responsible for encoding valence, the biological value of aversive and rewarding stimuli; it plays a critical role in the formation and storage of emotional memories, assessment of threat-related stimuli, and fear consolidation. As a step toward identifying how specific cell populations contribute to these functions, we sought to systematically identify the cell types of the human amygdala, the genes (mRNAs) expressed by each cell type, and their relationships to cell types in mouse and marmoset amygdala. Methods: We used single-cell RNA-seq (Drop-seq and the 10X Genomics platform) to profile mRNA expression in nuclei sampled from the amygdala of n¼4 postmortem healthy donors. We used computational methods we have described previously (Sanders et al., Cell 2018; Krienen et al., Nature 2020) to organize these profiles into clusters of transcriptionally similar cells corresponding to cell types and cell states. We also analyzed these data in relationship to similar data we generated from mouse and marmoset amygdala. Results: We profiled mRNA expression in more than 100,000 nuclei sampled from the 4-human amygdala. Computational analysis revealed at least 30 transcriptionally distinct types of neurons in the amygdala, including 17 GABAergic and 13 glutamatergic types. We found homologs for these principal cell types in the amygdala of marmoset and mouse. However, the expression patterns of many individual genes (across these neuronal types) diverged considerably among the three species. Conclusions: Availability of this data resource will help enable diverse scientific investigations of the amygdala and its relationship to post-traumatic stress disorder and other psychiatric disorders.

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of H2O2. By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of H2O2 maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.

Vesicoureteral reflux (VUR) is a common, familial genitourinary disorder, and a major cause of pediatric urinary tract infection (UTI) and kidney failure. The genetic basis of VUR is not well understood. A diagnostic analysis sought rare, pathogenic copy number variant (CNV) disorders among 1737 patients with VUR. A GWAS was performed in 1395 patients and 5366 controls, of European ancestry. Altogether, 3% of VUR patients harbored an undiagnosed rare CNV disorder, such as the 1q21.1, 16p11.2, 22q11.21, and triple X syndromes ((OR, 3.12; 95% CI, 2.10 to 4.54; P=6.35×10-8) The GWAS identified three study-wide significant and five suggestive loci with large effects (ORs, 1.41-6.9), containing canonical developmental genes expressed in the developing urinary tract (WDPCP, OTX1, BMP5, VANGL1, and WNT5A). In particular, 3.3% of VUR patients were homozygous for an intronic variant in WDPCP (rs13013890; OR, 3.65; 95% CI, 2.39 to 5.56; P=1.86×10-9). This locus was associated with multiple genitourinary phenotypes in the UK Biobank and eMERGE studies. Analysis of Wnt5a mutant mice confirmed the role of Wnt5a signaling in bladder and ureteric morphogenesis. These data demonstrate the genetic heterogeneity of VUR. Altogether, 6% of patients with VUR harbored a rare CNV or a common variant genotype conferring an OR >3. Identification of these genetic risk factors has multiple implications for clinical care and for analysis of outcomes in VUR.

Background: Malnutrition has historically been a main clinical consequence of CF. Consensus recommendations have encouraged a highcalorie, high-fat diet but with little guidance related to nutrient density or quality of foods consumed to meet elevated metabolic needs. Highly effective modulators are associated with improved growth and increases in weight and body mass index (BMI) in subsets of the CF population. Recently elexacaftor/tezacaftor/ivacaftor was approved for use in up to 90% of people with CF. PROMISE is an open-label observational cohort study designed to longitudinally assess the effectiveness of elexacaftor/tezacaftor/ivacaftor in the clinical setting. Our single-center sub-study aimed to explore the changes in BMI, dietary intake, muscle strength, pancreatic enzyme replacement therapy (PERT), and resting energy expenditure with use of elexacaftor/tezacaftor/ivacaftor. Methods: Participants were enrolled and had baseline (V1) measurements performed prior to taking their first dose of elexacaftor/tezacaftor/ ivacaftor. Follow-up measurements were obtained at 2 visits. Short-term follow occurred at 28 days (V2) on elexacaftor/tezacaftor/ivacaftor and long-term follow measurements were obtained at > 6 months on drug (V3). Measurements at each time point included: resting energy expenditure as percent of predicted (REE%) using indirect calorimetry, hand grip strength (HGS), dietary intake using 3-day diet records, and PERT dosage. Diet records were reviewed by the study dietitian and were analyzed using NDSR software. The Healthy Eating Index (HEI) is a validated measure of diet quality based on the Dietary Guidelines for Americans 2015-2020. HEI-2015 scores range from 0 to 100, with a score of 100 indicating the best diet quality. Lung function and QOL (CFQ-r) were assessed at each visit as part of the parent PROMISE protocol. Wilcoxon sign rank tests were used to compare changes in outcomes at each time point. Results: A total of 22 participants enrolled and completed baseline assessments. Patients were 16-54 years of age (mean age 26 years), 68% were female, and 50% were not previously on CFTR modulators. V2 was completed by 20 participants and 17 participants completed all assessments through V3. Mean (± SD) BMI improved by 0.46 ± 0.93 kg/m² (P < 0.05) at V2 and 0.92 ± 0.88 kg/m² at V3 compared to V1 (P < 0.0001). REE% decreased by 6.6 ± 15.3 from V1 to V3 (P < 0.05). Total caloric intake increased by 297 ± 766 kcal/day (P < 0.05) and total fat intake increased by 19 ± 37 grams/day between V1 and V3 (P < 0.05). The average HEI for the cohort at baseline was 52.1 ± 10.7 and did not significantly change over the course of the study. There were no significant changes in HGS, PERT dosage, and intake of other macro and micro-nutrients, including fat-soluble vitamins. Conclusion: Elexacaftor/tezacaftor/ivacaftor improved BMI status rapidly, and this improvement was sustained through 6 or more months. Decreased energy expenditure combined with increased caloric intake are mechanisms of weight gain on elexacaftor/tezacaftor/ivacaftor. HEI in this CF cohort was similar to what is observed in adults in the general U.S. population. Diet quality did not improve with use of elexacaftor/tezacaftor/ ivacaftor therapy, despite increases in total caloric and fat intake. These findings highlight the need for individualized nutritional counseling to improve diet quality and manage weight changes on elexacaftor/tezacaftor/ ivacaftor in the clinical setting. Ongoing analyses are examining correlations between nutrition QOL domains (body image and eating disturbances) and changes in dietary intake on elexacaftor/tezacaftor/ivacaftor, as well as correlations between decreased REE% and lung function improvements.

Introduction: Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, albeit their limited translatability and predictivity for the in vivo situation. The feasibility of more sophisticated assays is often reduced by complex protocols and long lasting procedures. We aimed to establish and validate an easy-to-use but yet (patho-) physiologically relevant 3D cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Methods: We utilized the 3-lane OrganoPlate system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker, that facilitates continuous gravity-triggered bidirectional perfusion in all channels. Thereafter the cells form a leaktight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of C2 and formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells (WBCs) were isolated and seeded into C3 (figure A). Results: The leak-tightness of the 3D-tubule increased significantly over time, as measured by tracing the diffusion of a 150 kDa FITClabeled dextran from C1 to C2 (time-to-leakage day 1: 3.3 2.6 min; day 9: 36.2 10.7 min), indicating the stability of the co-culture system as well as a cellular maturation resulting in significant barrier functionality as seen in vivo (figure B). In accordance with this and other studies, the primary human tubular cells expressed higher levels of functionally relevant proteins in 3D than under 2D, no-flow conditions, as indicated by cell-number normalized mean fluorescence intensity measured by immunofluorescence, e..g ZO-1 (2.1 0.4 vs. 82.2 20.8) and Na-KATPase (2.3 0.3 vs. 52.8 5.4). Additionally, the growth conditions of the OrganoPlate rendered the cells more resilient to stimuli of acute tubular necrosis, e.g. extracellular histones, as compared to standard cell culture, indicated by cell number normalized lactate dehydrogenase release.The primary WBCs seeded inside the endothelial lumen (C3) did not leave the compartment under normal culture conditions, but displayed extravasation and directed migration from C3 through C2 towards C1 when attracted by chemokines released from tubular cells in C1. This effect was inhibitable by pre-emptive treatment of the endothelium with an selective monoclonal anti-P-selectin antibody (percent migrated cells, medium: 0 0, chemokines: 4.59 0.6, chemokines + Pselectin AB: 1.0 0.5, figure C). This serves as a proof of principle, that the system is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Conclusions: The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited for in depth mechanistic studies in vitro.