Gut Tissue Research Articles

Global population genetics and diversity in the TAS2R bitter taste receptor family.

Bitter taste receptors (TAS2Rs) are noted for their role in perception, and mounting evidence suggests that they mediate responses to compounds entering airways, gut, and other tissues. The importance of these roles suggests that TAS2Rs have been under pressure from natural selection. To determine the extent of variation in TAS2Rs on a global scale and its implications for human evolution and behavior, we analyzed patterns of diversity in the complete 25 gene repertoire of human TAS2Rs in ∼2,500 subjects representing worldwide populations. Across the TAS2R family as a whole, we observed 721 single nucleotide polymorphisms (SNPs) including 494 nonsynonymous SNPs along with 40 indels and gained and lost start and stop codons. In addition, computational predictions identified 169 variants particularly likely to affect receptor function, making them candidate sources of phenotypic variation. Diversity levels ranged widely among loci, with the number of segregating sites ranging from 17 to 41 with a mean of 32 among genes and per nucleotide heterozygosity (π) ranging from 0.02% to 0.36% with a mean of 0.12%. F ST ranged from 0.01 to 0.26 with a mean of 0.13, pointing to modest differentiation among populations. Comparisons of observed π and F ST values with their genome wide distributions revealed that most fell between the 5th and 95th percentiles and were thus consistent with expectations. Further, tests for natural selection using Tajima’s D statistic revealed only two loci departing from expectations given D’s genome wide distribution. These patterns are consistent with an overall relaxation of selective pressure on TAS2Rs in the course of recent human evolution.

The Hepatic Mitochondrial Alterations Exacerbate Meta-Inflammation in Autism Spectrum Disorders.

The role of the liver in autism spectrum disorders (ASD), developmental disabilities characterized by impairments in social interactions and repetitive behavioral patterns, has been poorly investigated. In ASD, it has been shown a dysregulation of gut–brain crosstalk, a communication system able to influence metabolic homeostasis, as well as brain development, mood and cognitive functions. The liver, with its key role in inflammatory and metabolic states, represents the crucial metabolic organ in this crosstalk. Indeed, through the portal vein, the liver receives not only nutrients but also numerous factors derived from the gut and visceral adipose tissue, which modulate metabolism and hepatic mitochondrial functions. Here, we investigated, in an animal model of ASD (BTBR mice), the involvement of hepatic mitochondria in the regulation of inflammatory state and liver damage. We observed increased inflammation and oxidative stress linked to hepatic mitochondrial dysfunction, steatotic hepatocytes, and marked mitochondrial fission in BTBR mice. Our preliminary study provides a better understanding of the pathophysiology of ASD and could open the way to identifying hepatic mitochondria as targets for innovative therapeutic strategies for the disease.

Oxidative stress, DNA damage, and histological alterations in Bombyx mori exposed orally to pesticide dimethoate

AbstractPesticides are an essential part of agricultural practices that ward off pathogens and diseases from the agricultural crop. However, apart from target organisms, these chemicals also have adverse effects on non‐target organisms. Dimethoate is an insecticide used extensively in agriculture and horticulture practices worldwide. We used the silkworm Bombyx mori as a model organism to study the effect of commercial formulation of dimethoate (Dimethoate‐30% EC) on the gut, silk gland, and fat body tissues. LD50 of dimethoate‐30% EC on silkworm (B. mori) was 997 ppm, as reported in a previous study. We used concentrations of 25, 50, and 100 ppm in our experiments. Our results showed that sub‐lethal doses of dimethoate caused weight loss and induced damage at the histological level to the mid‐gut, silk gland, and fat body of B. mori. It also caused a decrease in the level of antioxidants like CAT, SOD, GPx, GSH, and GST, indicating that dimethoate has produced a shift of ROS balance towards free radical generation and therefore resulted in overall damage to this organism. Sub‐lethal doses of this pesticide also caused lipid peroxidation in the silk gland, gut, and fat body of B. mori, damaging these tissues. The disruption was also seen in the mid‐gut and middle silk gland at the DNA level, where it caused single‐strand breaks, as was revealed by single cell gel electrophoresis studies. Damage at histological, biochemical, and molecular levels was most extreme at a concentration of 100 ppm, the highest sub‐lethal concentration given to B. mori.

GLP-2 as an indicator and modulator of acute inflammation improves cardiac function and survival in sepsis

Abstract Purpose GLP-1 and GLP-2 (glucagon-like peptide-1/2) are gut hormones secreted in response to food. While GLP-1 controls glucose metabolism, GLP-2 is a local gut growth factor regulating intestinal nutrient absorption. GLP-2 has been found to be upregulated in patients with colitis. We hypothesize that beyond its local intestinal function GLP-2 might be involved in systemic immune responses. Methods and results To analyze whether GLP-2 secretion is modulated by the immune system, we measured circulating GLP-2 levels in 2 clinical cohorts. In the first cohort (n=34) GLP-2 levels increased over time following cardiac surgery as an inflammatory stimulus. In the second cohort 223 patients with sepsis had a 3.9 fold increase of GLP-2 plasma levels vs. 53 healthy controls (3.0 ng/mL vs. 11.4 ng/mL; p<0.001). High GLP-2 levels were associated with markers of inflammation (IL-6, PCT, CRP), septic cardiomyopathy (NT-proBNP) and independently predicted mortality in humans with sepsis. Induction of sepsis in mice by endotoxin or cecal ligation puncture strongly increased GLP-2 levels independent from food intake. By injecting various proinflammatory cytokines and inducing sepsis in IL1R−/− and IL6−/− mice we identified that inflammation upregulates GLP-2 secretion through IL-6. To identify the source of GLP-2 secretion under inflammation, we induced sepsis in Gcg−/− mice lacking endogenous GLP-2 production with a tissue-specific reactivation of Gcg in gut L-cells (GcgRAΔvilCre) or pancretic alpha cells (GcgRAΔPDX1-Cre). We observed sepsis-induced GLP-2 secretion to be derived from the pancreas and not from the gut. Additional in-vitro and ex-vivo approaches revealed that IL-6 directly activates GLP-2 secretion from pancreatic alpha cells. Gcg−/− mice lacking GLP-2 production and Glp2r−/− mice show aggravated sepsis indicating that endogenous upregulation of GLP-2 is protective. Finally, we analyzed whether inflammatory upregulation of GLP-2 has immunomodulatory relevance. We administered GLP-2 or saline as control per central jugular vein catheter mice who underwent CLP. GLP-2 treatment improved LV-contractility (dp/dtmax) in septic cardiomyopathy (control 7361 vs. GLP-2 9500 mmHg/s; p<0.01), inhibited sepsis-induced hypotension and reduced mortality (p=0.018). Mechanistically GLP-2 reduced myeloid immune cell infiltration into heart and liver tissue and decreased proinflammatory cytokine levels in various organs and the blood (TNF-α, IL-6 and IL-1β). After broad GLP-2 receptor profiling we found maximum mRNA expression in gut tissues with no expression on immune cells. By further mechanistic studies we found GLP-2 to protect against sepsis-induced gut barrier dysfunction. Conclusions Here we identified a counter-regulatory control system in which IL-6 derived upregulation of GLP-2 secretion limits excessive innate immune responses and protects against sepsis. These findings might open new avenues for the treatment of patients with inflammatory diseases. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): European Foundation for the Study of Diabetes, European Research Area Network on Cardiovascular Diseases (ERA-CVD and BMBF), Deutsche Forschungsgemeinschaft (DFG)

Melatonin alleviates lead-induced intestinal epithelial cell pyroptosis in the common carps (Cyprinus carpio) via miR-17-5p/TXNIP axis

Lead (Pb) has been concerned as one of the most severe hazardous contaminants, because it can cause pyroptosis in multiple tissues of mammals and birds. Melatonin (Mel) has attracted much interest for its role in governing intestinal injury via microRNAs (miRNAs). To explore the effect of Mel on Pb exposure-induced intestinal epithelial cell pyroptosis in common carps by regulating miR-17-5p/TXNIP axis, the Pb exposure and Pb-Mel treated models were constructed in vivo. The results elucidated that the suppressed expression of miR-17-5p and intensified level of TXNIP were primarily detected in Pb-exposed gut tissues, and both abolished with Mel addition, along with downregulated Pb-mediated elevated expression of NLRP3, CASP1, IL1β and GSDMD. Additionally, the targeting relationship between miR-17-5p and TXNIP were demonstrated by dual-luciferase reporter assay, and on this basis, miR-17-5p NC, mimic and inhibitor cell models were established. Thereby, Thereby, the expression of TXNIP in the miR-17-5p mimic groups was significant lower in the Pb-exposure but still elevated than the Control group, and the expression of NLRP3 and NLRP3-dependent pyrotposis-related genes performed consistent alterations. Noticeably, the expression of TXNIP suppressed with Mel addition even in the miR-17-5p inhibitor cell model, resulting in the inactivation of NLRP3 inflammasome-dependent pyroptosis. Overall, we draw the conclusion as Mel attenuates Pb-induced intestinal epithelial cell pyroptosis via miR-17-5p/TXNIP axis. The present study provides a novel perspective for toxicological mechanism of Pb, and new insights for the detoxification mechanism of Mel.

Evaluation of a droplet digital PCR assay for quantification of Mycobacterium avium subsp. paratuberculosis DNA in whole-blood and fecal samples from MAP-infected Holstein cattle.

Bovine paratuberculosis (PTB) is an infectious disease that affects ruminants worldwide and is a burden on the dairy industry. PTB control measures include culling of Mycobacterium avium subsp. paratuberculosis (MAP)-infected animals from the herd and the enhancement of farm-biosecurity measures. Diagnostics tools for the direct detection of MAP are fecal real-time qPCR and bacteriological culture, the last one being considered the gold standard. However, both show limitations for detecting subclinical MAP-infected cattle with low bacterial load in feces and gut tissues. Droplet digital polymerase chain reaction (ddPCR) is a third-generation PCR method that shows high reproducibility for the quantification of low DNA copy numbers. The objective of this study was to design a ddPCR assay to detect and quantify a fragment of the F57 MAP-specific sequence in samples of naturally MAP-infected Holstein cattle. DNA was isolated from whole-blood and fecal samples from control cows with a negative ELISA and qPCR result (N = 75) and from cows with PTB-associated focal (N = 32), multifocal (N = 21), and diffuse lesions (N = 17) in gut tissues. After ddPCR, the DNA extracted from fecal samples of cows with diffuse lesions showed higher mean copies per microliter (13,791.2 copies/μl) than samples from cows with multifocal lesions (78.8 copies/μl), focal lesions (177.1 copies/μl) or control cows (4.8 copies/μl) (P ≤ 0.05). Significant differences in mean DNA copies/μl were also observed in the blood samples from cows with focal lesions (47.7 copies/μl) when compared with cows with multifocal and diffuse lesions; 18.1 and 12.4 copies/μl, respectively. Using a principal component analysis, the results of the fecal ddPCR clustered together with the results of a commercial ELISA for the specific detection of MAP antibodies, fecal and tissue qPCR, and bacteriological culture results. In contrast, blood ddPCR results clustered together with the results of an ELISA for the detection of a biomarker of subclinical PTB, the ABCA13 transporter. Blood ddPCR was the most sensitive tool (sensitivity 71%, specificity 100%) of all the quantitative methods used in the study for the detection of subclinical cows with focal lesions.

Microbiome and metabolome dysbiosis of the gut-lung axis in pulmonary hypertension

Previous studies have suggested that dysbiosis of the gut microbiota is associated with the development of pulmonary hypertension (PH). In this study, we established a left pulmonary artery ligation (LPAL)-induced PH rat model due to high flow and hemodynamic stress and investigated the association between gut microbiota composition and host metabolome signatures (in both gut and lung tissues) by using multiomics and correlation analysis. The results showed that LPAL successfully induced PH, characterized by increased right ventricular systolic pressure, right ventricular hypertrophy and pulmonary vascular remodelling. Moreover, gut pathological abnormalities were observed in association with dramatic alterations in the gut microbiome and metabolome as well as the lung metabolome. The increased bacterial genus Sporobacter and decreased genera Eubacterium, Eubacteriaceae, Deltaproteobacteria and Desulfovibrio featured the altered gut microbiome in LPAL-PH versus SHAM rats. Moreover, imbalanced abundance of protective metabolites (e.g., butyrate, propionate) and pathogenic metabolites (e.g., proinflammatory mediators) were seen in the gut metabolome of LPAL-PH versus SHAM rats. In addition, the altered gut microbiome strongly correlated with the altered metabolome patterns in both the gut and lung of LPAL-PH rats. In conclusion, this study revealed significant gut dysbiosis in LPAL-PH rats, characterized by altered gut microbiota composition, in association with specific changes in gut and lung metabolome profiles. These findings enriched our understanding of the unique signature of the gut microbiome and the close association of the “gut-lung axis” in LPAL-PH induced by long-term high flow, leading to novel therapeutic, diagnostic or management paradigms for this subtype of PH.

MHC class II-restricted-presentationMHC class II and DM molecules in chickens: different yet again

The chicken MHC is simple and compact, with strong genetic associations for resistance to infectious disease, due at least in part to high expression of a single classical class I gene, BF2. The closely-linked TAP and tapasin genes are highly polymorphic, and co-evolve with the dominantly-expressed BF2 gene, due to the rarity of recombination between genes of this system. The classical class II system has some similarities to the class I system, with two class II B (BLB), one DMA and two DMB genes, all of which are closely-linked and polymorphic. Like β2-microglobulin, the class II A gene is monomorphic and located distant to the MHC. Only BLB2 is well-expressed in spleen and blood. The objective is to determine to what extent the chicken class II system follows the same principles as the class I system. RNA levels were determined by RT-PCR and RT-qPCR, proteins were detected by Western blot, flow cytometry and tissue section staining, and genes were knocked out (KO) by CRISPR-Cas9. RNAs for BLB2 and DMB2 are widely expressed, while BLB1 and DMB1 are only strongly expressed in gut tissues. Tissue staining shows very high DMB1 (and BLB1) staining in intestinal epithelial cells (IECs), while sorted IECs show similar levels for DMB1 and DMB2 by RT-PCR, but only DMB1 by western blot. Both BLB1 and BLB2 class II molecules mostly bear CLIP peptides in DMA KO cells, but some alleles are exquisitely DM-dependent and others less so. Both BLB1 and BLB2 are affected in DMB1 and DMB2 KO cells. The presence of high level DMB1 (and BLB1) expression in IECs but not elsewhere is unprecedented for class II molecules, and the exact ways in which the different DM loci and alleles affect the two class II molecules remain interesting questions still to answer.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates β-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

Background: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. β-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. Results: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. β-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. Conclusions: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces β-catenin expression in ob/ob mice.

Intestinal permeability and gene expression after polyethylene and polyamide microplastic ingestion in Wistar rats

Microplastic particles are ubiquitous in the environment. However, little is known about their uptake and effects in humans or mammalian model organisms. Here, we studied the effects of pristine polyamide (15–20 µm) and polyethylene (40–48 µm) particles after oral ingestion in rats. The animals received feed containing microplastic particles (0.1% polyamide or polyethylene, or a mixture of both polymers) or a control diet without microplastic particles, for 5 weeks. The permeability of the duodenum was investigated in an Ussing chamber, whereas gene expression and concentration of tight junction proteins were measured in gut tissue and plasma. Microplastic particles were quantified by pyrolysis-gas chromatography/mass spectrometry in rats’ feces. Rats fed with microplastic particles had higher duodenal permeability. Expression of gene coding for the tight junction protein occludin (OCLN) was higher in PE treated animals compared to control or the PA group. No changes in the expression of the gene coding for zonula occludens protein 1 were detected. Occludin protein concentrations were below the limit of detection of the applied method in both gut and plasma. Zonula occludens protein 1 concentrations in the gut were significantly higher in groups exposed to PA and PE as compared to control, while zonula occludens protein 1 concentrations in plasma did not show significant changes. These results demonstrated that short-term exposure to a dose of 0.1% (w/w) microplastic particles in feed had limited effects on duodenal permeability, expression of pro-inflammatory protein genes and tight junction protein genes in the duodenum.

Localized efficacy of environmental RNAi in Tetranychus urticae

Environmental RNAi has been developed as a tool for reverse genetics studies and is an emerging pest control strategy. The ability of environmental RNAi to efficiently down-regulate the expression of endogenous gene targets assumes efficient uptake of dsRNA and its processing. In addition, its efficiency can be augmented by the systemic spread of RNAi signals. Environmental RNAi is now a well-established tool for the manipulation of gene expression in the chelicerate acari, including the two-spotted spider mite, Tetranychus urticae. Here, we focused on eight single and ubiquitously-expressed genes encoding proteins with essential cellular functions. Application of dsRNAs that specifically target these genes led to whole mite body phenotypes—dark or spotless. These phenotypes were associated with a significant reduction of target gene expression, ranging from 20 to 50%, when assessed at the whole mite level. Histological analysis of mites treated with orally-delivered dsRNAs was used to investigate the spatial range of the effectiveness of environmental RNAi. Although macroscopic changes led to two groups of body phenotypes, silencing of target genes was associated with the distinct cellular phenotypes. We show that regardless of the target gene tested, cells that displayed histological changes were those that are in direct contact with the dsRNA-containing gut lumen, suggesting that the greatest efficiency of the orally-delivered dsRNAs is localized to gut tissues in T. urticae.

Hypergastrinemia in Children : A Case Report

Gastrin is an important hormone in the gastrointestinal system that promotes gastric acid secretion. Gastrin hormone is produced by the G-cells in the antrum of the stomach. Besides stimulating gastric acid secretion, gastrin also induces the proliferation of the gut epithelial cells, tissue remodelling, and angiogenesis. Gastrin levels higher than 100-150 pg/ml are known as hypergastrinemia. Hypergastrinemia may cause the hypersecretion of stomach acid, which, if not treated properly, may leads to refractory peptic ulcer, severe gastroesophageal reflux disease (GERD), diarrhea, or death due to complications of refractory peptic ulcer. This case presented a 12 years old boy with a chief complaint of severe epigastric pain in the past month, accompanied by nausea, especially during supine position. The patient had a previous history of esophagitis. He showed no significant changes upon empirical PPI treatment. However, slight improvements were observed after the administration of Helicobacter pylori treatment. The gastrin level in this patient was 198 pg/mL. Upon discharge, the patient was still given PPI treatment. During the follow-up visitation, patient complaints had improved significantly, and the patient was planned to undergo routine evaluations of gastrin.

Genoprotective activity of the Pleurotus eryngii mushrooms following their in vitro and in vivo fermentation by fecal microbiota.

Pleurotus eryngii mushrooms are commercially cultivated and widely consumed due to their organoleptic properties, and the low caloric and high nutritional value. In addition, they contain various biologically active and health-promoting compounds; very recently, their genoprotective effect in Caco-2 cells after their fermentation by the human fecal microbiota was also documented. In the current study, the effect of P. eryngii pre- and post-fermentation supernatants in micronuclei formation was evaluated in human lymphocytes. In addition, the genoprotective properties of increasing concentrations of aqueous extracts from P. eryngii mushrooms (150, 300, 600 mg/kg) against the cyclophosphamide-induced DNA damage were studied in young and elderly female and male mice in bone marrow and whole blood cells. The ability of the highest dose (600 mg/kg) to regulate the main cellular signaling pathways was also evaluated in gut and liver tissues of female animals by quantifying the mRNA expression of NrF2, Nfkβ, DNMT1, and IL-22 genes. P. eryngii post-fermentation, but not pre-fermentation, supernatants were able to protect human lymphocytes from the mitomycin C-induced DNA damage in a dose-dependent manner. Similarly, genoprotection was also observed in bone marrow cells of mice treated by gavage with P. eryngii extract. The effect was observed in all the experimental groups of mice (young and elderly, male and female) and was more potent in young female mice. Overexpression of all genes examined was observed in both tissues, mainly among the elderly animals. In conclusion, P. eryngii mushrooms were shown to maintain genome integrity through protecting cells from genotoxic insults. These beneficial effects can be attributed to their antioxidant and immunomodulatory properties, as well as their ability to regulate the cell’s epigenetic mechanisms and maintain cell homeostasis.

PARP1 inhibition enhances reactive oxygen species on gut microbiota.

Poly(ADP-ribose) polymerase 1 (PARP1) plays a key role in genome stability by modulating DNA-damage responses. Activated by DNA interruptions through ultraviolet (UV) exposure, PARylation is synthesized by PARP1 and serves as a survival mechanism for cancer and metabolic diseases. Several strategies including ROS and antimicrobial peptides(AMPs) function in host defenses, while the targeted tissue and mechanism under DNA damage are unknown. Here, we show that DNA damage induces responses specifically in the gut tissue. The knockdown of PARP1 reduces the activation of PARylation. Parp1 knockdown under DNA damage results in over-accumulated ROS and secretion of AMPs through the regulation of Relish, a subunit of nuclear factor-κB(NF-κB). Double-knockdown of Parp1 and Relish specifically in the gut inhibits AMP secretion. In conclusion, the host defense is achieved through ROS accumulation rather than the AMPs under DNA damage. In contrast, the knockdown of PARP1 exacerbates ROS accumulation to a harmful level. Under this circumstance, NF-κb targeted AMP secretion is provoked for host defense. Microbiome and functional analysis provide evidence for the hazard of DNA damage and show variations in the metabolic pathways following Parp1 inhibition. Our findings suggest the notion that PARP1 inhibition contributes to ROS accumulation under DNA damage and its role in NF-κb activation for host defense.

RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin

BackgroundDue to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella.ResultsPresent study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein–protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process.ConclusionSince the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.

Repetitive transcranial direct current stimulation modulates the brain–gut–microbiome axis in obese rodents

BackgroundComplex interactions between the brain, gut and adipose tissue allow to recognize obesity as a neurometabolic disorder. The recent data have shown that gut microbiota can play a potential role in obesity development. Transcranial direct current stimulation (tDCS) is a safe and non-invasive technique to modulate the activity of cerebral cortex and other connected brain areas also in context of appetite control. The objective of this study was to evaluate the effects of repetitive anodal tDCS (AtDCS) of prefrontal cortex on feeding behavior, metabolic status and selected phyla of gut microbiota in rats with obesity induced by high-calorie diet (HCD).Methods32 female Wistar rats were equally divided into 4 subgroups depending on diet effect (lean versus obese) and type of stimulation (active versus sham tDCS versus no stimulation). Feed intake, body weight, blood lipoproteins and leptin levels as well as Firmicutes and Bacteroidetes in intestines and stool were examined.ResultsHCD changed feeding behavior and metabolic parameters typically for obesity-related ranges and resulted in an abundance of Firmicutes at the expanse of Bacteroidetes in the large intestine and stool. AtDCS decreased appetite, body weight, and cholesterol levels. In addition, AtDCS reduced ratio of the average number of Firmicutes to average number of Bacteroidetes in all examined tissues.ConclusionsRepetitive AtDCS is not only effective for appetite restriction but can also modulate gut microbiome composition which demonstrates the existence of the brain–gut–microbiome axis and points at this technique as a promising complementary treatment for obesity. However, the effects should be further replicated in human studies.

Anatomical Laser Microdissection of the Ileum Reveals mtDNA Depletion Recovery in A Mitochondrial Neuro-Gastrointestinal Encephalomyopathy (MNGIE) Patient Receiving Liver Transplant.

mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE) is a rare genetic disorder characterized by thymidine phosphorylase (TP) enzyme defect. The absence of TP activity induces the imbalance of mitochondrial nucleotide pool, leading to impaired mitochondrial DNA (mtDNA) replication and depletion. Since mtDNA is required to ensure oxidative phosphorylation, metabolically active tissues may not achieve sufficient energy production. The only effective life-saving approach in MNGIE has been the permanent replacement of TP via allogeneic hematopoietic stem cell or liver transplantation. However, the follow-up of transplanted patients showed that gut tissue changes do not revert and fatal complications, such as massive gastrointestinal bleeding, can occur. The purpose of this study was to clarify whether the reintroduction of TP after transplant can recover mtDNA copy number in a normal range. Using laser capture microdissection and droplet-digital-PCR, we assessed the mtDNA copy number in each layer of full-thickness ileal samples of a naive MNGIE cohort vs. controls and in a patient pre- and post-TP replacement. The treatment led to a significant recovery of gut tissue mtDNA amount, thus showing its efficacy. Our results indicate that a timely TP replacement is needed to maximize therapeutic success before irreversible degenerative tissue changes occur in MNGIE.

Novel Antimicrobial Peptide "Octoprohibitin" against Multidrug Resistant Acinetobacter baumannii.

Octoprohibitin is a synthetic antimicrobial peptide (AMP), derived from the prohibitin-2 gene of Octopus minor. It showed substantial activity against multidrug resistant (MDR) Acinetobacter baumannii with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 200 and 400 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of octoprohibitin against A. baumannii. The morphology and ultrastructure of A. baumannii were altered by treatment with octoprohibitin at the MIC and MBC levels. Furthermore, propidium iodide-fluorescein diacetate (PI-FDA) staining and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining of octoprohibitin-treated A. baumannii revealed membrane permeability alterations and reactive oxygen species (ROS) generation, respectively. Agarose gel retardation results confirmed the DNA-binding ability of octoprohibitin to the genomic DNA of A. baumannii. Furthermore, octoprohibitin showed concentration-dependent inhibition of biofilm formation and eradication. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of octoprohibitin were 1000 and 1460 µg/mL, respectively. Octoprohibitin produced no significant cytotoxicity up to 800 µg/mL, and no hemolysis was observed up to 400 µg/mL. Furthermore, in vivo analysis in an A. baumannii-infected zebrafish model confirmed the effective bactericidal activity of octoprohibitin with higher cumulative survival percent (46.6%) and fewer pathological signs. Histological analysis showed reduced alterations in the gut, kidney, and gill tissues in the octoprohibitin-treated group compared with those in the phosphate-buffered saline (PBS)-treated group. In conclusion, our results suggest that octoprohibitin is a potential antibacterial and antibiofilm agent against MDR A. baumannii.

Gut Microbiota-Derived Tryptophan Metabolites Maintain Gut and Systemic Homeostasis.

Tryptophan is an essential amino acid from dietary proteins. It can be metabolized into different metabolites in both the gut microbiota and tissue cells. Tryptophan metabolites such as indole-3-lactate (ILA), indole-3-acrylate (IAC), indole-3-propionate (IPA), indole-3-aldehyde (IAID), indoleacetic acid (IAA), indole-3-acetaldehyde and Kyn can be produced by intestinal microorganisms through direct Trp transformation and also, partly, the kynurenine (Kyn) pathway. These metabolites play a critical role in maintaining the homeostasis of the gut and systematic immunity and also potentially affect the occurrence and development of diseases such as inflammatory bowel diseases, tumors, obesity and metabolic syndrome, diseases in the nervous system, infectious diseases, vascular inflammation and cardiovascular diseases and hepatic fibrosis. They can not only promote the differentiation and function of anti-inflammatory macrophages, Treg cells, CD4+CD8αα+ regulatory cells, IL-10+ and/or IL-35+B regulatory cells but also IL-22-producing innate lymphoid cells 3 (ILC3), which are involved in maintaining the gut mucosal homeostasis. These findings have important consequences in the immunotherapy against tumor and other immune-associated diseases. We will summarize here the recent advances in understanding the generation and regulation of tryptophan metabolites in the gut microbiota, the role of gut microbiota-derived tryptophan metabolites in different immune cells, the occurrence and development of diseases and immunotherapy against immune-associated diseases.

756 Skin wounding alters the gut microbiome by inducing antimicrobial peptide and mucin production from intestinal epithelia

Intestinal and skin microflora can influence several systemic health behaviors. In this study, we hypothesized that skin and gut tissue microbiota are linked, and that skin health can control the gut microbiota. To test this, we compared intestinal responses in control mice to co-housed littermates with full-thickness incisions on back skin or K14Cre-targeted expression of HYAL-1 in the epidermis. The latter mice represent a skin-specific model of the hyaluronidase activity produced by wounding but without a systemic inflammatory or stress response.