Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly

AbstractThe gut is the most extensive, interactive, and complex interface between the human host and the environment and therefore a critical site of immunological activity. Non-invasive methods to assess the host response in this organ are currently lacking. Feces are the available analyte which have been in proximity to the gut tissue.We applied a method of concentrating host transcripts from fecal specimens using a existing bead-based affinity separation method for nucleic acids and quantified transcripts using droplet digital PCR (ddPCR) to determine the copy numbers of a variety of key transcripts in the gut immune system. ddPCR compartmentalizes the reaction in a small aqueous droplet suspended in oil, and counts droplets as either fluorescent or non-fluorescent. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize transcript concentration.This method was applied to 799 fecal samples from rural Malawian children, and over 20,000 transcript concentrations were quantified. Host mRNA was detected in >99% samples, a threshold for target detection was established at an average expression of 0.02 copies target/GAPDH, above which correlation coefficient between duplicate measurements is >0.95. Quantities of transcript detected using ddPCR were greater than standard qPCR. Fecal sample preservation at the time of collection did not require immediate freezing or the addition of buffers or enzymes. Measurements of transcripts encoding immunoactive proteins correlated with a measure of gut inflammation in the study children, thereby substantiating their relevance. This method allows investigators to interrogate gene expression in the gut

Growth velocity in children with environmental enteric dysfunction is associated with specific bacterial and viral taxa of the gastrointestinal tract in Malawian children

Environmental enteric dysfunction (EED) is characterized by diffuse villous atrophy of the small bowel. EED is strongly associated with stunting, a major public health problem linked to increased childhood morbidity and mortality. EED and subsequent stunting of linear growth are surmised to have microbial origins. To interrogate this relationship, we defined the comprehensive virome (eukaryotic virus and bacteriophage) and bacterial microbiome of a longitudinal cohort of rural Malawian children with extensive metadata and intestinal permeability testing at each time point. We found thirty bacterial taxa differentially associated with linear growth. We detected many eukaryotic viruses. Neither the total number of eukaryotic families nor a specific viral family was statistically associated with improved linear growth. We identified 3 differentially abundant bacteriophage among growth velocities. Interestingly, there was a positive correlation between bacteria and bacteriophage richness in children with subsequent adequate/moderate growth which children with subsequent poor growth lacked. This suggests that a disruption in the equilibrium between bacteria and bacteriophage communities might be associated with subsequent poor growth. Future studies of EED and stunting should include the evaluation of viral communities in addition to bacterial microbiota to understand the complete microbial ecology of these poorly understood entities

Environmental enteric dysfunction includes a broad spectrum of inflammatory responses and epithelial repair processes

Background & AimsEnvironmental enteric dysfunction (EED), a chronic diffuse inflammation of the small intestine, is associated with stunting in children in the developing world. The pathobiology of EED is poorly understood because of the lack of a method to elucidate the host response. This study tested a novel microarray method to overcome limitation of RNA sequencing to interrogate the host transcriptome in feces in Malawian children with EED.MethodsIn 259 children, EED was measured by lactulose permeability (%L). After isolating low copy numbers of host messenger RNA, the transcriptome was reliably and reproducibly profiled, validated by polymerase chain reaction. Messenger RNA copy number then was correlated with %L and differential expression in EED. The transcripts identified were mapped to biological pathways and processes. The children studied had a range of %L values, consistent with a spectrum of EED from none to severe.ResultsWe identified 12 transcripts associated with the severity of EED, including chemokines that stimulate T-cell proliferation, Fc fragments of multiple immunoglobulin families, interferon-induced proteins, activators of neutrophils and B cells, and mediators that dampen cellular responses to hormones. EED-associated transcripts mapped to pathways related to cell adhesion, and responses to a broad spectrum of viral, bacterial, and parasitic microbes. Several mucins, regulatory factors, and protein kinases associated with the maintenance of the mucous layer were expressed less in children with EED than in normal children.ConclusionsEED represents the activation of diverse elements of the immune system and is associated with widespread intestinal barrier disruption. Differentially expressed transcripts, appropriately enumerated, should be explored as potential biomarkers

Is it true coeliacs do not digest gliadin ?. Degradation pattern of gliadin in coeliac disease small intestinal mucosa

Prolyl-endopeptidase supplementation has been proposed to favour gliadin degradation as an alternative treatment for coeliac disease (CD), although the real usefulness of this therapy in vivo is still under discussion. 1 However, our data point to alternative treatments aiming to modify the intestinal microbiota in patients with CD by the use of probiotics and/or prebiotics. We propose that the induction of gliadin proteolysis in the human gut might not be the solution but the origin of CD

The effect of dietary resistant starch type 2 on the microbiota and markers of gut inflammation in rural Malawi children

BACKGROUND: Resistant starch (RS) decreases intestinal inflammation in some settings. We tested the hypothesis that gut inflammation will be reduced with dietary supplementation with RS in rural Malawian children. Eighteen stunted 3–5-year-old children were supplemented with 8.5 g/day of RS type 2 for 4 weeks. The fecal samples were analyzed for the microbiota, the microbiome, short chain fatty acids, metabolome, and proteins indicative of inflammation before and after the intervention. Subjects served as their own controls. RESULTS: The consumption of RS changed the composition of the microbiota; at the phylum level Actinobacteria increased, while Firmicutes decreased. Among the most prevalent genera, Lactobacillus was increased and Roseburia, Blautia, and Lachnospiracea incertae sedis were decreased. The Shannon H index at the genus level decreased from 2.02 on the habitual diet and 1.76 after the introduction of RS (P < 0.01). Fecal acetate concentration decreased, and fecal propionate concentration increased after RS administration (−5.2 and 2.0 μmol/g, respectively). Fecal calprotectin increased from 29 ± 69 to 89 ± 49 μg/g (P = 0.003) after RS was given. The lipopolysaccharide biosynthesis pathway was upregulated. CONCLUSIONS: Our findings do not support the hypothesis that RS reduces gut inflammation in rural Malawian children. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40168-015-0102-9) contains supplementary material, which is available to authorized users

A 27 kDa protein binds to a positive and a negative regulatory sequence in the promoter of the ICL1 gene from Saccharomyces cerevisiae.

IsocitrateICL1, is one of the key enzymes of the glyoxylate pathway, which operates as an anaplerotic route for replenishing the tricarboxylic acid cycle; it is required for growth of Saccharomyces cerevisiae on carbon sources such as ethanol, but is dispensable when fermentable carbon sources are available. The positive regulation of the ICL1 gene by an upstream activating sequence (UAS) element located between -397 and -388 has been previously reported. In this paper we show that the ICL1 promoter sequence 5'-AGTCCGGACTAGCATCCCAG-3' located between -261 and -242 contains an upstream repressing sequence (URS) element. We have identified and partially purified a 27 kDa protein that binds specifically to both the UAS and URS sequences of the ICL1 promoter. For both UAS and URS, binding requires the protein Snf1 (Cat1), a protein kinase essential for the derepression of genes repressed by glucose. Binding does not take place with extracts from glucose-grown strains, unless they lack Mig1, a negative regulatory protein involved in glucose repression

Factores que influyen en los daños producidos por el oso en la Cordillera Cantábrica y los Pirineos

XV Congreso de la Sociedad Española para la Conservación y Estudio de los Mamíferos (SECEM), Córdoba, 4-7 diciembre de 2021.Peer reviewe