The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn's Disease.

BACKGROUND & AIMS: The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. METHODS: We performed single-cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. RESULTS: We identified 5 distinct FOXP3+ regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor-α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. CONCLUSIONS: We identified a novel, proinflammatory FOXP3+ T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration-approved drug vorinostat.

G9a Modulates Lipid Metabolism in CD4 T Cells to Regulate Intestinal Inflammation.

BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Microrchidia 2/histone deacetylase 1 complex regulates E-cadherin gene expression and function.

Although Microrchidia 2 (MORC2) is widely overexpressed in human malignancies and linked to cancer cell proliferation, metabolism, and metastasis, the mechanism of action of MORC2 in cancer cell migration and invasion is yet undeciphered. Here, we identified for the first time that MORC2, a chromatin remodeler, regulates E-cadherin expression and, subsequently regulates breast cancer cell migration and invasion. We observed a negative correlation between the expression levels of MORC2 and E-cadherin in breast cancer. Furthermore, the overexpression of MORC2 resulted in decreased expression levels of E-cadherin. In addition, co-immunoprecipitation and chromatin immunoprecipitation assays revealed that MORC2 interacts with HDAC1 and gets recruited onto the E-cadherin promoter to inhibit its transcription, thereby suppress its expression. Consequently, knockdown of HDAC1 in MORC2-overexpressing cells led to reduced cancer cell migration and invasion. Interestingly, we noticed that MORC2-regulated glucose metabolism via c-Myc, and LDHA, also modulates the expression of E-cadherin. Collectively, these results demonstrate for the first time a mechanistic role for MORC2 as an upstream regulator of E-cadherin expression and its associated functions in breast cancer.

Comparison of biochemical, microbial and mucosal mRNA expression in bile acid diarrhoea and irritable bowel syndrome with diarrhoea.

OBJECTIVE: There are altered mucosal functions in irritable bowel syndrome with diarrhoea (IBS-D); ~30% of patients with IBS-D have abnormal bile acid (BA) metabolism (ABAM) and diarrhoea (summarised as BAD). AIM: To compare biochemical parameters, gastrointestinal and colonic transit, rectal sensation and pathobiological mechanisms in IBS-D without ABAM and in BAD (serum 7C4>52 ng/mL). DESIGN: In patients with Rome III criteria of IBS-D, we compared biochemical features, colonic transit, rectal sensation, deep genotype of five BA-related genes, ileal and colonic mucosal mRNA (differential expression (DE) analysis) and stool dysbiosis (including functional analysis of microbiome). Results in BAD were compared with IBS-D without ABAM. RESULTS: Compared with 161 patients with IBS-D without ABAM, 44 patients with BAD had significantly faster colonic transit, lower microbial alpha diversity, different compositional profile (beta diversity) and higher Firmicutes to Bacteroidetes ratio with evidence of decreased expression of bile acid thiol ligase (involved in transformation of primary to secondary BAs) and decreased sulfatases. In BAD (compared with IBS-D without ABAM), terminal ileal biopsies showed downregulation of SLC44A5 (a BA transporter), and ascending colon biopsies showed upregulation in barrier-weakening genes (CLDN2), serine protease inhibitors, immune activation, cellular differentiation and a cellular transporter (FABP6; BA binding). No DE of genes was documented in descending colon biopsies. The two groups had similar rectal sensation. CONCLUSION: Though sharing clinical symptoms with IBS-D, BAD is associated with biological differences and mechanisms that have potential to enhance diagnosis and treatment targeting barrier dysfunction, inflammatory and microbial changes.

Zika virus alters osteogenic lineage progression of human mesenchymal stromal cells.

Arboviruses target bone forming osteoblasts and perturb bone remodeling via paracrine factors. We previously reported that Zika virus (ZIKV) infection of early-stage human mesenchymal stromal cells (MSCs) inhibited the osteogenic lineage commitment of MSCs. To understand the physiological interplay between bone development and ZIKV pathogenesis, we employed a primary in vitro model to examine the biological responses of MSCs to ZIKV infection at different stages of osteogenesis. Precommitted MSCs were infected at the late stage of osteogenic stimulation (Day 7) with ZIKV (multiplicity of infection of 5). We observe that MSCs infected at the late stage of differentiation are highly susceptible to ZIKV infection similar to previous observations with early stage infected MSCs (Day 0). However, in contrast to ZIKV infection at the early stage of differentiation, infection at a later stage significantly elevates the key osteogenic markers and calcium content. Comparative RNA sequencing (RNA-seq) of early and late stage infected MSCs reveals that ZIKV infection alters the mRNA transcriptome during osteogenic induction of MSCs (1251 genes). ZIKV infection provokes a robust antiviral response at both stages of osteogenic differentiation as reflected by the upregulation of interferon responsive genes (n > 140). ZIKV infection enhances the expression of immune-related genes in early stage MSCs while increasing cell cycle genes in late stage MSCs. Remarkably, ZIKA infection in early stage MSCs also activates lipid metabolism-related pathways. In conclusion, ZIKV infection has differentiation stage-dependent effects on MSCs and this mechanistic understanding may permit the development of new therapeutic or preventative measures for bone-related effects of ZIKV infection.

Lipoblastoma-Like Tumor and Fibrosarcoma-Like Lipomatous Neoplasm Represent the Same Entity: A Clinicopathologic and Molecular Genetic Study of 23 Cases Occurring in Both Men and Women at Diverse Locations.

Lipoblastoma-like tumor (LLT) is a benign soft tissue tumor demonstrating mixed morphologic features of lipoblastoma, myxoid liposarcoma, and spindle cell lipoma but lacking genetic alterations associated with those tumors. LLT was originally thought to be specific to the vulva but has since been reported in the paratesticular region. The morphologic features of LLT overlap with those of "fibrosarcoma-like lipomatous neoplasm" (FLLN), a rare, indolent adipocytic neoplasm considered by some to form part of the spectrum of atypical spindle cell and pleomorphic lipomatous tumor. We compared the morphologic, immunohistochemical, and genetic features of 23 tumors previously classified as LLT (n = 17) and FLLN (n = 6). The 23 tumors occurred in 13 women and 10 men (mean age, 42 years; range, 17 to 80 years). Eighteen (78%) cases arose in the inguinogenital region, whereas 5 tumors (22%) involved noninguinogenital soft tissue, including the flank (n = 1), shoulder (n = 1), foot (n = 1), forearm (n = 1), and chest wall (n = 1). Microscopically, the tumors were lobulated and septated, with variably collagenized fibromyxoid stroma, prominent thin-walled vessels, scattered univacuolated or bivacuolated lipoblasts, and a minor component of mature adipose tissue. Using immunohistochemistry, 5 tumors (42%) showed complete RB1 loss, with partial loss in 7 cases (58%). RNA sequencing, chromosomal microarray, and DNA next-generation sequencing study results were negative for significant alterations. There were no clinical, morphologic, immunohistochemical, or molecular genetic differences between cases previously classified as LLT or FLLN. Clinical follow-up (11 patients [48%]; range, 2-276 months; mean, 48.2 months) showed all patients were alive without disease, and only one patient had experienced a single local recurrence. We conclude that LLT and FLLN represent the same entity, for which "LLT" seems most appropriate. LLT may occur in either sex and any superficial soft tissue location. Careful morphologic study and appropriate ancillary testing should allow for the distinction of LLT from its potential mimics.

An Unusual Presentation of Superior Mesenteric Venous Occlusion in Mild COVID-19.

SARS-CoV-2, an etiological agent of COVID-19, has been reported to inflict remarkably diverse manifestations in different subjects across the globe. Though patients with COVID-19 predominantly have fever, respiratory and constitutional symptoms, atypical presentations are becoming increasingly evident. COVID-19 may predispose to both venous and arterial thromboembolism due to excessive inflammation, hypoxia, immobilization, and diffuse intravascular coagulation in moderate to severe symptomatic cases. In this case report, we are reporting thromboembolic complications of COVID-19 in a mild symptomatic subject incidentally diagnosed with mesenteric venous occlusion with no abdominal symptoms. Early recognition of the abdominal symptoms, diagnosis, initiation of anticoagulants, and timely surgical intervention may improvise the outcome in a patient with COVID-19 infection-induced mesenteric thrombosis. Superior mesenteric artery and venous thrombosis may lead to subsequent ischemia necessitating emergency laparotomy. Thus, the usage of low-dose anticoagulants in all the patients of COVID-19 irrespective of the categorization into mild, moderate, and severe COVID-19 disease should be considered.

Differential mRNA expression in ileal and colonic biopsies in irritable bowel syndrome with diarrhea or constipation.

Altered mucosal functions are documented in jejunal or colorectal mucosa from patients with irritable bowel syndrome (IBS). Our aim was to quantify ileal, ascending, and rectosigmoid colon mucosal expression of genes in IBS-diarrhea (D) and IBS-constipation (C). Forty-four patients with IBS-D, 30 with IBS-C, and 30 healthy volunteers underwent colonoscopic ileal, ascending, and rectosigmoid colon biopsies. Biopsies were stored in RNAlater at -80 °C, purified with on-column DNase, cDNA libraries prepared from 100-200 ng of total RNA, sequenced on Illumina NovaSeq 6000, and analyzed on Illumina's RTA version 3.4.4. Normalized mRNA expression was obtained using MAP-RSeq bioinformatics pipeline. Differential expressions in the groups (Log2-fold change) were measured using the bioinformatics package edgeR 2.6.2, corrected for false discovery rate (PADJ <0.05). There were 30 females with IBS-C and 31 females and 13 males with IBS-D. In IBS-D and IBS-C groups, there were differential expressions of 181 genes in ascending colon and 199 genes in rectosigmoid colon. The majority were gene upregulations in IBS-D with functions reflecting activation of inflammation genes, TRPV1 (visceral hypersensitivity) and neurotransmitters/receptors (specifically purinergic, GABA, and cannabinoid). Although gene differential expressions in the ascending and rectosigmoid colon mucosa of the two groups were different, the diverse upregulated genes involved immune functions, receptors, transmitters, ion channels, and transporters. Conversely, there was reduced expression of PI15 and PI16 genes that inhibit proteases. In patients with IBS-D and IBS-C, differential expressions of genes related to immune, transmitter, nociceptive, protease inhibition, channel, and transporter functions suggest opportunities to reverse the pathobiology and treat patients with IBS.NEW & NOTEWORTHY This study compares gene expression in mucosa of the terminal ileum, right colon, and left colon in patients with diarrhea- or constipation-predominant irritable bowel syndrome (IBS) and contrasts expression between these two disease entities and also between each entity and mucosa from healthy controls. The study shows there is differential expression of genes related to immune, transmitter, nociceptive, ion channel, and transporter functions, as well as reduced serine protease inhibition, in patients with IBS.

EWSR1-WT1 gene fusions in neoplasms other than desmoplastic small round cell tumor: a report of three unusual tumors involving the female genital tract and review of the literature.

Desmoplastic small round cell tumor (DSRCT) is a high-grade round cell sarcoma that typically arises in the abdominopelvic cavity of young males, co-expresses keratins and desmin, and carries a pathognomonic EWSR1-WT1 gene fusion. The EWSR1-WT1 gene fusion is generally considered specific for DSRCT, although there are two reports of this fusion in tumors otherwise lacking features of DSRCT. We report three female genital tract tumors with EWSR1-WT1 fusions but showing morphologic and immunohistochemical features incompatible with DSRCT. The tumors occurred in the uterine cervix, uterine corpus/ovaries, and vagina, respectively, of 46, 30, and 20-year-old women. Two tumors consisted of a sheet-like to fascicular proliferation of relatively uniform spindled to occasionally more epithelioid cells arrayed about thick-walled, hyalinized, and capillary-sized vessels, with distinctive areas of pseudovascular change, and absence of desmoplastic stroma. The third tumor resembled a monomorphic spindle cell sarcoma with necrosis. All had diffuse desmin and variable but more limited keratin expression, two of three expressed smooth muscle actin, and all were negative for h-caldesmon, CD10, estrogen receptor, myogenin, N-terminus WT-1, and S100 protein. One patient received neoadjuvant chemotherapy and radiation therapy followed by resection and is disease-free 42 months after diagnosis. Another patient was managed by resection only and is disease-free 9 months after initial diagnosis. The remaining patient recently underwent resection of multifocal pelvic disease. Comprehensive differential gene expression analysis on two tumors compared to two classic DSRCTs with known EWSR1-WT1 fusions resulted in 1726 genes that were differentially expressed (log2 fold change >2 or < -2) and statistically significant (FDR < 5%). In combination with previous reports, our findings suggest pleiotropy of the EWSR1-WT1 fusion is possible and not limited to DSRCT. Subsets of non-DSRCT EWSR1-WT1 positive tumors may represent discrete entities, but further study is necessary.

Effects of Colesevelam on Bowel Symptoms, Biomarkers, and Colonic Mucosal Gene Expression in Patients With Bile Acid Diarrhea in a Randomized Trial.

BACKGROUND & AIMS: Approximately one-third of patients with IBS-diarrhea (IBS-D) have increased bile acid (BA) synthesis or excretion. An open-label study showed benefits of colesevelam on bowel functions, consistent with luminal BA sequestration by colesevelam. We compared the effects of colesevelam vs placebo on symptoms and gene expression patterns in the sigmoid colon mucosa in patients with BA diarrhea associated with IBS-D. METHODS: We performed a double-blind, parallel-group study of 30 adults with IBS-D and evidence of increased BA synthesis or fecal excretion, from December 2017 through December 2018 at a single center. Patients were randomly assigned (1:1) to groups given colesevelam (3 tablets, 625 mg each) or matching placebo, orally twice daily for 4 weeks. Stool diaries documented bowel functions for 8 days before and 28 days during colesevelam or placebo. Stool and fasting serum samples were collected for analyses of fecal BAs and serum levels of C4 and FGF19. We measured colonic transit by scintigraphy, mucosal permeability by in vivo excretion of saccharide probes, and mRNA levels in rectosigmoid biopsies. All measurements were made at baseline and on the last days of treatment. The primary endpoints were change in total fecal BA concentration and stool consistency. RESULTS: Compared with placebo, colesevelam was associated with significant changes in sequestered fecal total BA excretion (P < .001) and serum levels of C4 and FGF19 (both P < .001), and with a mean increase in fecal level of deoxycholic acid (10%; P = .07) compared to placebo. Colesevelam decreased colon mucosal expression of NR1H4 and P2RY4 and increased expression of GPBAR1, compared with baseline. Stool frequency and consistency, colonic transit, and permeability did not differ significantly between groups. Colesevelam was well tolerated. CONCLUSIONS: In a randomized trial, we found that colesevelam increases delivery of total and secondary BAs to stool, hepatic BA synthesis, and colonic mucosal expression of genes that regulate BA, farnesoid X, and GPBAR1 receptors. Larger studies are needed to determine the effects on clinical responses. ClinicalTrials.gov no: NCT03270085.

Gene Expression Signatures Characterized by Longitudinal Stability and Interindividual Variability Delineate Baseline Phenotypic Groups with Distinct Responses to Immune Stimulation.

Human immunity exhibits remarkable heterogeneity among individuals, which engenders variable responses to immune perturbations in human populations. Population studies reveal that, in addition to interindividual heterogeneity, systemic immune signatures display longitudinal stability within individuals, and these signatures may reliably dictate how given individuals respond to immune perturbations. We hypothesize that analyzing relationships among these signatures at the population level may uncover baseline immune phenotypes that correspond with response outcomes to immune stimuli. To test this, we quantified global gene expression in peripheral blood CD4+ cells from healthy individuals at baseline and following CD3/CD28 stimulation at two time points 1 mo apart. Systemic CD4+ cell baseline and poststimulation molecular immune response signatures (MIRS) were defined by identifying genes expressed at levels that were stable between time points within individuals and differential among individuals in each state. Iterative differential gene expression analyses between all possible phenotypic groupings of at least three individuals using the baseline and stimulated MIRS gene sets revealed shared baseline and response phenotypic groupings, indicating the baseline MIRS contained determinants of immune responsiveness. Furthermore, significant numbers of shared phenotype-defining sets of determinants were identified in baseline data across independent healthy cohorts. Combining the cohorts and repeating the analyses resulted in identification of over 6000 baseline immune phenotypic groups, implying that the MIRS concept may be useful in many immune perturbation contexts. These findings demonstrate that patterns in complex gene expression variability can be used to define immune phenotypes and discover determinants of immune responsiveness.

Extracorporeal Membrane Oxygenation Support After Heart Transplantation in Children-Outcomes of a Single Center Cohort.

OBJECTIVES: Extracorporeal membrane oxygenation is used for postcardiotomy low cardiac output but is less established following heart transplantation. We characterized outcomes for children supported with extracorporeal membrane oxygenation after heart transplantation. DESIGN: Single-center retrospective study. SETTING: Large pediatric cardiac referral center. PATIENTS: All patients who received heart transplantation and were cannulated to extracorporeal membrane oxygenation between 1995 and 2016. INTERVENTIONS: Primary outcome measure was mortality 12 months postextracorporeal membrane oxygenation. Patient characteristics were analyzed for association with outcome according to early graft failure (extracorporeal membrane oxygenation ≤ 7 d after heart transplantation), or late graft failure. MEASUREMENTS AND MAIN RESULTS: There were 246 heart transplants during the study period and 50 extracorporeal membrane oxygenation runs in 44 patients. Median time from transplant to extracorporeal membrane oxygenation was 1 day (range, 0-11.7 yr), with early graft failure in 28 patients (median 1, range 0-2 d) and 22 extracorporeal membrane oxygenation runs in 20 late graft failure patients (median, 0.8 yr; range, 8 d to 11.7 yr), including four patients with prior extracorporeal membrane oxygenation for early graft failure. Twenty-six patients (59%) survived to hospital discharge, and survival 12 months postextracorporeal membrane oxygenation was 24 patients (55%), lower in those with late graft failure (40% vs 67%; p 0.02). Independent risk factors for 12-month mortality were congenital heart disease, higher pulmonary vascular resistance indexed to body surface area (> 2.2 Woods U/m), and higher creatinine. Higher panel reactive antibody levels were associated with 12-month mortality in the late graft failure group only. CONCLUSIONS: Extracorporeal membrane oxygenation can be effectively used to rescue patients with graft dysfunction after heart transplantation but is associated with high early mortality. Factors associated with mortality within 12 months include presence of congenital heart disease, renal dysfunction, elevated pulmonary vascular resistance indexed to body surface area and in those supported with extracorporeal membrane oxygenation late after heart transplantation, significant human leukocyte antigen sensitization.

RNA sequencing identifies a novel USP9X-USP6 promoter swap gene fusion in a primary aneurysmal bone cyst.

Primary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full-length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell-rich primary bone tumors. In this report, we present a case of a 16-year-old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X-USP6 promoter swap fusion. This result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin-specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate-specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.

The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD).

Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a pivotal role in maintaining immunologic self-tolerance. We and others have shown previously that EZH2 is recruited to the FOXP3 promoter and its targets in Treg cells. To further address the role for EZH2 in Treg cellular function, we have now generated mice that lack EZH2 specifically in Treg cells (EZH2Δ/ΔFOXP3+). We find that EZH2 deficiency in FOXP3+ T cells results in lethal multiorgan autoimmunity. We further demonstrate that EZH2Δ/ΔFOXP3+ T cells lack a regulatory phenotype in vitro and secrete proinflammatory cytokines. Of special interest, EZH2Δ/ΔFOXP3+ mice develop spontaneous inflammatory bowel disease. Guided by these results, we assessed the FOXP3 and EZH2 gene networks by RNA sequencing in isolated intestinal CD4+ T cells from patients with Crohn's disease. Gene network analysis demonstrates that these CD4+ T cells display a Th1/Th17-like phenotype with an enrichment of gene targets shared by FOXP3 and EZH2. Combined, these results suggest that the inflammatory milieu found in Crohn's disease could lead to or result from deregulation of FOXP3/EZH2-enforced T cell gene networks contributing to the underlying intestinal inflammation.

Thiostrepton degrades mutant p53 by eliciting an autophagic response in SW480 cells.

Mutations in p53 gene are one of the hallmarks of tumor development. Specific targeting of mutant p53 protein has a promising role in cancer therapeutics. Our preliminary observation showed destabilization of mutant p53 protein in SW480, MiaPaCa and MDAMB231 cell lines upon thiostrepton treatment. In order to elucidate the mechanism of thiostrepton triggered mutant p53 degradation, we explored the impact of proteasome inhibition on activation of autophagy. Combined treatment of thiostrepton and cycloheximide/chloroquine prevented the degradation of mutant p53 protein, reinforcing autophagy as the means of mutant p53 destabilization. Our initial studies suggested that mutant p53 degradation post THSP treatment was carried out by BAG3 mediated autophagy, based on the evidence of BAG1 to BAG3 switching. Subsequent interactome analysis performed post thiostrepton treatment revealed an association of p53 with autophagosome complex associated proteins such as BAG3, p62 and HSC70. Reaccumulation of p53 was seen in BAG3 silenced cells treated with thiostrepton, thereby confirming the role of BAG3 in destabilization of this molecule. Further, localization of p53 into the lysosome upon THSP treatment substantiated our findings that mutant p53 was degraded by an autopahgic process.

Comprehensively evaluating cis-regulatory variation in the human prostate transcriptome by using gene-level allele-specific expression.

The identification of cis-acting regulatory variation in primary tissues has the potential to elucidate the genetic basis of complex traits and further our understanding of transcriptomic diversity across cell types. Expression quantitative trait locus (eQTL) association analysis using RNA sequencing (RNA-seq) data can improve upon the detection of cis-acting regulatory variation by leveraging allele-specific expression (ASE) patterns in association analysis. Here, we present a comprehensive evaluation of cis-acting eQTLs by analyzing RNA-seq gene-expression data and genome-wide high-density genotypes from 471 samples of normal primary prostate tissue. Using statistical models that integrate ASE information, we identified extensive cis-eQTLs across the prostate transcriptome and found that approximately 70% of expressed genes corresponded to a significant eQTL at a gene-level false-discovery rate of 0.05. Overall, cis-eQTLs were heavily concentrated near the transcription start and stop sites of affected genes, and effects were negatively correlated with distance. We identified multiple instances of cis-acting co-regulation by using phased genotype data and discovered 233 SNPs as the most strongly associated eQTLs for more than one gene. We also noted significant enrichment (25/50, p = 2E-5) of previously reported prostate cancer risk SNPs in prostate eQTLs. Our results illustrate the benefit of assessing ASE data in cis-eQTL analyses by showing better reproducibility of prior eQTL findings than of eQTL mapping based on total expression alone. Altogether, our analysis provides extensive functional context of thousands of SNPs in prostate tissue, and these results will be of critical value in guiding studies examining disease of the human prostate.

Autologous Mesenchymal Stem Cells, Applied in a Bioabsorbable Matrix, for Treatment of Perianal Fistulas in Patients With Crohn's Disease.

In patients with Crohn's disease, perianal fistulas recur frequently, causing substantial morbidity. We performed a 12-patient, 6-month, phase 1 trial to determine whether autologous mesenchymal stem cells, applied in a bioabsorbable matrix, can heal the fistula. Fistula repair was not associated with any serious adverse events related to mesenchymal stem cells or plug placement. At 6 months, 10 of 12 patients (83%) had complete clinical healing and radiographic markers of response. We found placement of mesenchymal stem cell-coated matrix fistula plugs in 12 patients with chronic perianal fistulas to be safe and lead to clinical healing and radiographic response in 10 patients. ClinicalTrials.gov Identifier: NCT01915927.

Spindle cell rhabdomyosarcoma of bone with FUS-TFCP2 fusion: confirmation of a very recently described rhabdomyosarcoma subtype.

AIMS: Rhabdomyosarcomas of bone are extremely rare, with fewer than 10 reported cases. A very rare subtype of spindle cell/sclerosing rhabdomyosarcoma harbouring a FUS-TFCP2 fusion and involving both soft tissue and bone locations has been reported very recently. We report only the fourth case of this unusual, clinically aggressive rhabdomyosarcoma. MATERIAL AND RESULTS: A previously well 72-year-old male presented with a destructive lesion of the mandible. Morphological and immunohistochemical study of a needle biopsy and the subsequent resection showed a spindle cell rhabdomyosarcoma. RNA-seq, RT-PCR and FISH confirmed the presence of the FUS-TFCP2 fusion. CONCLUSIONS: Spindle cell rhabdomyosarcomas carrying the FUS-TFCP2 fusion are very rare rhabdomyosarcoma variants with osseous predilection. The classification and differential diagnosis of this unusual molecular variant of spindle cell/sclerosing rhabdomyosarcoma are discussed.

Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression.

BACKGROUND: Smurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. METHODS: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation. RESULTS: CNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines. CONCLUSIONS: Altogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.