Fecal Extracts Research Articles

Prediction of functional proteins associated with the gut microbiome of an adult population in Lagos State, Nigeria

BackgroundThe human gut microbiome is implicated in health and disease. Yet, its functions in healthy adults from a divergent population like those seen in Africa are quite unclear. We set to reveal the roles of the gut microbiome of a healthy rural and urban Yoruba population of Lagos State. MethodologySociodemographic and clinical data, and fecal samples were obtained from rural (n = 10) and urban (n = 10) participants. fecal DNA extraction was done using ZR Fecal DNA MiniPrep™ D6010 and metagenomic next generation sequencing performed on Illumina Miseq platform. Functional roles prediction and antibiotics resistance genes (ARGs) were done using the Clusters of Orthologous Groups (COG) and Comprehensive Antibiotic Resistance Database (CARD) tools, respectively. The potential link between phenotypes and COG abundance were inferred using principal component analysis (PCA). ResultsWe found 12 (urban) and 11 (rural) COG classes of proteins from the gut microbes. Class E protein which denotes amino acid transport and metabolism was unique to urban respondents and was dominated by 5-enolpyruvylshikimate-3-phosphate synthase (45%) and asparagine synthase (30%), implying a high protein-based diet. Class S was found in both groups. COG families of proteins revealed the presence of exo-beta-1,3-glucanase (31%), pyruvate-decarboxylase, and thiamine pyrophosphate-requiring enzymes (22%) among the rural respondents’ indicative of carbohydrate-rich diets. In addition, uncharacterized protein linked to hypothetical functions was found in both. The COG proteins clustered with female sex, BMI (body mass index) and BP (blood pressure) for urban while for the rural populace, it clustered with height, BMI, and BP. CARD analysis showed ARGs in both groups (urban 90%; rural 10%) and all were Escherichia coli 16S rRNA (rrsB) mutation conferring resistance to streptomycin with gene variant model A523C n/a to aminoglycoside antibiotics. ConclusionThe study defined the classes of proteins from gut microbes of urban and rural respondents and highlighted the presence of uncharacterized proteins linked to hypothetical and unknown functions. Differences seen among the predicted roles appear to be driven by diet, clinical and sociodemographic factors. The higher abundance of ARGs observed in the urban population poses a serious public health concern with regards to the spread of antibiotic resistance.

Reevaluation of Piezo1 as a gut RNA sensor.

Piezo1 is a stretch-gated ion channel required for mechanosensation in many organ systems. Recent findings point to a new role for Piezo1 in the gut, suggesting that it is a sensor of microbial single-stranded RNA (ssRNA) rather than mechanical force. If true, this would redefine the scope of Piezo biology. Here, we sought to replicate the central finding that fecal ssRNA is a natural agonist of Piezo1. While we observe that fecal extracts and ssRNA can stimulate calcium influx in certain cell lines, this response is independent of Piezo1. Additionally, sterilized dietary extracts devoid of gut biome RNA show similar cell line-specific stimulatory activity to fecal extracts. Together, our data highlight potential confounds inherent to gut-derived extracts, exclude Piezo1 as a receptor for ssRNA in the gut, and support a dedicated role for Piezo channels in mechanosensing.

An Optimised Monophasic Faecal Extraction Method for LC-MS Analysis and Its Application in Gastrointestinal Disease.

Liquid chromatography coupled with mass spectrometry (LC-MS) metabolomic approaches are widely used to investigate underlying pathogenesis of gastrointestinal disease and mechanism of action of treatments. However, there is an unmet requirement to assess faecal metabolite extraction methods for large-scale metabolomics studies. Current methods often rely on biphasic extractions using harmful halogenated solvents, making automation and large-scale studies challenging. The present study reports an optimised monophasic faecal extraction protocol that is suitable for untargeted and targeted LC-MS analyses. The impact of several experimental parameters, including sample weight, extraction solvent, cellular disruption method, and sample-to-solvent ratio, were investigated. It is suggested that a 50 mg freeze-dried faecal sample should be used in a methanol extraction (1:20) using bead beating as the means of cell disruption. This is revealed by a significant increase in number of metabolites detected, improved signal intensity, and wide metabolic coverage given by each of the above extraction parameters. Finally, we addressed the applicability of the method on faecal samples from patients with Crohn's disease (CD) and coeliac disease (CoD), two distinct chronic gastrointestinal diseases involving metabolic perturbations. Untargeted and targeted metabolomic analysis demonstrated the ability of the developed method to detect and stratify metabolites extracted from patient groups and healthy controls (HC), highlighting characteristic changes in the faecal metabolome according to disease. The method developed is, therefore, suitable for the analysis of patients with gastrointestinal disease and can be used to detect and distinguish differences in the metabolomes of CD, CoD, and HC.

Predictors of testosterone in zoo-managed male African elephants (Loxodonta Africana).

Reproductive complications for both male and female zoo-managed African elephants (Loxodonta africana) contribute to the rapidly declining population. In zoo-managed bull elephants, few studies have explored the potential physiological, physical, social, and environmental factors that influence bull fertility, particularly, androgen production. Testosterone is the essential steroid hormone for male sexual maturation and inadequate concentrations can be detrimental for spermatogenesis. Testosterone, fecal glucocorticoid metabolites, leptin, glucose, insulin, and triglycerides were analyzed from weekly fecal and blood serum samples taken over 6 months from six zoo-managed African elephant bulls (10-19 years of age). Testosterone levels were compared to endocrine factors, weekly social and environmental variables, daily musth signs, and body condition scores (BCS). The glucose-to-insulin ratio (G:I) was the only physiological biomarker found to be positively associated with testosterone. Predictive physical variables included Musth Score (+) and Moderate Exercise (+). Bulls with BCSsignifying overweight (BCS 4) had lower testosterone (36.6 ± 1.6 ng/g fecal extraction [FE]) than bulls with healthyBCS 3; 51.2 ± 4.9 ng/g FE). Numerous social variables influenced testosterone concentrations, including Total Contact Day (+), Female Interaction Day (+), Indirect Contact Day (+), Indirect Contact Night (+) and Total No Contact (-). Both percentage of Time Outdoor and Time Mixed positively influenced testosterone, whereas testosterone decreased for percentage of Time Indoors. Each additional daily browse opportunity increased testosterone by approximately 7 ng/g FE. In managed care, the emphasis should be placed on optimizing these predictors of testosterone production to promote bull reproductive health.

Comparative analysis of different fecal DNA extraction methods

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.

Polysaccharide, fecal microbiota, and curcumin-based novel oral colon-targeted solid self-nanoemulsifying delivery system: formulation, characterization, and in-vitro anticancer evaluation

The use of polysaccharides in preparation of microbially triggered colon-targeted delivery system has been explored extensively to deliver drugs at the colonic site. This type of targeting is attributed to inherent property of polysaccharides by virtue of which they are digested in the gut only by the colonic microflora. In last two decades, the use of fecal microbiota transplantation (FMT) has been reported widely to treat colonic diseases such as Crohn's disease, irritable bowel syndrome, and recurrent Clostridium difficile infection. It has been established that FMT downregulates inflammatory proliferation and pro carcinogenic pathways. Although a few reports exist on the use of colon-targeted FMT capsules in increasing the microbial diversity of gut microflora, they have not been able to establish clinical superiority of the colon-targeted capsules over conventional orally administered capsules. Hence, in the present study, an attempt has been made to utilize a multipronged strategy through a single formulation, wherein an anticancer phytoconstituent, polysaccharides, fecal microbiota extract (FME), and nanodelivery systems have been designed for the effective treatment of colon cancer. Overall, the study is aimed toward the development of an oral colon-targeted solid self-nanoemulsifying drug delivery system (S-SNEDDS) in which curcumin (CCM) was loaded. These SNEDDS were added to polysaccharide mixture of guar gum, pectin, lactose along with aerosol 200 (A-200) and then to the slurry of fecal microbiota extract. The liquid was then freeze dried and developed into solid SNEDDS powder. The process of formulating liquid SNEDDS was optimized using ternary phase diagram and Box Behnken Design. The developed formulation was characterized for physicochemical, physicomechanical, drug release and cytotoxic potential studies. The droplet size of reconstituted S-SNEDDS was found to be 78.46 ± 0.87 nm. The site-specific release of S-SNEDDS powder was confirmed by dissolution studies in medium containing rat caecal contents, wherein, the formulation containing guar gum and pectin in 1% w/w ratio showed less than 10% drug release in initial 5 h and burst release between 5th and 10th h indicating colon-specific release of CCM. Significant cytotoxic effect was seen for CCM-loaded S-SNEDDS as compared to control formulations. The use of 16s metagenomic technique confirmed no significant change in fecal microbiota composition of formulation pre- and postlyophilization. Overall, the study suggested a novel, oral colon-targeted SNEDDS-based formulation containing fecal microbiota and CCM for the treatment of colon cancer.

Stability of Saxitoxin in 50% Methanol Fecal Extracts and Raw Feces from Bowhead Whales (Balaena mysticetus).

In recent decades, harmful algal blooms (HABs) producing paralytic shellfish toxins (including saxitoxin, STX) have become increasingly frequent in the marine waters of Alaska, USA, subjecting Pacific Arctic and subarctic communities and wildlife to increased toxin exposure risks. Research on the risks of HAB toxin exposures to marine mammal health commonly relies on the sampling of marine mammal gastrointestinal (GI) contents to quantify HAB toxins, yet no studies have been published testing the stability of STX in marine mammal GI matrices. An understanding of STX stability in test matrices under storage and handling conditions is imperative to the integrity of toxin quantifications and conclusions drawn thereby. Here, STX stability is characterized in field-collected bowhead whale feces (stored raw in several treatments) and in fecal extracts (50% methanol, MeOH) over multiple time points. Toxin stability, as the percent of initial concentration (T0), was reported for each storage treatment and time point. STX was stable (mean 99% T0) in 50% MeOH extracts over the 8-week study period, and there was no significant difference in STX concentrations quantified in split fecal samples extracted in 80% ethanol (EtOH) and 50% MeOH. STX was also relatively stable in raw fecal material stored in the freezer (mean 94% T0) and the refrigerator (mean 93% T0) up to 8 weeks. STX degraded over time in the room-temperature dark, room-temperature light, and warm treatments to means of 48 ± 1.9, 38 ± 2.8, and 20 ± 0.7% T0, respectively, after 8 weeks (mean ± standard error; SE). Additional opportunistically analyzed samples frozen for ≤4.5 years also showed STX to be relatively stable (mean 97% T0). Mean percent of T0 was measured slightly above 100% in some extracts following some treatments, and (most notably) at some long-term frozen time points, likely due to evaporation from samples causing STX to concentrate, or variability between ELISA plates. Overall, these results suggest that long-term frozen storage of raw fecal samples and the analysis of extracts within 8 weeks of extraction in 50% MeOH is sufficient for obtaining accurate STX quantifications in marine mammal fecal material without concerns about significant degradation.

Host‐marking pheromone of the South American fruit fly Anastrepha fraterculus and cross‐recognition by the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae)

AbstractFollowing oviposition, females of many fruit flies deposit on the fruit surface a host‐marking pheromone (HMP) that can deter oviposition by conspecifics and heterospecifics, thereby minimizing overexploitation of hosts. We describe the oviposition behaviour of two economically important fruit flies (Anastrepha fraterculus and Ceratitis capitata) in agar spheres marked with methanolic and aqueous faeces extracts that contain the HMP. Laboratory bioassays revealed that A. fraterculus mated female produces an HMP that can be extracted from faeces, and causes a significant reduction in fruit infestation by both conspecifics and heterospecifics (cross‐recognition). Furthermore, mated female faeces extract contains higher amounts of methyl (9E)‐hexadecenoate, methyl hexadecanoate, methyl linoleate and methyl (9Z)‐octadecenoate than male faeces extract. Our results provide evidence for the potential use of this ovipositional deterrent in crop protection and pest management programmes for A. fraterculus and C. capitata.

Verification of the fCAL turbo immunoturbidimetric assay for measurement of the fecal calprotectin concentration in dogs and cats.

The concentration of calprotectin in feces (fCal) is a clinically useful marker of chronic gastrointestinal inflammation in humans and dogs. No commercial assay is widely available to measure fCal in small animal medicine, to date. Thus, we verified the immunoturbidimetric fCAL turbo assay (Bühlmann) of fCal for canine and feline fecal extracts by determining linearity, spiking and recovery, and intra-assay and inter-assay variability. We determined RIs, temporal variation over 3 mo, and effect of vaccination and NSAID treatment. Observed:expected (O:E) ratios (x̄ ± SD) for serial dilutions of feces were 89–131% (106 ± 9%) in dogs and 77–122% (100 ± 12%) in cats. For spiking and recovery, the O:E ratios were 90–118% (102 ± 11%) in dogs and 83–235% (129 ± 42%) in cats. Intra- and inter-assay CVs for canine samples were ≤19% and ≤7%, and for feline samples ≤22% and ≤21%. Single-sample RIs were <41 μg/g for dogs and <64 μg/g for cats. With low reciprocal individuality indices, using population-based fCal RIs is appropriate, and moderate fCal changes between measurements (dogs 44.0%; cats: 43.2%) are considered relevant. Cats had significant (but unlikely relevant) fCal increases post-vaccination. Despite individual fCal spikes, no differences were seen during NSAID treatment. The fCAL turbidimetric assay is linear, precise, reproducible, and sufficiently accurate for measuring fCal in dogs and cats. Careful interpretation of fCal concentrations is warranted in both species during the peri-vaccination period and for some patients receiving NSAID treatment.

Orlistat, a competitive lipase inhibitor used as an antiobesity remedy, enhances inflammatory reactions in the intestine

Alterations in secondary gut metabolites derived from the microbial fermentation of food in the gut have significant effects on various aspects of host physiology. Our recent studies on obese mice treated with Orlistat, an antiobesity treatment, revealed a significantly altered gut microbial profile marked by an over-abundance of Proteobacteria and alterations in secondary gut metabolites. In this study, we determined effect of fecal metabolites from high-fat diet fed mice treated with Orlistat (HFDOrl) on colonic epithelial cells in relation to inflammation, barrier function, mitochondrial activity, reactive oxygen species (ROS) levels, and oxidative stress. Quantitative PCR was used to measure intestinal mRNA expression of oxidative stress, inflammation, apoptosis, and gut barrier function genes in mice on a high fat diet with and without Orlistat treatment versus those fed a low-fat diet (HFDOrl, HFD, Normal diet-fed [ND] respectively). Alterations to antioxidant function in HCT-116-ARE-luciferase stable cell line and mitochondrial function in Caco-2 cells was analyzed under oxidative stress with exposure to aqueous fecal extracts from HFDOrl, HFD, and ND groups. The results of this study indicate that a significant increase in anti-oxidative response was observed based on the luciferase activity of HCT-116-ARE-luciferase stable cells. Increased maximal respiration and mitochondrial ROS under oxidative stress was also detected in confluent Caco-2 cells resulting from exposure to fecal extracts from the HFDOrl group compared with the HFD group and pure Orlistat. Furthermore, mice from the HFDOrl group exhibited a significant increase in colonic epithelial expression of oxidative markers (Nrf-2 and SOD-2), inflammation-related markers (IL-6 and TNF-α), and gut barrier function markers (Muc-2 and Occludin). Taken together, the results suggest that Orlistat treatment in the HFD group causes changes in secondary gut metabolites which affect the colonic redox state and may eventually lead to the development of inflammatory, oxidative, and mitochondrial dysfunction at the cellular level.

Faecal Concentrations of Progesterone and 17β-Oestradiol of Female Malayan Tapir from Different Faecal Hormone Extraction Methods

Non-invasive hormone monitoring of endangered wildlife provides an essential tool to optimize breeding strategies, mainly in captive management. However, the protocol of reproductive hormone monitoring on pregnancy status using non-invasive faecal samples in Malayan tapir is still inconclusive. Therefore, we compared the metabolites reproductive hormones; 17β-oestradiol and progesterone in the faecal samples extracted using methods of Schwarzenberger et al. (1996) (Methods A and B), Brown et al. (2001) (Method C) and Shutt et al. (2012) (Method D) from pregnant and non-pregnant captive Malayan Tapir. Faecal samples from four female of Malayan Tapir (Tapirus indicus) were collected for five months at Sungai Dusun Wildlife Reserve (n = 2), Zoo Taiping (n = 1) and Zoo Negara (n = 1), Malaysia. Analysis by liquid chromatography-mass spectrometry (LCMS) confirmed that progesterone was detected in all fecal sample extracted by extraction Method A, while 17β-oestradiol was undetectable in all methods. The measurement of reproductive hormones for pregnancy status via Enzyme-Linked Immunosorbent Assay (ELISA) analysis showed no significant difference (p &gt; 0.05) for progesterone between the fecal extraction methods. Yet the data obtained were not able to validate the pregnancy status, due to similarity in concentration for both pregnant and non-pregnant tapirs. While for 17β-oestradiol, significant results were observed in all extraction methods and in pregnancy status (p &lt; 0.05). Method C was found to be the most reliable extraction method (p &lt; 0.05) to indicate pregnancy status. From the results, it showed that solvent used, boiling process and multiphase extraction plays important role in fecal extraction process in Malayan Tapir.

Fecal extract from obese horses induces an inflammatory response by murine macrophages in vitro.

To compare the inflammatory response of murine macrophages exposed to the enteric microbiome of obese horses versus nonobese horses. Fecal samples from 12 obese horses (body condition score ≥ 7/9) and 12 nonobese horses (body condition score 4 to 5/9) with similar dietary management. Fecal supernatant was prepared from frozen fecal samples. RAW 264.7 macrophage cells were exposed to the fecal extract. Inflammatory cytokine (interleukin-1β, tumor necrosis factor-α, and interleukin-6) gene expression was quantified via real-time quantitative reverse transcription PCR assay, and cytokine concentration was quantified via ELISA. Lipopolysaccharide was evaluated in fecal extract via chromo-limulus amoebocyte lysate assay. Compared with fecal extracts from nonobese horses, fecal extracts from obese horses presented higher concentrations of lipopolysaccharide and induced a heightened expression of the proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, and interleukin-6 from macrophages. The increased levels of inflammatory markers induced in murine macrophages by the microbiome of obese horses in vitro suggested important differences in the enteric microbial composition of these horses, compared with nonobese horses. Overall, this study showed that the microbiome may play a role in mediating an inflammatory response within the gastrointestinal tract of obese horses. Mechanisms of obesity in the horse have not been fully elucidated. Improved understanding of the pathophysiology of disease will guide future research into potential diagnostic and therapeutic interventions for equine obesity.

Orlistat, an anti‐obesity remedy as a competitive lipase inhibitor, enhanced inflammatory reaction in the intestine

Secondary gut metabolites derived from microbial fermentation of food in the gut have broad impacts on various aspects of host physiology. Our recent studies on obese mice treated with Orlistat, an anti‐obesity treatment, showed significantly altered gut microbial profile marked by an overabundance of Proteobacteria and possible alterations in secondary gut metabolites. Consequently, aqueous fecal extracts from treated and untreated mice groups were analyzed for anti‐oxidant and mitochondrial altering function using HCT‐116‐ARE and Caco2 cells under oxidative stress. The results of this study showed that fecal extracts in groups containing increased cecal concentraations of butyrate and propionate had reduced fold change in luciferase activity, thus, alleviating oxidative stress. Conversely, fecal extracts from high fat/orlistat treated mice resulted in a significant increase in fold change in the luciferase activity and maximal ATP production of cells under oxidative stress. Changes in secondary gut metabolites during obesity treatment may provide the necessary insight into the possible side effects seen during intervention.

Paneth cells regulate allergic sensitization in the gastrointestinal tract

Abstract The gut immune homeostasis plays a major role in controlling sensitization to food allergens and the development of allergic responses in the gastrointestinal tract, but also to skin or lung. Epithelial cells sensing of commensal microbes is crucial for the maturation of the gut immune system and antibiotic treatment during early life is believed to have contributed to the increased incidence of food allergy. Paneth cells secrete antimicrobial products and cytokines in the crypts of the small intestine, but their role in food allergy remains elusive. We found that SOX9ΔIEC mice, which lack Paneth cells, display dysbiosis with increased microbial diversity. Interestingly, Paneth cell deficiency increased the of numbers of ILC2 in intestinal lymphoid tissues. Upon oral sensitization with a model food antigen in the presence of cholera toxin, SOX9ΔIEC mice developed significantly higher allergen-specific serum IgE responses, and enhanced signs of allergic responses (i.e., swelling, hypothermia, impairment of lung functions) after nasal allergen challenge. Transplantation of fecal materials from SOX9ΔIEC mice was able to transfer the hyper-allergenic phenotype to control wild-type mice. In addition, bacteria-free fecal extract from SOX9ΔIEC mice increased the number of IgE producing B cells than fecal extract from control mice with treatment of anti-CD40 and IL-4 in vitro. Ongoing studies are assessing the metabolites present in the intestinal lumen of SOX9ΔIEC mice and their effect of intestinal epithelial cells and myeloid cells to elucidate the mechanisms by which impaired or defective anti-microbial functions of Paneth cells regulate allergic sensitization of the GI tract. Supported by Ohio State Univerisity NIH

Identification of conserved coronavirus epitopes targeted by antibodies after SARS-CoV-2 infection or vaccination

Abstract SARS-CoV-2 is a novel betacoronavirus that causes coronavirus disease 2019 and has resulted in millions of deaths worldwide. Novel coronavirus infections in humans have steadily become more common. Understanding antibody responses to SARS-CoV-2, and identifying conserved, cross-reactive epitopes among coronavirus strains could inform the design of vaccines and therapeutics with broad application. Here, we determined that individuals with previous SARS-CoV-2 infection or vaccinated with the Pfizer-BioNTech BNT162b2 vaccine produced antibody responses that cross-reacted with related betacoronaviruses. Moreover, we designed a peptide-conjugate vaccine with a conserved SARS-CoV-2 S2 spike epitope, immunized mice and determined cross-reactive antibody binding to SARS-CoV-2 and other related coronaviruses. This conserved spike epitope also shared sequence homology to proteins in commensal gut microbiota and antibodies targeting this region reacted with proteins in human fecal protein extracts. This indicated that the composition of the gut microbiota could prime immune responses in humans. Thus, SARS-CoV-2 conserved epitopes elicited cross-reactive immune responses to both related coronaviruses and host bacteria that could serve as future targets for broad coronavirus therapeutics and vaccines. This work was supported through internal institutional funds from Children’s Mercy Research Institute and Children’s Mercy Kansas City.

Taxonomic and Functional Fecal Microbiota Signatures Associated With Insulin Resistance in Non-Diabetic Subjects With Overweight/Obesity Within the Frame of the PREDIMED-Plus Study.

ObjectiveAn altered gut microbiota has been associated with insulin resistance, a metabolic dysfunction consisting of cellular insulin signaling impairment. The aim of the present study is to determine the taxonomic and functional fecal microbiota signatures associated with HOMA-IR index in a population with high cardiovascular risk.MethodsA total of 279 non-diabetic individuals (55–75 years aged) with overweight/obesity and metabolic syndrome were stratified according to tertiles of HOMA-IR index. Blood biochemical parameters, anthropometric measurements and fecal samples were collected at baseline. Fecal microbial DNA extraction, 16S amplicon sequencing and bioinformatics analysis were performed.Results Desulfovibrio, Odoribacter and Oscillospiraceae UCG-002 were negatively associated with HOMA-IR index, whereas predicted total functional abundances revealed gut metabolic modules mainly linked to amino acid degradation. Butyricicoccus, Erysipelotrichaceae UCG-003, Faecalibacterium were positively associated with HOMA-IR index, whereas predicted total functional abundances revealed gut metabolic modules mainly linked to saccharide degradation. These bacteria contribute differentially to the gut metabolic modules, being the degree of contribution dependent on insulin resistance. Both taxa and gut metabolic modules negatively associated to HOMA-IR index were linked to mechanisms involving sulfate reducing bacteria, improvement of intestinal gluconeogenesis and production of acetate. Furthermore, both taxa and gut metabolic modules positively associated to HOMA-IR index were linked to production and mechanisms of action of butyrate.ConclusionsSpecific taxonomic and functional fecal microbiota signatures associated with insulin resistance were identified in a non-diabetic population with overweight/obesity at high cardiovascular risk. These findings suggest that tailoring therapies based on specific fecal microbiota profiles could be a potential strategy to improve insulin sensitivity.

Clinical significance and intestinal microbiota composition in immunocompromised children with norovirus gastroenteritis.

BackgroundNorovirus (NoV) infection is common in pediatric patients with immunodeficiency and is more likely to cause severe disease. Objective Our study aims to figure out the clinical differences and distribution of intestinal microbiota in immunocompromised children with NoV gastroenteritis.MethodsPediatric patients admitted to Shang-Ho Hospital with diagnosis of acute gastroenteritis including different immune status were enrolled and their medical records were reviewed. NoV gastroenteritis was validated using RT-PCR molecular methods. Viral shedding period was determined by real-time RT-PCR assays. Intestinal microbiota enrichment analysis was carried out by next generation sequencing after fecal DNA extraction and subsequent Linear Discriminant Analysis (LDA) Effect Size (LEfSe) method.ResultsSignificantly higher frequency of diarrhea [mean, (IQR), 3.8 (3–5) /day] and longer viral shedding time [mean, IQR, 8.5 (5–13) days] was found in immunocompromised NoV infections than in immunocompetent patients without NoV infections (p = 0.013*) and immunocompetent patients with NoV infections (p = 0.030**). The fever prevalence was significantly lower in immunocompromised NoV infections than in different immune or infection status. Intestinal microbiota metagenomics analysis showed no significant community richness difference while the LEfSe analysis showed a significant difference in commensal richness at the phylum level, the family level, and the genus level in patients under different immune status.ConclusionWe evaluated the clinical significances and microbiota composition in immunocompromised children with norovirus gastroenteritis. This will further facilitate studies of the interaction between the intestinal microbiota in such patients with precise determination of their bacterial infection control and probiotic supplements strategy.

Technical report: In-gel sample preparation prior to proteomic analysis of bovine faeces increases protein identifications by removal of high molecular weight glycoproteins

Bovine faecal composition is complex and a knowledge gap exists in the understanding of the bovine faecal proteome. In the present study, in-gel sample preparation (IGSP) of faecal samples prior to proteomics showed an increase in the number of proteins identified in faecal samples compared to those processed by filter-aided sample preparation (FASP). The optimised sample preparation method removed high molecular weight glycoproteins as part of the clean-up process of the faecal samples, and in combination with in-gel digestion before liquid chromatography with tandem mass spectrometry (LC-MS/MS). The use of IGSP led to enhanced protein identification with increases in the number of peptides identified and in the percent coverage of proteins in the bovine faecal samples. SignificanceCharacterization of faecal proteins has the potential to increase our understanding of host responses to changes such as diet, disease and drug-treatment. In-gel sample preparation prior to proteomics can be used to remove high molecular weight glycoproteins and reduce protein/peptide loss in FASP. This method of sample preparation will have application not only in the investigation of bovine faecal extracts but also in studies where large molecules such as glycoproteins or oligosaccharides could have detrimental influences on sample preparation involving ultrafiltration.

Validation of an enzyme immunoassay for measurement of fecal dehydroepiandrosterone sulfate in gibbons and siamangs.

Monitoring wildlife stress levels is essential to ensure their quality of life in captivity or in the wild. One promising method to assess the stress response is the comeasurement of glucocorticoids (GC) and dehydroepiandrosterone sulfate (DHEAS), adrenal hormones involved in the modulation of the stress response. Although noninvasive methods to measure GCs have been validated in several species, only a few studies have validated DHEAS assays. The aims of this study were (1) to describe an enzyme immunoassay (EIA) to measure DHEAS levels, (2) to validate this assay for fecal samples in gibbons and siamangs, and (3) to test hormonal stability after one freeze-thaw cycle and over time at two freezer temperatures (-20°C and -80°C). Subjects included 32 gibbons and siamangs from U.S. zoological parks. The EIA was validated analytically by parallelism and accuracy tests, and biologically by confirming a DHEAS response 1-2 days after a stressful event (accident, vaccination, or transportation) in three individuals. In addition, fecal DHEAS levels in a pregnant female were above nonpregnant/nonlactating levels and declined progressively the following parturition. The hormonal stability experiments revealed no significant changes in fecal DHEAS levels after one freeze-thaw cycle. Hormonal levels in fecal extracts were stable for 2 months, regardless of the storage temperature, with no significant differences between -20°C and -80°C conditions. The EIA described has high sensitivity and it is suitable for fecal DHEAS measurement in gibbons and siamangs, with a potential to be applied to other species.

The Gut Microbiome, Seleno-Compounds, and Acute Myocardial Infarction.

Background: Gut microbiome alterations might be considered a metabolic disorder. However, the relationship between the microbiome and acute myocardial infarction (AMI) has not been properly validated. Methods: The feces of 44 subjects (AMI: 19; control: 25) were collected for fecal genomic DNA extraction. The variable region V3–V4 of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. The metabolite amounts were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Results: The bacteria were more enriched in the AMI group both in the observed operational taxonomic units (OTUs) and faith phylogenetic diversity (PD) (p-value = 0.01 and <0.001 with 95% CI, individually). The Selenomonadales were less enriched in the AMI group at the family, genus, and species levels (all linear discriminant analysis (LDA) scores > 2). Seleno-compounds were more abundant in the AMI group at the family, genus, and species levels (all LDA scores > 2). Conclusions: This is the first study to demonstrate the association of Selenomonadales and seleno-compounds with the occurrence of AMI. Our findings provide an opportunity to identify a novel approach to prevent and treat AMI.