BACKGROUND AND AIMS:Intestinal fibrosis is a common complication of Inflammatory Bowel Disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), but the precise mechanism by which it occurs is incompletely understood hampering the development of effective therapeutic strategies. Here, we aimed at inducing and characterizing an inflammation-mediated fibrosis in patient-derived organoids (PDOs) issued from crypts isolated from colonic mucosal biopsies of IBD pediatric patients and age matched-control subjects (CTRLs). METHODS:Inflammatory-driven fibrosis was induced by exposing CTRL-, CD- and UC-PDOs to the pro-inflammatory cytokine TNF-α for one day, followed by a co-treatment with TNF-α and TGF-β1 for three days. Fibrotic response was proven by analyzing inflammatory and fibrotic markers by RT-qPCR and immunofluorescence. Transcriptomic changes were assessed by RNA-sequencing. RESULTS:Co-treatment with TNF-α and TGF-β1 caused in CTRL- and IBD-PDOs morphological changes towards a mesenchymal-like phenotype and up-regulation of inflammatory, mesenchymal, and fibrotic markers. Transcriptomic profiling highlighted that in all intestinal PDOs, regardless of the disease, the co-exposure to TNF-α and TGF-β1 regulated EMT genes and specifically increased genes involved in positive regulation of cell migration. Finally, we demonstrated that CD-PDOs display a specific response to fibrosis compared to both CTRL- and UC-PDOs, mainly characterized by upregulation of nuclear factors controlling transcription. CONCLUSIONS:This study demonstrates that intestinal PDOs may develop an inflammatory-derived fibrosis thus representing a promising tool to study fibrogenesis in IBD. Fibrotic PDOs show increased expression of EMT genes. In particular, fibrotic CD-PDOs display a specific gene expression signature compared to UC and CTRL-PDOs.

OBJECTIVE AND RATIONALE:Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, manifests with chronic intestinal inflammation and frequent sequential fibrosis. Current pharmacological therapies may show harmful side effects and are not useful for prevention or resolution of fibrosis. Thus, the use of alternative therapies is emerging as a novel useful approach. Previous results suggest that Scutellaria baicalensis Georgi (SBG) and Boswellia serrata (BS) display anti-inflammatory properties. The aim of this study was to investigate in intestinal epithelial cells and fibroblasts the anti-inflammatory and anti-fibrotic potential of SBG and BS, alone or in combination. METHODS:Human colorectal adenocarcinoma cells (HT29), human intestinal epithelial cells (HIEC6) and human colon fibroblasts (CCD-18Co) were used. Cells were pretreated with SBG and BS and then exposed to pro-inflammatory and pro-fibrotic cytokines. RESULTS:SBG and BS extracts significantly decreased pro-inflammatory cytokine expression and improved epithelial restitution in HT29 and HIEC6 cells. Besides, fibrotic marker expression, including SNAIL, ACTA2, ZNF281, was strongly reduced. Colon myofibroblasts treated with SBG and BS showed a significant decrease of fibrotic markers as well. CONCLUSIONS:SBG and BS extracts significantly reduce inflammation and impair fibrosis in intestinal epithelial cells and colon myofibroblasts. No cooperative effect is observed.

Extracellular High-mobility group box 1 (HMGB1) contributes to the pathogenesis of inflammatory disorders, including inflammatory bowel diseases (IBD). Poly (ADP-ribose) polymerase 1 (PARP1) has been recently reported to promote HMGB1 acetylation and its secretion outside cells. In this study, the relationship between HMGB1 and PARP1 in controlling intestinal inflammation was explored. C57BL6/J wild type (WT) and PARP1−/− mice were treated with DSS to induce acute colitis, or with the DSS and PARP1 inhibitor, PJ34. Human intestinal organoids, which are originated from ulcerative colitis (UC) patients, were exposed to pro-inflammatory cytokines (INFγ + TNFα) to induce intestinal inflammation, or coexposed to cytokines and PJ34. Results show that PARP1−/− mice develop less severe colitis than WT mice, evidenced by a significant decrease in fecal and serum HMGB1, and, similarly, treating WT mice with PJ34 reduces the secreted HMGB1. The exposure of intestinal organoids to pro-inflammatory cytokines results in PARP1 activation and HMGB1 secretion; nevertheless, the co-exposure to PJ34, significantly reduces the release of HMGB1, improving inflammation and oxidative stress. Finally, HMGB1 release during inflammation is associated with its PARP1-induced PARylation in RAW264.7 cells. These findings offer novel evidence that PARP1 favors HMGB1 secretion in intestinal inflammation and suggest that impairing PARP1 might be a novel approach to manage IBD.

Virus-encoded microRNAs were first reported in the Epstein–Barr virus in 2004. Subsequently, a few hundred viral miRNAs have been identified, mainly in DNA viruses belonging to the herpesviridae family. To date, only 30 viral miRNAs encoded by RNA viruses are reported by miRBase. Since the outbreak of the SARS-CoV-2 pandemic, several studies have predicted and, in some cases, experimentally validated miRNAs originating from the positive strand of the SARS-CoV-2 genome. By integrating NGS data analysis and qRT-PCR approaches, we found that SARS-CoV-2 also encodes for a viral miRNA arising from the minus (antisense) strand of the viral genome, in the region encoding for ORF1ab, herein referred to as SARS-CoV-2-miR-AS1. Our data show that the expression of this microRNA increases in a time course analysis of SARS-CoV-2 infected cells. Furthermore, enoxacin treatment enhances the accumulation of the mature SARS-CoV-2-miR-AS1 in SARS-CoV-2 infected cells, arguing for a Dicer-dependent processing of this small RNA. In silico analysis suggests that SARS-CoV-2-miR-AS1 targets a set of genes which are translationally repressed during SARS-CoV-2 infection. We experimentally validated that SARS-CoV-2-miR-AS1 targets FOS, thus repressing the AP-1 transcription factor activity in human cells.

BACKGROUND:Faecal biomarkers have emerged as important tools in managing of inflammatory bowel disease [IBD], which includes Crohn's disease [CD] and ulcerative colitis [UC].AIM:To identify new biomarkers of gut inflammation in the stools of IBD patients using a proteomic approach.METHODS:Proteomic analysis of stools was performed in patients with both active CD and CD in remission and in controls by 2-DIGE and MALDI-TOF/TOF MS. An ELISA was used to confirm results in a second cohort of IBD patients and controls.RESULTS:2-DIGE analysis detected 70 spots in the stools of patients with active CD or patients in remission CD and in controls. MALDI-TOF/TOF MS analysis identified 21 proteins with Chymotrypsin C, Gelsolin and Rho GDP-dissociation inhibitor 2 [RhoGDI2] best correlating with the levels of intestinal inflammation. Results were confirmed in a second cohort of IBD patients and controls [57 CD, 60 UC, 31 controls]. The identified faecal markers significantly correlated with the severity of intestinal inflammation in IBD patients [SES-CD in CD, Mayo endoscopic subscore in UC] [CD; Chymotrypsin-C: r = 0.64, p < 0.001; Gelsolin: r = 0.82, p < 0.001; RhoGDI2: r = 0.64, p < 0.001; UC; Chymotrypsin-C: r = 0.76, p < 0.001; Gelsolin: r = 0.75, p < 0.001; RhoGDI2: r = 0.63, p < 0.001]. Moreover, ROC analysis showed that Gelsolin [p < 0.0002] and RhoGDI2 [p < 0.0001] in CD, and RhoGDI2 [p = 0.0004] in UC, have higher sensitivity and specificity than faecal calprotectin in discriminating between patients and controls.CONCLUSIONS:We show for the first time that 2-DIGE is a reliable method to detect proteins in human stools. Three novel faecal biomarkers of gut inflammation have been identified that display good specificity and sensitivity for identifying IBD and significantly correlate with IBD severity.

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract. Chronic inflammation is the main factor leading to intestinal fibrosis, resulting in recurrent stenosis, especially in CD patients. Currently, the underlying molecular mechanisms of fibrosis are still unclear. ZNF281 is a zinc-finger transcriptional regulator that has been characterized as an epithelial-to-mesenchymal transition (EMT)-inducing transcription factor, suggesting its involvement in the regulation of pluripotency, stemness, and cancer. The aim of this study is to investigate in vivo and in vitro the role of ZNF281 in intestinal fibrogenesis. Intestinal fibrosis was studied in vivo in C57BL/6J mice with chronic colitis induced by two or three cycles of administration of dextran sulfate sodium (DSS). The contribution of ZNF281 to gut fibrosis was studied in vitro in the human colon fibroblast cell line CCD-18Co, activated by the pro-fibrotic cytokine TGFβ1. ZNF281 was downregulated by siRNA transfection, and RNA-sequencing was performed to identify genes regulated by TGFβ1 in activated colon fibroblasts via ZNF281. Results showed a marked increase of ZNF281 in in vivo murine fibrotic colon as well as in in vitro human colon fibroblasts activated by TGFβ1. Moreover, abrogation of ZNF281 in TGFβ1-treated fibroblasts affected the expression of genes belonging to specific pathways linked to fibroblast activation and differentiation into myofibroblasts. We demonstrated that ZNF281 is a key regulator of colon fibroblast activation and myofibroblast differentiation upon fibrotic stimuli by transcriptionally controlling extracellular matrix (ECM) composition, remodeling, and cell contraction, highlighting a new role in the onset and progression of gut fibrosis.

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak demonstrates the potential of coronaviruses, especially bat-derived beta coronaviruses to rapidly escalate to a global pandemic that has caused deaths in the order of several millions already. The huge efforts put in place by the scientific community to address this emergency have disclosed how the implementation of new technologies is crucial in the prepandemic period to timely face future ecological crises. In this context, we argue that metagenomics and new approaches to understanding ecosystems and biodiversity offer veritable prospects to innovate therapeutics and diagnostics against novel and existing infectious agents. We discuss the opportunities and challenges associated with the science of metagenomics, specifically with an eye to inform and prevent future ecological crises and pandemics that are looming on the horizon in the 21st century.

In the last decade, the field of epitranscriptomics highlighted a wide array of post-transcriptional modifications in human RNAs, including microRNAs (miRNAs). Recent reports showed that human miRNAs undergo cytosine methylation. We describe the first high-throughput NGS-based method (BS-miRNA-seq) and an analysis pipeline (MAmBA) to attain high-resolution mapping of (hydroxy)-methyl-5-cytosine ((h)m5C) modifications in human miRNAs. Our method uncovers that miRNAs undergo widespread cytosine modification in various sequence contexts. Furthermore, validation of our data with specific antibodies reveals both m5C and hm5C residues in human mature miRNAs. BS-miRNA-seq and MAmBA may contribute to the precise mapping of (h)m5C on miRNAs in various cell types and tissues, a key achievement towards the understanding of the functional implications of this modification in miRNAs. MAmBA is available for download at https://github.com/flcvlr/MAmBA

Background 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. Patients and methods Fecal and colonic samples from pediatric IBD (Crohn’s disease or ulcerative colitis) and controls were analyzed. The relative abundance of bacteria at phylum and genus/species levels and bacterial diversity were determined through 16S rRNA sequence-based of fecal and mucosal microbiota analysis. Results A total of 59 children with IBD (26 Crohn’s disease, 33 ulcerative colitis) and 39 controls were analyzed. A clear separation between IBD and controls in the overall composition of fecal and mucosal microbiota was found, as well as a reduced bacterial richness in the fecal microbiota of IBD. At the phylum level, abundance of Proteobacteria and Actinobacteria occurred in fecal microbiota of IBD, while species with anti-inflammatory properties (i.e., Ruminococcus) were reduced. Fusobacterium prevailed in inflamed IBD areas in comparison to noninflamed and controls samples. Conclusion Significant alterations in gut microbiota profile were shown in our IBD pediatric patients, in whom an abundance of species with a proinflammatory mucosal activity was clearly detected. An analysis of gut microbiota could be incorporated in designing personalized IBD treatment scenarios in future.

MicroRNAs are pervasive regulators of gene expression at the post-transcriptional level in metazoan, playing key roles in several physiological and pathological processes. Accordingly, these small non-coding RNAs are also involved in cancer development and progression. Furthermore, miRNAs represent valuable diagnostic and prognostic biomarkers in malignancies. In the last twenty years, the role of RNA modifications in fine-tuning gene expressions at several levels has been unraveled. All RNA species may undergo post-transcriptional modifications, collectively referred to as epitranscriptomic modifications, which, in many instances, affect RNA molecule properties. miRNAs are not an exception, in this respect, and they have been shown to undergo several post-transcriptional modifications. In this review, we will summarize the recent findings concerning miRNA epitranscriptomic modifications, focusing on their potential role in cancer development and progression.

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 disease.

OMICS: A Journal of Integrative BiologyVol. 24, No. 3 CommentaryFree AccessMetagenomics in Italy and Europe: Three Actionable Challenges/Prospects in 2020Ilaria Laudadio, Vincenzo Cesi, and Claudia CarissimiIlaria LaudadioDepartment of Molecular Medicine, “Sapienza” University of Rome, Rome, Italy.Search for more papers by this author, Vincenzo CesiDepartment of Molecular Medicine, “Sapienza” University of Rome, Rome, Italy.Search for more papers by this author, and Claudia CarissimiAddress correspondence to: Claudia Carissimi, PhD, Department of Molecular Medicine, “Sapienza” University of Rome, Vle Regina Elena, 324, Rome 00161, Italy E-mail Address: claudia.carissimi@uniroma1.itDepartment of Molecular Medicine, “Sapienza” University of Rome, Rome, Italy.Search for more papers by this authorPublished Online:13 Mar 2020https://doi.org/10.1089/omi.2020.0006AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail IntroductionMetagenomics has gained worldwide prominence as one of the indispensable omics technologies for the past several years in particular. Metagenomics applications in clinical medicine, ecology, planetary health, and built environments are increasingly reported in the literature. Many countries are both striving and struggling to cultivate metagenomics scholarship as one of the drivers of life sciences innovation in early 21st century. Italy is no exception and has made important strides in metagenomics for the past decade.As we shift our gaze toward the new year 2020, we highlight what we consider to be three actionable emerging challenges in the field of metagenomics, specifically in Italy, that can transform to prospects for clinical, translational, ecological, and/or discovery science innovation globally.Actionable Challenge 1Antimicrobial resistance and metagenomicsAntimicrobial resistance (AMR) is a global public health crisis that is impeding our ability to successfully treat infectious diseases. A recent report from the Italian Higher Health Institute highlights that Italy is one of the European Member states with the highest level of deaths caused by AMR, accounting for almost one-third of deaths in Europe due to resistance to antibiotics each year (Italian Government, Piano Nazionale di Contrasto AMR, 2017–2020).The human microbiome is an important reservoir of AMR and a key player in the dissemination of AMR genes (Brinkac et al., 2017). A complete understanding of the structural and functional complexity of the microbial communities in humans, animals, and the environment is mandatory for halting and reversing the current AMR predicament in Italy and on a planetary scale. In this context, single-cell metagenomics offers a promising tool to improve our ability to taxonomically characterize human microbiota. Moreover, functional analyses of metagenomic data might enhance our comprehension of the rise of multidrug resistance. We believe that metagenomics will help in understanding the mechanisms underlying the development and spread of AMR and in successfully governing this threat to the worldwide public health.Actionable Challenge 2Novel molecules and strategies to fight against bacterial resistanceA report of the European Centre for Disease Prevention and Control concluded: “The levels of carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii have now reached hyper-endemic levels and, together with meticillin-resistant Staphylococcus aureus, this situation causes Italy to be one of the Member States with the highest level of resistance in Europe” (Cassini et al., 2017). To address the bacterial resistance crisis in Italy and Europe, there is an urgent need to develop more selective and safe biological therapies. Bacteriocins, which are antimicrobial peptides/proteins synthesized by certain bacteria, are interesting alternatives to conventional antibiotics (Cotter et al., 2013).The increasing interest in bacteriocins is due to their diversified mechanisms of action that overcome bacterial resistance to antimicrobials while preserving commensal bacteria in microbiota. To date, these antimicrobial peptides have a long history of safe use in dairy industry as preservatives (Silva et al., 2018). This makes them quite appealing and promising as alternative therapeutics. We envision that functional metagenomics analyses of various Holobionts, delineated as a community of species that are closely associated and have complex interactions, will offer great promise for discovering novel human microbial bacteriocins gene clusters and for studying bacteriocins to manipulate and express them in host microbes.Actionable Challenge 3ProbioticsFor the past decade, the growing interest in promoting health in ways attuned to the nature led to intensification of research in the field of probiotics on a global scale. During the last “Probiotics, Prebiotics and New Foods” symposium an overview about “Probiotics: market, technical, scientific and quality topics in Italy and in Europe,” report that the Italian probiotic market is one of the most notable in Europe; today it is worth >500 million euros, >50% of the European total (Probiotics, Prebiotics & New Foods, Nutraceuticals and Botanicals for Nutrition & Human and Microbiota Health 1st Science & Business Symposium, 2019).The potential applications of probiotics are continuously widening and offer a promising opportunity in the management of a wide range of human diseases in the near future. Therefore, monitoring effectiveness and safety of gut microbiota manipulation by prebiotics deserves further efforts. Taking advantage of microbial meta-pathways analyses based on metagenomics, characterization of community members, functions, and interactions, in specific ecosystems will be possible (Laudadio et al., 2019). This knowledge is crucial for optimal development of functional probiotics that requires not only a detailed insight into specific probiotic member strains, dosage, and long-term safety, but also the elucidation of the exact mechanisms by which probiotics produce health benefits.OutlookAMR poses a growing challenge to health care, in particular in Italy. Reducing such planetary health threats caused by uncontrolled antibiotic use through microbiome-based approaches is an ambitious but important research goal in each and every country, including in Italy. In this context, metagenomics is an invaluable tool to empower local public health systems around the world. Indeed, metagenomics is not only one of the newest omics technologies but also the one harboring the broadest set of applications and impacts. Metagenomics has rapidly expanded our understanding of the structure and function of environmental and clinical microbial communities, allowing to address previously unattainable biological questions as well as accelerating genome-based discovery of novel microbial genes associated with AMR.We envision that this approach will revolutionize infectious disease treatments in the future by allowing the development of novel diagnostics and precision therapeutics. The challenges and possible solutions offered here highlight the growing importance of metagenomics in Italy, Europe, and worldwide.ReferencesBrinkac L, Voorhies A, Gomez A, and Nelson KE. (2017). The threat of antimicrobial resistance on the human microbiome. Microb Ecol 74, 1001–1008. Crossref, Medline, Google ScholarCassini A, Monnet DL, Mancarella G, et al.; European Centre for Disease Prevention and Control. (2017). ECDC Country Visit to Italy to Discuss Antimicrobial Resistance Issues. Stockholm: ECDC. Google ScholarCotter PD, Ross RP, and Hill C. (2013). Bacteriocins—a viable alternative to antibiotics? Nat Rev Microbiol 11, 95–10. Crossref, Medline, Google ScholarItalian Government, Piano Nazionale di Contrasto AMR, 2017–2020. www.salute.gov.it/imgs/C_17_pubblicazioni_2660_allegato.pdf Google ScholarLaudadio I, Fulci V, Stronati L, and Carissimi C. (2019). Next-generation metagenomics: Methodological challenges and opportunities. Omics J Integr Biol 23, 327–333. Link, Google ScholarProbiotics, Prebiotics & New Foods, Nutraceuticals and Botanicals for Nutrition & Human and Microbiota Health 1st Science & Business Symposium (2019). Rome, Italy, p18. https://probiotics-prebiotics-newfood.com (Last viewed on Feb. 3, 2020.) Google ScholarSilva CCG, Silva SPM, and Ribeiro SC. (2018). Application of bacteriocins and protective cultures in dairy food preservation. Front Microbiol 19, 594. Crossref, Google ScholarAbbreviation UsedAMRantimicrobial resistanceFiguresReferencesRelatedDetails Volume 24Issue 3Mar 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:Ilaria Laudadio, Vincenzo Cesi, and Claudia Carissimi.Metagenomics in Italy and Europe: Three Actionable Challenges/Prospects in 2020.OMICS: A Journal of Integrative Biology.Mar 2020.122-123.http://doi.org/10.1089/omi.2020.0006Published in Volume: 24 Issue 3: March 13, 2020Online Ahead of Print:February 19, 2020PDF download

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 describes 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.

Background: Topical steroids are effective in eosinophilic esophagitis (EoE), but patients often show different tendencies to relapse. We assessed whether gene expression is associated with a sort of steroid dependency in EoE children. Methods: Biopsy samples were prospectively collected on EoE children responding to topical steroids. Patients treated with viscous budesonide for 24 weeks were subsequently classified as early (6 months) or late (>6 months) relapsing. RNA was isolated from esophageal biopsies at the time of the relapse and analyzed by NGS for transcriptome profiling. Results: Of 40 patients, 22 patients were considered for mRNA expression profile. Thirteen were included in the early-relapse group, and 9 were in the late-relapse. No significant difference was observed in the two groups for clinical, endoscopic or histological features. Using the mRNA expression profile we performed supervised clustering using the 10 top differentially expressed genes between early and late relapsing patients. The heatmap and PCA show a proper segregation among patients. SERPINB12 is the only gene attaining a significant differential expression between the two groups (FDR < 0.05). Conclusions: Different tendencies to relapse in EoE children responding to topical steroids might be related to altered mRNA expressions. SERPINB12 presented a significantly higher expression in the late relapse group and it deserves further investigations. (C) 2019 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

Metagenomics is not only 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 health care 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.

Background and Aims Recent evidence implicates gut microbiota (GM) and immune alterations in autism spectrum disorders (ASD). We assess GM profile and peripheral levels of immunological, neuronal and bacterial molecules in ASD children and controls. Alarmin HMGB1 was explored as a non-invasive biomarker to monitor gastrointestinal (GI) symptoms. Methods Thirty ASD children and 14 controls entered into the study. GM metagenomic analysis was performed for 16 ASD patients and 7 controls. GM functional profile was assessed by GO term analysis. Blood levels of IL-1β, TNFα, TGFβ, IL-10, INFγ, IL-8, lipopolysaccharide, Neurotensin, Sortilin1 and GSSG/GSH ratio were analyzed in all subjects by ELISA. Fecal HMGB1 was analyzed by Western blot. Results We observed a significant decrease in bacterial diversity. Furthermore, 82 GO terms underrepresented in ASD. Four of them pointed at 3,3 phenylpropionate catabolism and were imputable to Escherichia coli (E. coli) group. Serum levels of TNFα, TGFβ, NT, and SORT-1 increased in ASD patients. Fecal levels of HMGB1 correlated with GI sign severity in ASD children. Conclusions We suggest that a decrease of E. coli might affect the propionate catabolism in ASD. We report occurrence of peripheral inflammation in ASD children. We propose fecal HMGB1 as a non-invasive biomarker to detect GI symptoms.

Metagenomics is not only 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 health care 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.

Next-generation sequencing has uncovered novel classes of small RNAs (sRNAs) in eukaryotes, in addition to the well-known miRNAs, siRNAs, and piRNAs. In particular, sRNA species arise from transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a detailed characterization of these new classes of sRNAs is still lacking. Here, we present a comprehensive study of sRNAs derived from TTSs of expressed genes (TTSa-RNAs) in human cell lines and primary tissues. Taking advantage of sRNA-sequencing, we show that TTSa-RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2, and their biogenesis does not require DICER and AGO2 endonucleolytic activity. TTSa-RNAs display a strong bias against a G residue in the first position at 5' end, a known feature of AGO-bound sRNAs, and a peculiar oligoA tail at 3' end. AGO-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation and DNA integrity checkpoints. Finally, we provide evidence that TTSa-RNAs can be detected by sRNA-Seq in primary human tissue, and their expression increases in tumor samples as compared to nontumor tissues, suggesting that in the future, TTSa-RNAs might be explored as biomarker for diagnosis or prognosis of human malignancies.