CRITICAL STEPS FOR HUMAN GUT EXFOLIOME RNA PROFILING ANALYSIS USING NON-INVASIVE STOOL SAMPLES

Abstract Introduction: Dietary exposure and drug treatments influence gut cellular pathways and hence growth and potentially even the gut-brain-microbiome axis. Since eukaryotic mRNA presents poly A sequence that distinguishes them from the prokaryotes mRNA, we could analyze the gene expression of human gut cells using exfoliated gut cells available in stool samples. However, the impact of the critical steps of these non-invasive methods must be analyzed. Methods: We tested prokaryote contamination in all the steps of different procedures to analyze human exfoliome by microarrays and the influence of the fecal sampling collection process. Results & Conclusion: The least bacterial contamination was found using RNA amplified with oligo dT from GeneChip 3´ IVT Pico Reagent kit or using RNA purified by both Oligotex® + oligodT. RNA later® collection of feces affects the microarray results compared to directly frozen fecal samples, although both methods produce similar cDNA quality. This technique is a potential non-invasive diagnostic tool that can be applied to larger studies to quantify intestinal gene expression in humans with non-invasive samples, but samples should always be collected and analyzed under the same procedure.

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Antisense Transcription across Nucleotide Repeat Expansions in Neurodegenerative and Neuromuscular Diseases: Progress and Mysteries

Genes ◽

10.3390/genes11121418 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1418

Author(s):

Ana F. Castro ◽

Joana R. Loureiro ◽

José Bessa ◽

Isabel Silveira

Keyword(s):

Gene Expression ◽

Neuromuscular Diseases ◽

Antisense Transcription ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 7 ◽

Repeat Expansions ◽

Cellular Pathways ◽

The Impact ◽

Lateral Sclerosis

Unstable repeat expansions and insertions cause more than 30 neurodegenerative and neuromuscular diseases. Remarkably, bidirectional transcription of repeat expansions has been identified in at least 14 of these diseases. More remarkably, a growing number of studies has been showing that both sense and antisense repeat RNAs are able to dysregulate important cellular pathways, contributing together to the observed clinical phenotype. Notably, antisense repeat RNAs from spinocerebellar ataxia type 7, myotonic dystrophy type 1, Huntington’s disease and frontotemporal dementia/amyotrophic lateral sclerosis associated genes have been implicated in transcriptional regulation of sense gene expression, acting either at a transcriptional or posttranscriptional level. The recent evidence that antisense repeat RNAs could modulate gene expression broadens our understanding of the pathogenic pathways and adds more complexity to the development of therapeutic strategies for these disorders. In this review, we cover the amazing progress made in the understanding of the pathogenic mechanisms associated with repeat expansion neurodegenerative and neuromuscular diseases with a focus on the impact of antisense repeat transcription in the development of efficient therapies.

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Amounts and Botanical Diversity of Dietary Fruits and Vegetables Affect Distinctly the Human Gut Microbiome

Current Developments in Nutrition ◽

10.1093/cdn/nzaa062_002 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1545-1545

Author(s):

Ayoub Al Othaim ◽

Noah Voreades ◽

Natalie Goodwin ◽

Jenae Curley ◽

Daya Marasini ◽

...

Keyword(s):

Fruits And Vegetables ◽

Illumina Miseq ◽

Future Research ◽

Dietary Interventions ◽

Human Gut ◽

Funding Sources ◽

Rrna Sequencing ◽

Stool Samples ◽

Botanical Diversity ◽

The Impact

Abstract Objectives While increasing fruit and vegetable (FV) intake is a near-universal recommendation for improved health outcomes, information on dietary FV amount and diversity impact on health biomarkers is scarce. FV are a major dietary source of gut microbiota (GM) accessible carbohydrates and phytochemicals, however, most studies have focused on single food items or their extracted components, with few holistic studies available. Here, two separate randomized dietary interventions were used to assess the impact of low vs high FV intake and low vs high botanical diversity on GM profiles in healthy adults. We hypothesized that increasing FV would result in beneficial modulations to GM with further increases benefits in those consuming FV from diverse botanical families. Methods Study 1 was a crossover design with, 11 males randomized to starting diets of low FV (L) or high FV (H) over 9 days. Stool samples were obtained at day 0, 3, 6 and 9 of each treatment period. In Study 2, 21 individuals were provided a low FV (L) lead-in diet for 4 days and then randomly assigned to a high FV diet with either low (LB; 11 families) or high botanical diversity (HB; 24 families) for an additional 4 days. Stool was collected at baseline, and after each diet intervention. GM was analyzed using 16S rRNA sequencing performed on an Illumina MiSeq. The Mothur pipeline was used for preliminary data analysis, followed by statistical analyses in PAST. Results In Study 1, the L treatment resulted in minimal microbiota alterations, while a significant increase in Bacteroidetes and decrease in Firmicutes (which peaked at day 6) was observed in the H group. Intriguingly, the L group experienced a short-term increase in Bifidobacterium and Lactobacillus, and both treatments incurred significant increases in Bacteroides and Akkermansia and a decline of Faecalibacterium. In study 2 the transition from low to high FV resulted in similar trends than in Study 1. However, the HB treatment resulted in a more diverse GM, characterized by increased relative abundances of beneficial Firmicutes (Lachnospiraceae, Faecalibacterium and Clostridium XIVa). Conclusions Our results suggest that both amounts of FV consumed and botanical diversity modulate the GM. Determining better FV combinations from a GM perspective thus appears as a possible task for future research. Funding Sources Colorado Agriculture Experiment Station.

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Synbiotic Supplementation Modulates Gut Microbiota, Regulates Beta-catenin Expression and Prevents Weight Gain in Ob/ob Mice

10.21203/rs.3.rs-655169/v1 ◽

2021 ◽

Author(s):

Sebastião Mauro Bezerra Duarte ◽

José Tadeu Stefano ◽

Lucas A. M. Franco ◽

Roberta C. Martins ◽

Bruna D. G. C. Moraes ◽

...

Keyword(s):

Gene Expression ◽

Weight Gain ◽

Gut Microbiota ◽

Body Weight Gain ◽

Standard Diet ◽

Beta Catenin ◽

Stool Samples ◽

Synbiotic Supplementation ◽

And Control ◽

The Impact

Abstract Background: Obesity is one of the main health problems in the world today and dysbiosis seem to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation in obesity and microbiota in ob/ob mice. 20 animals were divided into four groups: Obese Treated (OT) and Control (OC), Lean Treated (LT) and Control (LC). All animals received standard diet for 8 weeks. Treated groups received a synbiotic in water while nontreated groups received water. After 8 weeks, all animals were sacrificed and gut tissue mRNA isolation and stool samples by microbiota analysis were collected. Beta-catenin, occludin, cadherin and zonulin were analyzed in gut tissue by RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using the Ion PGM Torrent platform. Results: The synbiotic supplementation reduced body weight gain in OT group comparing with OC (p=0.0398), increase of Enterobacteriaceae (p=0.005) and decrease of Cyanobacteria (p=0.047), Clostridiaceae (p=0.026), Turicibacterales (p=0.005) and Coprococcus (p=0.047). In the other hand, a significant reduction of Sutterella bacteria (p=0.009) and Turicibacter (p=0.005) was observed in LT group compared to LC. Alpha and beta diversities were differ between all treated groups. Beta-catenin gene expression was significantly decreased in the gut tissue of OT group (p≤0.0001) when compared to other groups. No changes were observed in occludin, cadherin and zonulin gene expression in the gut tissue. Conclusion: The synbiotics supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces beta-catenin expression in ob/ob mice.

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Human gut microbiome: hopes, threats and promises

Gut ◽

10.1136/gutjnl-2018-316723 ◽

2018 ◽

Vol 67 (9) ◽

pp. 1716-1725 ◽

Cited By ~ 332

Author(s):

Patrice D Cani

Keyword(s):

Gut Microbiota ◽

Dietary Habits ◽

Current Knowledge ◽

Metagenomic Data ◽

Human Gut ◽

Drug Treatments ◽

Key Issues ◽

Number Of Publications ◽

The Impact

The microbiome has received increasing attention over the last 15 years. Although gut microbes have been explored for several decades, investigations of the role of microorganisms that reside in the human gut has attracted much attention beyond classical infectious diseases. For example, numerous studies have reported changes in the gut microbiota during not only obesity, diabetes, and liver diseases but also cancer and even neurodegenerative diseases. The human gut microbiota is viewed as a potential source of novel therapeutics. Between 2013 and 2017, the number of publications focusing on the gut microbiota was, remarkably, 12 900, which represents four-fifths of the total number of publications over the last 40 years that investigated this topic. This review discusses recent evidence of the impact of the gut microbiota on metabolic disorders and focus on selected key mechanisms. This review also aims to provide a critical analysis of the current knowledge in this field, identify putative key issues or problems and discuss misinterpretations. The abundance of metagenomic data generated on comparing diseased and healthy subjects can lead to the erroneous claim that a bacterium is causally linked with the protection or the onset of a disease. In fact, environmental factors such as dietary habits, drug treatments, intestinal motility and stool frequency and consistency are all factors that influence the composition of the microbiota and should be considered. The cases of the bacteria Prevotella copri and Akkermansia muciniphila will be discussed as key examples.

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