Micro-/bio-/nano-/syn-encapsulations and co-treatments of bioactive microbial feed supplementation in augmenting finfish health and aquaculture nutrition: a review.

Finfish and fish products are globally the most acknowledged health-promoting foods. The rising incidence of pathogenic and disease outbreaks have had a sizeable impact on aquaculture. Microbial supplementation of food in the form of probiotics, prebiotics, and their controlled release combinations (=co-encapsulations) as 'synbiotics' is noted for its significant biotherapeutic and health benefits. Supplementation of probiotic microbial feed additives in the fish diet claims to improve fish health by modulation of resident intestinal microbiota and by introducing healthy microbiota procured from an exogenous source, capable of combating pathogens, improving nutrient uptake, assimilation, growth as well as survival. Prebiotics are selectively digestible substrates beneficially used by host gut microbes to enhance probiotic effects. Formulating a fish diet with augmented probiotics and prebiotic microbial bio-supplements can ensure a sustainable alternative for establishing fish health in a naturally susceptible aquaculture scenario. Micro-encapsulation, co-encapsulation, and nano-encapsulation are novel strategies of biotechnical interventions in functional feeds for finfish. These aim to improve probiotic persistence, survivability, and efficacy in commercial formulations during probiotic transit through the host-gut environment. This review discusses the importance of co-treatment and encapsulation strategies for improving probiotic and prebiotic potential in aquafeed formulations, reliably improving finfish health and nutritional returns from aquaculture, and, consequently, for consumers.

Fibro-Adipogenic Progenitors: Versatile keepers of skeletal muscle homeostasis, beyond the response to myotrauma.

While Fibro-Adipogenic Progenitors (FAPs) have been originally identified as muscle-interstitial mesenchymal cells activated in response to muscle injury and endowed with inducible fibrogenic and adipogenic potential, subsequent studies have expanded their phenotypic and functional repertoire and revealed their contribution to skeletal muscle response to a vast range of perturbations. Here we review the emerging contribution of FAPs to skeletal muscle responses to motor neuron injuries and to systemic physiological (e.g., exercise) or pathological metabolic (e.g., diabetes) perturbations. We also provide an initial blueprint of discrete sub-clusters of FAPs that are activated by specific perturbations and discuss their role in muscle adaptation to these conditions.

The impact of reclassification of C3 predominant glomerulopathies on diagnostic accuracy, outcome and prognosis in patients with C3 glomerulonephritis.

BACKGROUND: C3 glomerulonephritis is a recently described entity with heterogeneous histopathological features. This study was conducted to assess the effect of reclassification of C3 glomerulopathies on renal outcomes, mortality, and response to therapy. METHODS: We undertook a retrospective analysis of 857 renal biopsies collected at The Canberra Hospital. Samples with predominant C3 staining were reviewed by a renal histopathologist. Of 31 biopsies with predominant C3 staining, 10 fulfilled histological criteria for C3 glomerulonephritis, while the remaining 21 cases were used as C3 Controls. RESULTS: Aside from a higher incidence of C3 glomerulonephritis in Torres Strait islanders (40% vs 5% C3 Controls, p = 0.04), presentation demographics were similar between the two groups. Median creatinine at diagnosis was higher in patients with C3 glomerulonephritis (253 umol/L IQR 103-333 vs 127 umol/L C3 Controls, IQR 105-182, p = 0.01). Prior to reclassification, a majority of C3 glomerulonephritis cases were diagnosed as membranoproliferative glomerulonephritis (60% vs 5% (C3 Controls) p < 0.01). Electron microscopy demonstrated all C3 glomerulonephritis patients had C3 deposition (100% vs 38% p = 0.02), these deposits were amorphous in nature (50% vs 5% respectively p = 0.007). C3 glomerulonephritis patients had shorter median follow-up (405 days IQR 203-1197 vs 1822 days respectively, IQR 1243-3948, p = 0.02). Mortality was higher in C3 glomerulonephritis patients (30% vs 14% in C3 Controls (log rank p = 0.02)). CONCLUSION: We have devised a diagnostic and treatment algorithm based on the results of literature review and our current study. Further prospective assessment is required to review diagnostic and treatment outcomes for this disease in Australian centres.

Genomic alterations driving psoriasis pathogenesis.

Psoriasis is an immune mediated inflammatory skin disease with complex etiology involving interplay between environmental and genetic risk factors as disease initiating event. Enhanced understanding on genetic risk factors, differentially expressed genes, deregulated proteins and pathway-targeted therapeutics have established multiple axis of psoriasis pathogenesis. So far, loci in 424 genes are reported to be associated with psoriasis alongside copy number variations and epigenetic alterations. From clinical perspective, presence of specific genetic trigger(s) in individual psoriasis patient could aid in devising a personalized therapeutic strategy. Therefore, the review presents an updates on reported genomic alterations and their subsequent course of cutaneous inflammations that potentially drive to psoriasis.

Inflammatory bowel disease in patients with Hirschsprung's disease: a systematic review and meta-analysis.

AIM AND OBJECTIVES: Hirschsprung-associated enterocolitis (HAEC) continues to be an important cause of morbidity in patients with Hirschsprung's disease (HSCR). HAEC can occur at any time during the course of the disease. The reported incidence of HAEC before surgery ranges from 6 to 50%, and after surgery, it ranges from 2 to 35%. HAEC and inflammatory bowel disease (IBD) have similar clinical presentation including diarrhea, hematochezia, and abdominal pain. In recent years, isolated cases of IBD have been reported in patients who had surgical treatment for HSCR. The exact pathogenesis of HAEC or IBD is not known. However, both conditions are characterized by an abnormal intestinal mucosal barrier function, which may be a common pathway. The purpose of this meta-analysis was to determine the clinical presentation and outcome in patients with HSCR who developed IBD after pull-through operation. MATERIALS AND METHODS: A systematic literature search for relevant articles was performed in four databases using the combinations of the following terms "inflammatory bowel disease", "Crohn/Crohn's disease", "ulcerative colitis", and "Hirschsprung disease/Hirschsprung's disease" for studies published between 1990 and 2017. The relevant cohorts of HSCR associated with IBD were systematically searched for clinical presentation and outcomes. RESULTS: 14 studies met defined inclusion criteria, reporting a total of 66 patients who had HSCR associated with IBD. Mean age at first operation for HSCR was 5.8 months, mean age at diagnosis of IBD was 7.7 years, and the majority of patients were male (73%). The extent of aganglionosis was total colonic aganglionosis in 41% of patients, long segment in 45%, and rectosigmoid in 14%. The majority of patients underwent a Duhamel procedure (84%) for HSCR. The distribution of IBD was Crohn's disease in 72.3% of patients, ulcerative colitis in 16.9%, and others in 10.8%. Eight articles (47 patients) reported about HAEC, and 22 patients (47%) had experienced HAEC after surgery for HSCR. CONCLUSION: Male patients with extensive colonic aganglionosis who continue to suffer from postoperative HAEC after a Duhamel procedure are more susceptible to develop IBD. Recognition of IBD may be important in the long-term follow-up of HSCR patients who have had postoperative HAEC.

Probiotics for the prevention of Hirschsprung-associated enterocolitis: a systematic review and meta-analysis.

BACKGROUND/PURPOSE: Hirschsprung-associated enterocolitis (HAEC) is a life-threatening complication of Hirschsprung's disease. HAEC is reported to occur in 6-50% of patients preoperatively and in 2-35% postoperatively. The exact cause of HAEC is not fully understood, but disturbances of intestinal microbiota have recently been reported in patients with HAEC. In recent years, the administration of probiotics has been proposed to reduce the incidence of HAEC. We conducted a systematic review and meta-analysis to determine the effect of probiotics on postoperative HAEC. METHODS: A systematic literature search for relevant articles was performed in four databases using the combinations of following terms "probiotics", "microbiota", "enterocolitis", "Lactobacillus", "Bifidobacterium", "Saccharomyces", "Streptococcus", and "Hirschsprung disease/Hirschsprung's disease" for studies published between 2002 and 2017. The relevant cohorts of the effect of probiotics in postoperative patients were systematically searched for clinical outcomes. Odds ratio (OR) or standard mean difference (SMD) with 95% confidence intervals (CI) were calculated using standardized statistical methodology. RESULTS: The search strategy identified 1274 reports. Overall, five studies met defined inclusion criteria, reporting a total of 198 patients. Two studies were prospective multicenter randomized control trials. Lactobacillus, Bifidobacterium, Streptococcus, and Enterococcus were used as probiotics. The incidence of HAEC with/without probiotics was 22.6 and 30.5%, respectively, but this was not statistically different (OR 0.72; 95% CI 0.37-1.39; P = 0.33). CONCLUSION: This study shows that the administration of probiotics was not associated with a significant reduction in the risk of HAEC. Additional studies are required to understand more fully the role of microbiota and complex interactions that cause HAEC. With increasing knowledge of the role of microbiota in HAEC, we are likely to understand better the potential benefits of probiotics in this disease.

Liver Function Tests Abnormalities and Hepatitis B Virus & Hepatitis C Virus Co-infection in Human Immunodeficiency Virus (HIV)-infected Patients in India.

BACKGROUND: While highly active anti-retroviral therapy (HAART) has improved survival of HIV-infected patients, there is increasing liver disease and progressive Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) related liver disease. AIMS: To study the liver function tests (LFT) and HBV and HCV co-infection in HIV-infected patients. MATERIAL AND METHODS: All HIV-positive patients presenting to a tertiary level hospital from April 2009 to April 2011 were evaluated. Baseline LFT, CD4/CD8 counts, ultrasound abdomen, HBsAg, IgG anti-HBc, HBVDNA, Anti-HCV and HCVRNA were done in all patients. LFT was repeated monthly or more frequently with anti-tubercular therapy (ATT)/HAART. RESULTS: Abnormal LFT were seen in 143/320 (44.6%) HIV-infected patients (n = 320; M-282, F-38; mean age-35.4 ± 7.3 years). Baseline LFT was abnormal in 48 (15%) [hepatotropic viruses-19, alcohol-24, NAFLD-1, disseminated TB-1, idiopathic-03). Subsequent LFT derangement developed in 95/272 (34.9%). In the majority, the LFT abnormality was mild (119/143-83.2%) and multi-factorial [HAART 132 (76.4%), alcohol 69 (48.2%), ATT 31 (21.7%), HBV 16 (11.2%), HCV 15 (10.4%)]. Using multivariate analysis, abnormal LFT were associated with HAART (OR, 5.92; 95%CI, 2.83-12.37), ATT (OR, 2.06; 95%CI, 1.06-3.99) or HCV infection (OR, 2.54; 95%CI, 1.03-6.26). Significant hepatotoxicity requiring drug modification was seen in only 7 cases. HBV, HCV and HBV + HCV co-infection were seen in 37 (11.6%), 28 (8.8%) and 2 (0.6%) respectively. Occult co-infections were rare [HBV-1 (0.3%); HCV-3 (0.9%)]. CONCLUSION: While LFT abnormalities in HIV are common, they are usually mild and multifactorial. HBV and HCV co-infections were seen in 11.6% and 8.8%, respectively. Occult HBV and HCV infections were rare.

Laparoscopic-assisted pull-through operation for Hirschsprung's disease: a systematic review and meta-analysis.

PURPOSE: In the last two decades, laparoscopic-assisted pull-through (LAPT) has gained much popularity in the treatment of Hirschsprung's disease. The aim of this meta-analysis was to determine the long-term outcome of patients treated laparoscopically. METHODS: A systematic literature-based search for relevant cohorts was performed using the terms "Hirschsprung's disease and Laparoscopy", "Laparoscopic-assisted pull-through outcome", "Laparoscopic-assisted Soave pull-through" "Laparoscopic-assisted Swenson pull-through" and Laparoscopic-assisted Duhamel pull-through. The relevant cohorts of laparoscopic operated HD were systematically searched for outcome regarding continence, constipation, secondary surgery related to the laparoscopic approach and enterocolitis. Pooled incidence rates and odds ratios (ORs) with 95 % confidence intervals (CI) were calculated using standardized statistical methodology. RESULTS: Sixteen studies met defined inclusion criteria, reporting a total of 820 patients. All studies were retrospective case series, with variability in outcome assessment quality and length of follow-up. The median cohort size consisted of 28 patients (range 15-218). In the long-term follow-up, 97 patients (11.14 %) experienced constipation (OR 0.06, 95 % CI 0.05-0.08, p < 0.00001), 53 (6.46 %) incontinence/soiling (OR 0.01 95 % CI 0.01-0.01, p < 0.00001), 75 (9.14 %) recurrent enterocolitis (OR 0.02 95 % CI 0.01-0.02, p < 0.00001) and 69 (8.4 %) developed complications requiring secondary surgery (OR 0.01 95 % CI 0.01-0.02, p < 0.00001). Overall events in long-term follow-up occurred in 225 (27.5 %) patients (OR 0.24 95 % CI 0.20-0.30, p < 0.00001). CONCLUSIONS: This meta-analysis shows that nearly one-third of the patients continue to have long-term bowel problems, such as constipation, soiling and recurrent enterocolitis following LAPT. Many patients treated by LAPT require secondary surgery. Large randomized studies with long-term follow-up are necessary to determine the difference in outcome between LAPT and completely transanal pull-through operation.

Autophagy regulates satellite cell ability to regenerate normal and dystrophic muscles.

Autophagy is emerging as a key regulatory process during skeletal muscle development, regeneration and homeostasis, and deregulated autophagy has been implicated in muscular disorders and age-related muscle decline. We have monitored autophagy in muscles of mdx mice and human Duchenne muscular dystrophy (DMD) patients at different stages of disease. Our data show that autophagy is activated during the early, compensatory regenerative stages of DMD. A progressive reduction was observed during mdx disease progression, in coincidence with the functional exhaustion of satellite cell-mediated regeneration and accumulation of fibrosis. Moreover, pharmacological manipulation of autophagy can influence disease progression in mdx mice. Of note, studies performed in regenerating muscles of wild-type mice revealed an essential role of autophagy in the activation of satellite cells upon muscle injury. These results support the notion that regeneration-associated autophagy contributes to the early compensatory stage of DMD progression, and interventions that extend activation of autophagy might be beneficial in the treatment of DMD. Thus, autophagy could be a 'disease modifier' targeted by interventions aimed to promote regeneration and delay disease progression in DMD.

Long-term results of transanal pull-through for Hirschsprung's disease: a meta-analysis.

INTRODUCTION: Several operative techniques have been developed for the treatment of Hirschsprung's disease (HD) in the past decades. Since one-stage transanal pull-through (TAPT) was first performed in 1998, multiple studies have shown favourable short-and midterm results compared to other techniques with shorter operation length, shorter hospital stay and lower complication rates. The aim of this meta-analysis was to determine the longterm results following TAPT for HD. METHODS: A systematic literature search for relevant articles was performed in four databases using the following terms "Hirschsprung/Hirschsprung's disease", "aganglionosis", "transanal", "pullthrough/pull-through", "longterm/long-term" "results", "follow-up" and "outcome". A meta-analysis was conducted for relevant articles for one-stage transanal pull-through for HD with a minimal follow-up of median 36 months regarding constipation, incontinence/soiling, enterocolitis and secondary operations. Odds ratio (OR) with 95 % confidence intervals (CI) were calculated. RESULTS: Six studies with 316 patients matched the set criteria and were included in this analysis. Overall 45 (14.2 %) patients had disturbances of bowel function (OR 0.05, 95 % CI 0.03-0.07, p < 0.00001). Of these, 24 (53.3 %) patients experienced constipation, 8 (17.8 %) incontinence/soiling and 13 (28.9 %) enterocolitis. 10 (3.2 %) patients developed complications requiring secondary surgery. Most patients had a daily defecation frequency of 1-3 bowel movements 3 years postoperatively, resembling the stooling patterns of healthy controls. CONCLUSION: Nearly 15 % of all patients operated with TAPT for HD continue to experience persistent bowel symptoms with constipation as the main problem. Further studies on the long-term outcome of children operated with this technique for HD are necessary to evaluate stooling patterns, urinary and sexual function as well as general quality of life during adolescence and adulthood.

RET gene is a major risk factor for Hirschsprung's disease: a meta-analysis.

PURPOSE: During the past two decades several genes have been identified that control morphogenesis and differentiation of the enteric neuron system (ENS). These genes, when mutated or deleted, interfere with ENS development. RET gene is the major gene causing Hirschsprung's disease (HD). Mutations in RET gene are responsible for 50% of familial HD cases and 15-20% of sporadic cases. The aim of this meta-analysis was to determine the incidence of RET gene mutations in patients with HD and to correlate RET mutations with the extent of aganglionosis. METHODS: A systematic literature-based search for relevant cohorts was performed using the terms "Hirschsprung's disease AND RET Proto-oncogene", "Hirschsprung's disease AND genetic polymorphism" and "RET Gene". The relevant cohorts of HD were systematically searched for reported mutations in the RET gene (RET+). Data on mutation site, phenotype, and familial or sporadic cases were extracted. Combined odds ratio (OR) with 95% CI was calculated to estimate the strength of the different associations. RESULTS: In total, 23 studies concerning RET with 1270 individuals affected with HD were included in this study. 228 (18%) of these HDs were RET+. Of these 228, 96 (42%) presented as rectosigmoid, 81 (36%) long segment, 18 (8%) as TCA, 16 (7%) as total intestinal aganglionosis and 17 (7%) individuals were RET+ but no extent of aganglionosis was not reported. In the rectosigmoid group, no significant association between phenotype and RET mutation could be shown (P = 0.006), whereas a clear association could be shown between long-segment disease, total colonic- and total intestinal aganglionosis and RET mutations (P = 0.0002). Mutations most often occurred in Exon 13 (24) and showed significant association with rectosigmoid disease (P = 0.004). No significance could be shown between RET+ and sporadic cases (P = 0.53), albeit a trend towards RET+ and Familial cases could be observed (P = 0.38). CONCLUSIONS: The association with the RET gene and HD is well recognized. This study showed a clear association between RET+ mutations and the long-segment, total colonic- and total intestinal aganglionosis. Exon 13 appears to be a mutational "hot spot" in rectosigmoid disease.

Interactions between muscle stem cells, mesenchymal-derived cells and immune cells in muscle homeostasis, regeneration and disease.

Recent evidence has revealed the importance of reciprocal functional interactions between different types of mononuclear cells in coordinating the repair of injured muscles. In particular, signals released from the inflammatory infiltrate and from mesenchymal interstitial cells (also known as fibro-adipogenic progenitors (FAPs)) appear to instruct muscle stem cells (satellite cells) to break quiescence, proliferate and differentiate. Interestingly, conditions that compromise the functional integrity of this network can bias muscle repair toward pathological outcomes that are typically observed in chronic muscular disorders, that is, fibrotic and fatty muscle degeneration as well as myofiber atrophy. In this review, we will summarize the current knowledge on the regulation of this network in physiological and pathological conditions, and anticipate the potential contribution of its cellular components to relatively unexplored conditions, such as aging and physical exercise.

Knockout mouse models of Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system, which occurs due to the failure of neural crest cell migration. Rodent animal models of aganglionosis have contributed greatly to our understanding of the genetic basis of HSCR. Several natural or target mutations in specific genes have been reported to produce developmental defects in neural crest migration, differentiation or survival. The aim of this study was to review the currently available knockout models of HSCR to better understand the molecular basis of HSCR. METHODS: A review of the literature using the keywords "Hirschsprung's disease", "aganglionosis", "megacolon" and "knockout mice model" was performed. Resulting publications were reviewed for relevant mouse models of human aganglionosis. Reference lists were screened for additional relevant studies. RESULTS: 16 gene knockout mouse models were identified as relevant rodent models of human HSCR. Due to the deletion of a specific gene, the phenotypes of these knockout models are diverse and range from small bowel dilatation and muscular hypertrophy to total intestinal aganglionosis. CONCLUSIONS: Mouse models of aganglionosis have been instrumental in the discovery of the causative genes of HSCR. Although important advances have been made in understanding the genetic basis of HSCR, animal models of aganglionosis in future should further help to identify the unknown susceptibility genes in HSCR.

The impact of Clostridium difficile on paediatric surgical practice: a systematic review.

PURPOSE: The pathogenic potential of Clostridium difficile in children remains a controversial subject as healthy infants can be colonised by this organism. However recent analyses have clarified that C. difficile is an important enteropath in paediatric populations, particularly in antibiotic-associated diarrhoea. Paediatric surgical patients including those with Hirschsprung's disease (HD) may be especially vulnerable to C. difficile infection (CDI) and complicated C. difficile enterocolitis such as pseudomembranous colitis may require surgical management if refractory to medical therapy. Reports of increasing prevalence and emergence of hyper-virulent strains of C. difficile worldwide prompted an examination of the literature to assess the impact of CDI on current paediatric surgical practise. METHODS: The literature was searched using a combination of the MESH terms "hirschsprung's disease", "enterocolitis", "clostridium difficile", and "children". Cases of Hirschsprung's associated enterocolitis (HAEC) investigated for C. difficile and complicated CDI in non HD patients were identified and analysed for clinical parameters, diagnostic evaluations, surgical interventions and outcome. RESULTS: Pathogen isolation in HAEC was infrequently described. Only 98 children have been reported with C. difficile during an episode of HAEC over the last 40 years and aetiology remains unclear as asymptomatic carriage of C. difficile in HD occurs. Nonetheless 34 confirmed cases of pseudomembranous colitis complicating HD are reported in the literature with an associated 50 % mortality rate. Over 20 % of non Hirschsprung's patients with reported severe or complicated CDI required operative intervention. The need for surgery was associated with the presence of co-morbidity and high mortality occurred in this group. CONCLUSION: Severe or complicated CDI in both HD and non HD paediatric patients is associated with high mortality and often requires surgical intervention. Although these patient cohorts represent a small number of cases, CDI should be suspected in children presenting with enterocolitis to enable early diagnosis and timely surgical intervention, particularly in patients with co-morbid conditions or preceding antibiotic use.

Randomised clinical trial: Lactobacillus GG modulates gut microbiome, metabolome and endotoxemia in patients with cirrhosis.

BACKGROUND: Safety of individual probiotic strains approved under Investigational New Drug (IND) policies in cirrhosis with minimal hepatic encephalopathy (MHE) is not clear. AIM: The primary aim of this phase I study was to evaluate the safety, tolerability of probiotic Lactobacillus GG (LGG) compared to placebo, while secondary ones were to explore its mechanism of action using cognitive, microbiome, metabolome and endotoxin analysis in MHE patients. METHODS: Cirrhotic patients with MHE patients were randomised 1:1 into LGG or placebo BID after being prescribed a standard diet and multi-vitamin regimen and were followed up for 8 weeks. Serum, urine and stool samples were collected at baseline and study end. Safety was assessed at Weeks 4 and 8. Endotoxin and systemic inflammation, microbiome using multi-tagged pyrosequencing, serum/urine metabolome were analysed between groups using correlation networks. RESULTS: Thirty MHE patients (14 LGG and 16 placebo) completed the study without any differences in serious adverse events. However, self-limited diarrhoea was more frequent in LGG patients. A standard diet was maintained and LGG batches were comparable throughout. Only in the LGG-randomised group, endotoxemia and TNF-α decreased, microbiome changed (reduced Enterobacteriaceae and increased Clostridiales Incertae Sedis XIV and Lachnospiraceae relative abundance) with changes in metabolite/microbiome correlations pertaining to amino acid, vitamin and secondary BA metabolism. No change in cognition was found. CONCLUSIONS: In this phase I study, Lactobacillus GG is safe and well-tolerated in cirrhosis and is associated with a reduction in endotoxemia and dysbiosis.

A new genome scan for primary nonsyndromic vesicoureteric reflux emphasizes high genetic heterogeneity and shows linkage and association with various genes already implicated in urinary tract development.

Primary vesicoureteric reflux (VUR), the retrograde flow of urine from the bladder toward the kidneys, results from a developmental anomaly of the vesicoureteric valve mechanism, and is often associated with other urinary tract anomalies. It is the most common urological problem in children, with an estimated prevalence of 1-2%, and is a major cause of hypertension in childhood and of renal failure in childhood or adult life. We present the results of a genetic linkage and association scan using 900,000 markers. Our linkage results show a large number of suggestive linkage peaks, with different results in two groups of families, suggesting that VUR is even more genetically heterogeneous than previously imagined. The only marker achieving P < 0.02 for linkage in both groups of families is 270 kb from EMX2. In three sibships, we found recessive linkage to KHDRBS3, previously reported in a Somali family. In another family we discovered sex-reversal associated with VUR, implicating PRKX, for which there was weak support for dominant linkage in the overall data set. Several other candidate genes are suggested by our linkage or association results, and four of our linkage peaks are within copy-number variants recently found to be associated with renal hypodysplasia. Undoubtedly there are many genes related to VUR. Our study gives support to some loci suggested by earlier studies as well as suggesting new ones, and provides numerous indications for further investigations.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.