Downexpression of miR-200c-3p Contributes to Achalasia Disease by Targeting the PRKG1 Gene.

Achalasia is an esophageal smooth muscle motility disorder with unknown pathogenesis. Taking into account our previous results on the downexpression of miR-200c-3p in tissues of patients with achalasia correlated with an increased expression of PRKG1, SULF1, and SYDE1 genes, our aim was to explore the unknown biological interaction between these genes and human miR-200c-3p and if this relation could unravel their functional role in the etiology of achalasia. To search for putative miR-200c-3p binding sites in the 3'-UTR of PRKG1, SULF1 and SYDE1, a bioinformatics tool was used. To test whether PRKG1, SULF1, and SYDE1 are targeted by miR-200c-3p, a dual-luciferase reporter assay and quantitative PCR on HEK293 and fibroblast cell lines were performed. To explore the biological correlation between PRKG1 and miR-200c-3p, an immunoblot analysis was carried out. The overexpression of miR-200c-3p reduced the luciferase activity in cells transfected with a luciferase reporter containing a fragment of the 3'-UTR regions of PRKG1, SULF1, and SYDE1 which included the miR-200c-3p seed sequence. The deletion of the miR-200c-3p seed sequence from the 3'-UTR fragments abrogated this reduction. A negative correlation between miR-200c-3p and PRKG1, SULF1, and SYDE1 expression levels was observed. Finally, a reduction of the endogenous level of PRKG1 in cells overexpressing miR-200c-3p was detected. Our study provides, for the first time, functional evidence about the PRKG1 gene as a direct target and SULF1 and SYDE1 as potential indirect substrates of miR-200c-3p and suggests the involvement of NO/cGMP/PKG signaling in the pathogenesis of achalasia.

Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer.

Fusion genes and epigenetic regulators (i.e., miRNAs and long non-coding RNAs) constitute essential pieces of the puzzle of the tumor genomic landscape, in particular in mechanisms behind the adenoma-to-carcinoma progression of colorectal cancer (CRC). In this work, we aimed to identify molecular signatures of the different steps of sporadic CRC development in eleven patients, of which synchronous samples of adenomas, tumors, and normal tissues were analyzed by RNA-Seq. At a functional level, tumors and adenomas were all characterized by increased activity of the cell cycle, cell development, cell growth, and biological proliferation functions. In contrast, organic survival and apoptosis-related functions were inhibited both in tumors and adenomas at different levels. At a molecular level, we found that three individuals shared a tumor-specific fusion named MRPS31-SUGT1, generated through an intra-chromosomal translocation on chromosome 13, whose sequence resulted in being 100% identical to the long non-coding RNA (lncRNA) MRPS31P5. Our analyses suggest that MRPS31P5 could take part to a competitive endogenous (ce)RNA network by acting as a miRNA sponge or/and as an interactor of other mRNAs, and thus it may be an important gene expression regulatory factor and could be used as a potential biomarker for the detection of early CRC events.

Multifaceted enrichment analysis of RNA-RNA crosstalk reveals cooperating micro-societies in human colorectal cancer.

Alterations in the balance of mRNA and microRNA (miRNA) expression profiles contribute to the onset and development of colorectal cancer. The regulatory functions of individual miRNA-gene pairs are widely acknowledged, but group effects are largely unexplored. We performed an integrative analysis of mRNA-miRNA and miRNA-miRNA interactions using high-throughput mRNA and miRNA expression profiles obtained from matched specimens of human colorectal cancer tissue and adjacent non-tumorous mucosa. This investigation resulted in a hypernetwork-based model, whose functional backbone was fulfilled by tight micro-societies of miRNAs. These proved to modulate several genes that are known to control a set of significantly enriched cancer-enhancer and cancer-protection biological processes, and that an array of upstream regulatory analyses demonstrated to be dependent on miR-145, a cell cycle and MAPK signaling cascade master regulator. In conclusion, we reveal miRNA-gene clusters and gene families with close functional relationships and highlight the role of miR-145 as potent upstream regulator of a complex RNA-RNA crosstalk, which mechanistically modulates several signaling pathways and regulatory circuits that when deranged are relevant to the changes occurring in colorectal carcinogenesis.

Association between polymorphisms of TAS2R16 and susceptibility to colorectal cancer.

BACKGROUND: Genetics plays an important role in the susceptibility to sporadic colorectal cancer (CRC). In the last 10 years genome-wide association studies (GWAS) have identified over 40 independent low penetrance polymorphic variants. However, these loci only explain around 1­4% of CRC heritability, highlighting the dire need of identifying novel risk loci. In this study, we focused our attention on the genetic variability of the TAS2R16 gene, encoding for one of the bitter taste receptors that selectively binds to salicin, a natural antipyretic that resembles aspirin. Given the importance of inflammation in CRC, we tested whether polymorphic variants in this gene could affect the risk of developing this neoplasia hypothesizing a role of TAS2R16 in modulating chronic inflammation within the gut. METHODS: We performed an association study using 6 tagging SNPs, (rs860170, rs978739, rs1357949, rs1525489, rs6466849, rs10268496) that cover all TAS2R16 genetic variability. The study was carried out on 1902 CRC cases and 1532 control individuals from four European countries. RESULTS: We did not find any statistically significant association between risk of developing CRC and selected SNPs. However, after stratification by histology (colon vs. rectum) we found that rs1525489 was associated with increased risk of rectal cancer with a (Ptrend of = 0.0071). CONCLUSIONS: Our data suggest that polymorphisms within TAS2R16 gene do not have a strong influence on colon cancer susceptibility, but a possible role in rectal cancer should be further evaluated in larger cohorts.

Deregulated expression of cryptochrome genes in human colorectal cancer.

BACKGROUND: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. METHODS: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. RESULTS: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes. CONCLUSION: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.

Peroxisome proliferator-activated receptor γ-mediated induction of microRNA-145 opposes tumor phenotype in colorectal cancer.

UNLABELLED: MicroRNAs (miRNAs) regulate diverse biological processes by inhibiting translation or inducing degradation of target mRNAs. miR-145 is a candidate tumor suppressor in colorectal carcinoma (CRC). Colorectal carcinogenesis involves deregulation of cellular processes controlled by a number of intertwined chief transcription factors, such as PPARγ and SOX9. Since PPAR family members are able to modulate complex miRNAs networks, we hypothesized a role of miRNA-145 in the interaction between PPARγ and SOX9 in colorectal carcinogenesis. To address this issue, we evaluated gene expression in tissue specimens of CRC patients and we took advantage of invitro models represented by CRC derived cell lines (CaCo2, SW480, HCT116, and HT-29), employing PPARγ activation and/or miRNA-145 ectopic overexpression to analyze how their interplay impact the expression of SOX9 and the development of a malignant phenotype. RESULTS: PPARγ regulates the expression of miR-145 by directly binding to a PPAR response element (PPRE) in its promoter at -1207/-1194bp from the transcription start site. The binding is essential for miR-145 upregulation by PPARγ upon rosiglitazone treatment. Ectopic expression of miR-145, in turn, regulates SOX9 expression through the binding to specific seed motifs. The PPARγ-miR-145-SOX9 axis overarches cell cycle progression, invasiveness and differentiation of CRC derived cell lines. Together, these results suggest that miR-145 is a novel target of PPARγ, acts as a tumor suppressor in CRC cell lines and is a key regulator of intestinal cell differentiation by directly targeting SOX9, a marker of undifferentiated progenitors in the colonic crypts.

Interplay between SOX9, ß-catenin and PPARγ activation in colorectal cancer.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and ß-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, ß-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, ß-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and ß-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, ß-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and ß-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, ß-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and ß-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

Mucosal Microbiota from Colorectal Cancer, Adenoma and Normal Epithelium Reveals the Imprint of Fusobacterium nucleatum in Cancerogenesis.

An increasing amount of evidence suggests the emerging role of the gut microbiota in the development of colorectal cancer (CRC). This study aimed to elucidate the architecture of microbial communities within normal and neoplastic colonic mucosa. METHODS: Microbiota were analyzed by NGS and by an ensemble of metagenomics analysis tools in a total of 69 tissues from 9 patients with synchronous colorectal neoplasia and adenomas (27 specimens: 9 from normal tissues, 9 adenomas, and 9 tumours), 16 patients with only colonic adenomas (32 specimens: 16 from normal tissues and 16 adenomas), and from healthy subjects (10 specimens of normal mucosa). RESULTS: Weak differences were observed in alpha and beta metrics among the synchronous tissues from CRC and controls. Through pairwise differential abundance analyses of sample groups, an increasing trend of Rikenellaceae, Pseudomonas and Fusobacterium, and decreasing trends of Staphylococcus, Actinobacillus and Gemmiger were observed in CRC, while Staphylococcus and Bifidobacterium were decreased in patients with only adenomas. At RT-qPCR analysis, Fusobacterium nucleatum was significantly enriched in all the tissues of subjects with synchronous colorectal neoplasia. CONCLUSION: Our findings provide a comprehensive view of the human mucosa-associated gut microbiota, emphasizing global microbial diversity mostly in synchronous lesions and proving the constant presence of Fusobacterium nucleatum, with its ability to drive carcinogenesis.

SIRT1 and the clock gene machinery in colorectal cancer.

SIRT1 and the clock genes are involved in carcinogenesis. We evaluated SIRT1 expression in 19 human colorectal cancer (CRC) specimens and clock gene expression in SIRT1-overexpressing CaCo2 and SW480 cells. In CRC, SIRT1 mean expression level was decreased. Compared to CaCo2 cells, SW480 cells displayed lower levels of SIRT1 and PER3 and higher levels of ARNTL1, CLOCK, PER1, PER2, CRY1, TIPIN, and CSNKIE. SIRT1 overexpression induced PER1 upregulation in CaCo2 and downregulation in SW480 cells. SIRT1 expression was heterogeneous in human CRC and in CRC cell lines. These results might have relevant implications for a better understanding of colorectal carcinogenesis.

Molecular pathways undergoing dramatic transcriptomic changes during tumor development in the human colon.

BACKGROUND: The malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations. METHODS: We used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa). RESULTS: Of the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics). CONCLUSIONS: Our analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.

A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application.

Background: Enterobius vermicularis (E. vermicularis) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Materials and methods: Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis. Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Results: Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. Conclusion: The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.

Germline Alterations in Patients With IBD-associated Colorectal Cancer.

BACKGROUND: Patients with inflammatory bowel diseases (IBD), both ulcerative colitis (UC) and Crohn's disease (CD), are at risk of developing a colorectal cancer (CRC). No information is available on the contribution of patients' genetic background to CRC occurrence. This study investigates germline alterations in patients with IBD-associated CRC. METHODS: We profiled a panel of 39 genes potentially involved in cancer predisposition and searched for germline variants in IBD patients with CRC or high-grade dysplasia. RESULTS: After clinical exclusion of genetic cancer syndromes, 25 IBD patients (4 CD and 21 UC) with CRC or high-grade dysplasia were studied. After excluding variants with low likelihood of pathogenicity (classes 1 or 2 according the International Agency for Research on Cancer [IARC]), the panel identified pathogenic variants, likely pathogenic, or variants with unknown significance in 18 patients (72%). Six patients (24%) carried pathogenic or likely variants (IARC class 5 or 4). Of the identified variants, 4 encompassed the APC region, 3 the MLH1 gene, and the remaining ones the MSH2, MSH3, monoallelic MUTYH, EPCAM, BRCA1, CHEK2, POLD1, POLE, CDKN2A, and PDGFRA genes. Four patients carried at least 2 variants in different genes. Duration of IBD was significantly shorter in carriers of 4 or 5 IARC variants (7 years; range 0-21; P = .002) and in those with variants with unknown significance (12 years; range 0-22; P = .005) compared with patients without or with only benign variations (23.5 years; range 15-34). CONCLUSIONS: In silico analysis and sequence-based testing of germline DNA from IBD patients with CRC or high-grade dysplasia detected 24% of variants positioned in pathogenic classes. In patients with type 3, 4, and 5 variants, the onset of high-grade dysplasia or CRC was significantly earlier than in patients with benign or unidentified variants. The screening for these genes could identify IBD patients requiring a more intensive endoscopic surveillance for earlier detection of dysplastic changes.

Adherence to Gluten-Free Diet Restores Alpha Diversity in Celiac People but the Microbiome Composition Is Different to Healthy People.

Celiac disease (CD) is an autoimmune disease with the destruction of small intestinal villi, which occurs in genetically predisposed individuals. At the present moment, a gluten-free diet (GFD) is the only way to restore the functionality of gut mucosa. However, there is an open debate on the effects of long-term supplementation through a GFD, because some authors report an unbalance in microbial taxa composition. METHODS: For microbiome analysis, fecal specimens were collected from 46 CD individuals in GFD for at least 2 years and 30 specimens from the healthy controls (HC). Data were analyzed using an ensemble of software packages: QIIME2, Coda-lasso, Clr-lasso, Selbal, PICRUSt2, ALDEx2, dissimilarity-overlap analysis, and dysbiosis detection tests. RESULTS: The adherence to GFD restored the alpha biodiversity of the gut microbiota in celiac people but microbial composition at beta diversity resulted as different to HC. The microbial composition of the CD subjects was decreased in a number of taxa, namely Bifidobacterium longum and several belonging to Lachnospiraceae family, whereas Bacteroides genus was found to be more abundant. Predicted metabolic pathways among the CD bacterial communities revealed an important role in tetrapyrrole biosynthesis. CONCLUSIONS: CD patients in GFD had a non-dysbiotic microbial composition for the crude alpha diversity metrics. We found significant differences in beta diversity, in certain taxon, and pathways between subjects with inactive CD in GFD and controls. Collectively, our data may suggest the development of new GFD products by modulating the gut microbiota through diet, supplements of vitamins, and the addition of specific prebiotics.

Healthy and pro-inflammatory gut ecology plays a crucial role in the digestion and tolerance of a novel Gluten Friendly™ bread in celiac subjects: a randomized, double blind, placebo control in vivo study.

Gluten Friendly™ (GF) is a new gluten achieved through a physicochemical process applied to wheat kernels. The goal of this research was to assess the in vivo effects of Gluten Friendly™ bread on celiac gut mucosa and microbiota. In a double-blind placebo-controlled intervention study, 48 celiac disease (CD) patients were randomized into 3 groups to eat 100 g of bread daily, containing different doses (0; 3 g; 6 g) of GF for 12 weeks. The small-bowel morphology (VH/CrD), intraepithelial densities of CD3+, celiac serology, MUC2, CB1, gut permeability, proinflammatory cytokines, gluten in stools, symptoms, and gut microbial composition were assessed. All 48 CD subjects experienced no symptoms. K-means analysis evidenced celiac subjects clustering around unknown parameters independent of GF dosage: K1 35%; K2 30%; K3 35%. VH/CrD significantly decreased in K1 and K2. VH/CrD did not correlate with IEL increase in K2. 33-mer was not detected in 47% and 73% of patients in both K1 and K2, respectively. VH/CrD and IEL did not change significantly and strongly correlated with the absence of 33-mer in K3. Inflammation and VH/CrD decrease are strongly related with the presence of proinflammatory species at the baseline. A boost in probiotic, butyrate-producing genera, is strongly related with GF tolerance at the end of the trial. Our research suggests that a healthy and proinflammatory ecology could play a crucial role in the digestion and tolerance of the new gluten molecule in celiac subjects. However, GF can be completely digested by gut microbiota of CD subjects and shapes it toward gut homeostasis by boosting healthy butyrate-producing populations. The clinical trial registry number is NCT03137862 (https://clinicaltrials.gov).

Circulating levels of cytokines, chemokines and growth factors in patients with achalasia.

Idiopathic achalasia is a disease that is characterized by the absence of peristalsis and incomplete relaxation of the lower esophageal sphincter, which is accompanied by dysphagia, regurgitation, chest pain and weight loss. The role of inflammatory infiltrates in the pathogenesis of achalasia remains controversial, although the infiltrating cell profile in the tissue has been previously characterized histologically and immunohistochemically. The present study aimed to evaluate the serum levels of 27 protein biomarkers to determine their association with achalasia and the clinical disease characteristics. The cytokine, chemokine and growth factor serum profiles of 68 patients with achalasia and 39 healthy individuals were explored using the 27-Bio-Plex Pro Human Cytokine assay. Reductions in the levels of inflammatory mediators IL-1ß, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, monocyte chemoattractant protein-1, macrophage inflammatory protein-1 (MIP-1)α and MIP-1ß, regulated upon activation normal T cell expressed and presumably secreted, TNF-α and VEGF were detected in the serum samples of patients with achalasia compared with those in the control group (P<0.05). However, significant associations between the expression in the levels of inflammatory factors and clinical characteristics of the patients were not found (P>0.05). These results suggest that achalasia is a disease that has a local but not a systemic inflammatory pattern. Further studies are required to improve the current understanding of the mechanism underlying this disease.

Microbiome Analysis of Mucosal Ileoanal Pouch in Ulcerative Colitis Patients Revealed Impairment of the Pouches Immunometabolites.

The pathogenesis of ulcerative colitis (UC) is unknown, although genetic loci and altered gut microbiota have been implicated. Up to a third of patients with moderate to severe UC require proctocolectomy with ileal pouch ano-anastomosis (IPAA). We aimed to explore the mucosal microbiota of UC patients who underwent IPAA. METHODS: For microbiome analysis, mucosal specimens were collected from 34 IPAA individuals. Endoscopic and histological examinations of IPAA were normal in 21 cases, while pouchitis was in 13 patients. 19 specimens from the healthy control (10 from colonic and 9 from ileum) were also analyzed. Data were analyzed using an ensemble of software packages: QIIME2, coda-lasso, clr-lasso, PICRUSt2, and ALDEx2. RESULTS: IPAA specimens had significantly lower bacterial diversity as compared to normal. The microbial composition of the normal pouch was also decreased also when compared to pouchitis. Faecalibacterium prausnitzii, Gemmiger formicilis, Blautia obeum, Ruminococcus torques, Dorea formicigenerans, and an unknown species from Roseburia were the most uncommon in pouch/pouchitis, while an unknown species from Enterobacteriaceae was over-represented. Propionibacterium acnes and Enterobacteriaceae were the species most abundant in the pouchitis and in the normal pouch, respectively. Predicted metabolic pathways among the IPAA bacterial communities revealed an important role of immunometabolites such as SCFA, butyrate, and amino acids. CONCLUSIONS: Our findings showed specific bacterial signature hallmarks of dysbiosis and could represent bacterial biomarkers in IPAA patients useful to develop novel treatments in the future by modulating the gut microbiota through the administration of probiotic immunometabolites-producing bacterial strains and the addition of specific prebiotics and the faecal microbiota transplantation.

Impact of the COVID-19 outbreak and the serum prevalence of SARS-CoV-2 antibodies in patients with inflammatory bowel disease treated with biologic drugs.

BACKGROUND: Patients receiving biologic therapies are at risk for viral infections. This study investigated the impact of the SARS-CoV-2 infection and the serum prevalence of SARS-CoV-2 antibodies in patients with inflammatory bowel disease (IBD) treated with biologic drugs. METHODS: Information on demography, co-morbidities, clinical data regarding IBD, symptoms suggestive of the SARS-CoV-2 infection, close contacts with SARS-CoV-2 positive patients, hospitalization, and therapies administered for COVID-19 was collected for all patients who were being treated with biologic drugs. All patients underwent SARS-CoV-2 antibody testing. RESULTS: Two hundred and fifty-nine patients (27 children) with a mean age of 42.2 ±â€¯16.7 years (range 9 - 88) and a mean duration of disease of 13.4 ±â€¯10 years (range 0.2 - 49) were enrolled. One hundred four patients (40.2%) had ulcerative colitis, and 155 (59.8%) had Crohn's disease. About the therapy: 62 patients were receiving infliximab, 89 adalimumab, 20 golimumab, 57 vedolizumab, 27 ustekinumab, 1 thalidomide, and 3 an experimental compound. The mean Charlson Comorbidity Index was 2. Thirty-two patients (12.3%) reported respiratory symptoms, and 2 of them were hospitalized (0.77%). Two patients resulted positive for IgG against SARS-CoV-2 (0.77%). CONCLUSIONS: In patients with IBD, treatment with biologic drug does not represent a risk factor for the SARS-CoV-2 infection.

False-positive results of SARS-CoV-2 IgM/IgG antibody tests in sera stored before the 2020 pandemic in Italy.

OBJECTIVES: Aside from the outbreak of the coronavirus disease 2019 (COVID-19), serological tests are not well known for their diagnostic value. We assessed the performance of serological tests using stored sera from patients with a variety of pathologic conditions, collected before the 2020 pandemic in Italy. METHODS: Rapid lateral flow tests and Enzyme-Linked Immunosorbent Assays (ELISA) that detect Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were carried out using 1150 stored human serum samples that had been collected in 2018 and 2019. The tests were also run using samples from 15 control patients who had positive or negative oral swab test results, as assessed using real-time reverse transcription-polymerase chain reaction (rRT-PCR). The urea dissociation test was employed to rule out false-positive reactivity in the two antibody detection methods. RESULTS: The lateral flow tests revealed 21 positive samples from the stored sera: 12 for IgM, four for IgG, and five for IgM/IgG. Among the nine rRT-PCR- positive controls, six individuals presented IgG and three IgM/IgG positivity. Using the urea (6 mol/L) dissociation test, two of the twelve stored samples that had shown IgM positivity were confirmed to be positive. The ELISA test detected four IgM-positive and three IgG-positive specimens. After treatment with 4 mol/L urea, the IgM-positive samples became negative, whereas the IgG positivity persisted. All of the rRT-PCR-positive controls were found to retain IgM or IgG positivity following the urea treatment. CONCLUSIONS: Our findings highlight the limited utility of serological testing for the SARS-CoV-2 virus based on the results of specimens collected before the outbreak of the infection.

microRNA-mRNA network model in patients with achalasia.

BACKGROUND: Achalasia is a rare idiopathic disease with a complex etio-pathogenesis still unknown. This study aimed to identify microRNA (miRNA)-mRNA regulatory networks underlying achalasia. METHODS: The investigation was performed in tissue specimens from 11 patients and five controls using the microarray technology followed by an integrated bioinformatics analysis. KEY RESULTS: One hundred and six miRNAs were significantly up-regulated and 64 were down-regulated in achalasia patients. The expression of the most 10 differential expressed miRNAs (miR-122-5p, miR-133a-3p, miR-504-5p, miR-187-3p, miR-133b, miR-200c-3p, miR-375, miR-200b-5p, miR-200b-3p, and miR203a) was confirmed by droplet digital PCR in an independent cohort. The interactions between the significant miRNAs and their targets uncovered 14 miRNA-mRNA interacting pairs with experimentally predicted genes (ie, FN1, ROCK2, DPYSL2), and 35 pairs with not experimentally target genes (ie, SULF1, MRVI1, PRKG1); all genes were involved in immune cell trafficking, skeletal and muscular system development, nervous system development macro-processes. CONCLUSION & INFERENCES: The mRNA-miRNA regulatory networks described in this study provide new insights in the genetic background of the disease, suggesting further investigations in novel pathogenic mechanisms.

Inflammatory Bowel Disease Meets Systems Biology: A Multi-Omics Challenge and Frontier.

The inflammatory bowel disease (IBD) is a systemic disease that is characterized by the inflammation of the gastrointestinal tract. It includes ulcerative colitis and the Crohn's disease. Presently, IBD is one of the most investigated common complex human disorders, although its causes remain unclear. Multi-omics mechanisms involving genomic, transcriptomic, proteomic, and epigenomic variations, not to forget the miRNome, together with environmental contributions, result in an impairment of the immune system in persons with IBD. Such interactions at multiple levels of biology and in concert with the environment constitute the actual engine of this complex disease, demanding a multifactorial and multi-omics perspective to better understand the root causes of IBD. This expert analysis reviews and examines the latest literature and underscores, from the perspective of systems biology, the value of multi-omics technologies as opportunities to unravel the "IBD integrome." We anticipate that multi-omics research will accelerate the new discoveries and insights on IBD in the near future. It shall also pave the way for early diagnosis and help clinicians and families with IBD to forecast and make informed decisions about the prognosis and, possibly, personalized therapeutics in the future.