Emerging roles of gut virome in pediatric diseases

In the last decade, the widespread application of shotgun metagenomics provided extensive characterization of the bacterial “dark matter” of the gut microbiome, propelling the development of dedicated, standardized bioinformatic pipelines and the systematic collection of metagenomic data into comprehensive databases. The advent of next-generation sequencing also unravels a previously underestimated viral population (virome) present in the human gut. Despite extensive efforts to characterize the human gut virome, to date, little is known about the childhood gut virome. However, alterations of the gut virome in children have been linked to pathological conditions such as inflammatory bowel disease, type 1 diabetes, malnutrition, diarrhea and celiac diseas

Role of age in dynamics of autoantibodies in pediatric celiac disease

Background. Celiac disease (CD) is characterized by elevated serum titers of autoantibodies IgA anti-tissue transglutaminase 2 (TGA-IgA) and IgA anti-endomysial (EMA), with small bowel mucosa atrophy. We evaluated age differences between CD children exhibiting variable antibody titers at diagnosis.MethodsCD children diagnosed between January 2014 and June 2019, according to 2012 ESPGHAN guidelines were studied. All had EMA and TGA-IgA measurements, while a proportion of them underwent esophagogastroduodenoscopy (EGD). Patients were grouped based on serum TGA-IgA titers normalized to the upper limit of normal (ULN) and differences in median age (years) assessed by analysis of variance (ANOVA) and creation of orthogonal contrasts.ResultsCD was diagnosed in 295 subjects (median age: 4.4 [IQR: 2.60-8.52]) with a biopsy sparing protocol (high titer: >= 10xULN) and in 204 by EGD biopsy. Of the latter, 142 (median age: 8.5 [IQR: 5.81-11.06]) and 62 (median age: 9.5 [IQR: 6.26-12.76]) had a low (< 5xULN) and a moderate (>= 5 < 10xULN) TGA-IgA titer, respectively. Potential CD was diagnosed in 20 patients (median age: 3.6 [IQR: 2.47-6.91]). The median age was significantly lower in the no-biopsy group (ANOVA: F-(3,F- 516) = 25.98, p < .001) than in low- and moderate titer groups (p < 0.0001), while there was no statistical difference between biopsy-sparing and potential CD groups.ConclusionCD patients with greatly elevated antibody titers (>= 10xULN) were diagnosed at an earlier age than those with lower titers. This may indicate that an increase in TGA-IgA is independent of age and suggests a polarization of autoimmunity in younger individuals with higher serum antibody levels

Comprehensive RNA dataset of AGO2 associated RNAs in Jurkat cells following miR-21 over-expression

AbstractWe set out to identify miR-21 targets in Jurkat cells using a high-throughput biochemical approach (10.1016/j.biochi.2014.09.021 [1]). Using a specific monoclonal antibody raised against AGO2, RISC complexes were immunopurified in Jurkat cells over-expressing miR-21 following lentiviral trasduction as well as in Jurkat control cells lines. A parallel immunoprecipitation using isotype-matched rat IgG was performed as a control. AGO2 associated mRNAs were profiled by microarray (GEO: GSE37212). AGO2 bound miRNAs were profiled by RNA-seq

ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites.

Argonaute (AGO) proteins have a well-established role in post-transcriptional regulation of gene expression as key component of the RNA silencing pathways. Recent evidence involves AGO proteins in mammalian nuclear processes such as transcription and splicing, though the mechanistic aspects of AGO nuclear functions remain largely elusive. Here, by SILAC-based interaction proteomics, we identify the chromatin-remodelling complex SWI/SNF as a novel AGO2 interactor in human cells. Moreover, we show that nuclear AGO2 is loaded with a novel class of Dicer-dependent short RNAs (sRNAs), that we called swiRNAs, which map nearby the Transcription Start Sites (TSSs) bound by SWI/SNF. The knock-down of AGO2 decreases nucleosome occupancy at the first nucleosome located downstream of TSSs in a swiRNA-dependent manner. Our findings indicate that in human cells AGO2 binds SWI/SNF and a novel class of sRNAs to establish nucleosome occupancy on target TSSs

Fecal and mucosal microbiota profiling in pediatric inflammatory bowel diseases

An altered gut microbiota profile has been widely documented in inflammatory bowel diseases (IBD). The intestinal microbial community has been more frequently investigated in the stools than at the level of the mucosa, while most of the studies have been performed in adults. We aimed to define the gut microbiota profile either by assessing fecal and colonic mucosa samples (inflamed or not) from pediatric IBD patients

Fecal High-Mobility Group Box 1 as a Marker of Early Stage of Necrotizing Enterocolitis in Preterm Neonates

Introduction: An early diagnosis of necrotizing enterocolitis (NEC), a major gastrointestinal emergency in preterm newborns, is crucial to improve diagnostic approach and prognosis. We evaluated whether fecal high-mobility group box protein 1 (HMGB1) may early identify preterms at risk of developing NEC.Materials and Methods: A case-control study including neonates admitted at the Neonatal Intensive Care Unit (NICU) of the Sapienza University Hospital “Umberto I” in Rome, from July 2015 to December 2016. Stool samples obtained from cases (preterm newborns with NEC) and controls (newborns without NEC) were collected at the enrolment (T0) and within 7–14 days after the first sample collection (T1). HMGB1, extracted and measured with western blot, was reported as densitometry units (DUS).Results: HMGB1 levels in 30 cases (n = 28—Bell stage 1, n = 2 Bell stage 2) were higher [T0: 21,462 DUS (95% CI, 16,370–26,553 DUS)—T1: 17,533 DUS (95% CI, 13,052–22,014 DUS)] than in 30 preterm controls [T0: 9,446 DUS (95% CI, 6,147–12,746 DUS)—T1: 9,261 DUS (95% CI, 5,126–13,396 DUS), p < 0.001). Preterm newborns showed significant higher levels of HMGB1 (15,690 DUS (95% CI, 11,929–19,451 DUS)] in comparison with 30 full-term neonates with birth weight >2,500 g [6,599 DUS (95% CI, 3,141–10,058 DUS), p = 0.003]. Multivariate analysis showed that the risk of NEC was significantly (p = 0.012) related to the HMGB1 fecal levels at T0.Conclusions: We suggest fecal HMGB1 as a reliable marker of early NEC in preterm neonates. This study supports further investigation on the role of fecal HMGB1 assessment in managing preterm newborns at risk of NEC. Further studies are advocated to evaluate diagnostic accuracy of this marker in more severe forms of the disease

A retrospective multicentric observational study of trastuzumab emtansine in HER2 positive metastatic breast cancer: A real-world experience

We addressed trastuzumab emtansine (T-DM1) efficacy in HER2+ metastatic breast cancer patients treated in real-world practice, and its activity in pertuzumab-pretreated patients. We conducted a retrospective, observational study involving 23 cancer centres, and 250 patients. Survival data were analyzed by Kaplan Meier curves and log rank test. Factors testing significant in univariate analysis were tested in multivariate models. Median follow-up was 15 months and median T-DM1 treatment-length 4 months. Response rate was 41.6%, clinical benefit 60.9%. Median progression-free and median overall survival were 6 and 20 months, respectively. Overall, no differences emerged by pertuzumab pretreatment, with median progression-free and median overall survival of 4 and 17 months in pertuzumab-pretreated (p=0.13), and 6 and 22 months in pertuzumab-na\uc3\uafve patients (p=0.27). Patients who received second-line T-DM1 had median progression-free and median overall survival of 3 and 12 months (p=0.0001) if pertuzumab-pretreated, and 8 and 26 months if pertuzumab-na\uc3\uafve (p=0.06). In contrast, in third-line and beyond, median progression-free and median overall survival were 16 and 18 months in pertuzumab-pretreated (p=0.05) and 6 and 17 months in pertuzumab-na\uc3\uafve patients (p=0.30). In multivariate analysis, lower ECOG performance status was associated with progression-free survival benefit (p < 0.0001), while overall survival was positively affected by lower ECOG PS (p < 0.0001), absence of brain metastases (p 0.05), and clinical benefit (p < 0.0001). Our results are comparable with those from randomized trials. Further studies are warranted to confirm and interpret our data on apparently lower T-DM1 efficacy when given as second-line treatment after pertuzumab, and on the optimal sequence order

Hepatic cell differentiation requires a HNF6 - miR-122 feedback loop

Hepatocyte differentiation and maturation during embryogenesis is controlled by a network of liver-enriched transcription factors (LETFs). Whether LETFs control microRNA (miRNA) expression during development, and whether such control is required for hepatocyte differentiation has not yet been investigated. Here we show that two LETFs, namely Hepatocyte Nuclear Factor (HNF) 6 and Onecut2 (OC2), redundantly stimulate the expression of several miRNAs during development, and that the hepatocyte-specific miR-122 is the most critically dependent on HNF6 and OC2. Using in vivo and in vitro gain- and loss-of-function experiments, we further demonstrate that accurate levels of miR-122 are required to ensure proper progression of hepatocyte differentiation and maturation from hepatoblasts: miR-122 stimulates the expression of hepatocyte-specific genes and of most LETFs, including HNF6. This reveals the existence of a HNF6 – miR-122 positive feedback loop. Moreover, stimulation of hepatocyte maturation by miR-122 is abolished in an HNF6-null background, revealing that a transcription factor can mediate microRNA function. Confirming direct regulation we provide evidence that all hepatocyte-specific genes whose expression is stimulated by miR-122 are occupied in vivo by HNF6. MiR-122 also promotes hepatocyte maturation by directly repressing specific mRNAs. Mir-122 participates to liver-specific disallowance of the enzyme 2-oxoacid CoA transferase (OXCT1) during hepatocyte maturation. OXCT1 is involved in ketone body catabolism and needs to be repressed in liver, where ketone bodies are produced, in order to avoid hepatic catabolism of these metabolites. We show that OXCT1 mRNA is a direct target of miR-122 and that the rise in miR-122 level during hepatocyte maturation induces repression of OXCT1 protein production during liver development and in adulthood. Together, our findings indicate that the expression level of the liver specific miR-122 is controlled by a HNF6 - miR-122 positive feedback loop and that miR-122 controls hepatocyte differentiation and maturation by indirect stimulation of hepatocyte-specific genes and by direct repression of a ubiquitous gene.(SBIM 3) -- UCL, 201

How RNAi machinery enters the world of telomerase

Human telomerase holoenzyme consists of the catalytic component TERT and the template RNA TERC. However, a network of accessory proteins plays key roles in its assembly, localization and stability. Defects in genes involved in telomerase biology affect the renewal of critical stem cell populations and cause disorders such as telomeropathies. Moreover, activation of telomerase in somatic cells allows neoplastic cells to proliferate indefinitely, thus contributing to tumorigenesis. For these reasons, identification of new players involved in telomerase regulation is crucial for the determination of novel therapeutic targets and biomarkers. In the very last years, increasing evidence describe components of the RNAi machinery as a new layer of complexity in human telomerase activity. In this review we will discuss how AGO2 and other proteins which collaborate with AGO2 in RNAi pathway play a pivotal role in TERC stability and function

Next Generation Metagenomics: Methodological Challenges and Opportunities

Metagenomics is one of the newest omics system science technologies but also one that has arguably the broadest set of applications and impacts globally. Metagenomics has found vast utility not only in environmental sciences, ecology and public health but also in clinical medicine and looking into the future, in planetary health. In line with the One Health concept, metagenomics solicits collaboration between molecular biologists, geneticists, microbiologists, clinicians, computational biologists, plant biologists, veterinarians and other healthcare professionals. Almost every ecological niche of our planet hosts an extremely diverse community of organisms that are still poorly characterized. Detailed characterization of the features of such communities is instrumental to our comprehension of ecological, biological and clinical complexity. This expert review article evaluates how metagenomics is improving our knowledge of microbiota composition from environmental to human samples. Furthermore, we offer an analysis of the common technical and methodological challenges and potential pitfalls arising from metagenomics approaches, such as metagenomics study design, data processing and interpretation. All in all, at this critical juncture of further growth of the metagenomics field, it is time to critically reflect on the lessons learned and the future prospects of next generation metagenomics science, technology and conceivable applications, particularly from the standpoint of a metagenomics methodology perspective