Enhanced mucosal mitochondrial function corrects dysbiosis and OXPHOS metabolism in IBD.

Background: Mitochondrial (Mito) dysfunction in IBD reduces mucosal O2 consumption and increases O2 delivery to the microbiome. Increased enteric O2 promotes blooms of facultative anaerobes (eg. Proteobacteria ) and restricts obligate anaerobes (eg. Firmicutes ). Dysbiotic metabolites negatively affect host metabolism and immunity. Our novel compound (AuPhos) upregulates intestinal epithelial cell (IEC) mito function, attenuates colitis and corrects dysbiosis in humanized Il10-/- mice. We posit that AuPhos corrects IBD-associated dysbiotic metabolism. Methods: Primary effect of AuPhos on mucosal Mito respiration and healing process was studied in ex vivo treated human colonic biopsies and piroxicam-accelerated (Px) Il10-/- mice. Secondary effect on microbiome was tested in DSS-colitis WT B6 and germ-free 129.SvEv WT or Il10-/- mice reconstituted with human IBD stool (Hu- Il10-/- ). Mice were treated orally with AuPhos (10- or 25- mg/kg; q3d) or vehicle, stool samples collected for fecal lipocalin-2 (f-LCN2) assay and microbiome analyses using 16S rRNA sequencing. AuPhos effect on microbial metabolites was determined using untargeted global metabolomics. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Effect of AuPhos on enteric oxygenation was assessed by E. coli Nissle 1917 WT (aerobic respiration-proficient) and cytochrome oxidase (cydA) mutant (aerobic respiration-deficient). Results: Metagenomic (16S) analysis revealed AuPhos reduced relative abundances of Proteobacteria and increased blooms of Firmicutes in uninflamed B6 WT, DSS-colitis, Hu-WT and Hu- Il10-/- mice. AuPhos also increased hypoxyprobe-1 staining in surface IECs suggesting enhanced O2 utilization. AuPhos-induced anaerobiosis was confirmed by a significant increase in cydA mutant compared to WT (O2-utlizing) E.coli . Ex vivo treatment of human biopsies with AuPhos showed significant increase in Mito mass, and complexes I and IV. Further, gene expression analysis of AuPhos-treated biopsies showed increase in stem cell markers (Lgr4, Lgr5, Lrig1), with concomitant decreases in pro-inflammatory markers (IL1ß,MCP1, RankL). Histological investigation of AuPhos-fed Px- Il10-/- mice showed significantly decreased colitis score in AuPhos-treated Px- Il10-/- mice, with decrease in mRNA of pro-inflammatory cytokines and increase in Mito complexes ( ND5 , ATP6 ). AuPhos significantly altered microbial metabolites associated with SCFA synthesis, FAO, TCA cycle, tryptophan and polyamine biosynthesis pathways. AuPhos increased pyruvate, 4-hydroxybutyrate, 2-hydroxyglutarate and succinate, suggesting an upregulation of pyruvate and glutarate pathways of butyrate production. AuPhos reduced IBD-associated primary bile acids (BA) with concomitant increase in secondary BA (SBA). AuPhos treatment significantly decreased acylcarnitines and increased L-carnitine reflective of enhanced FAO. AuPhos increases TCA cycle intermediates and creatine, energy reservoir substrates indicating enhanced OxPHOS. Besides, AuPhos also upregulates tryptophan metabolism, decreases Kynurenine and its derivatives, and increases polyamine biosynthesis pathway (Putresceine and Spermine). Conclusion: These findings indicate that AuPhos-enhanced IEC mitochondrial function reduces enteric O2 delivery, which corrects disease-associated metabolomics by restoring short-chain fatty acids, SBA, AA and IEC energy metabolism.

The Impact of Essential Amino Acids on the Gut Microbiota of Broiler Chickens.

The research involving the beneficial aspects of amino acids being added to poultry feed pertaining to performance, growth, feed intake, and feed conversion ratio is extensive. Yet currently the effects of amino acids on the gut microbiota aren't fully understood nor have there been many studies executed in poultry to explain the relationship between amino acids and the gut microbiota. The overall outcome of health has been linked to bird gut health due to the functionality of gastrointestinal tract (GIT) for digestion/absorption of nutrients as well as immune response. These essential functions of the GI are greatly driven by the resident microbiota which produce metabolites such as butyrate, propionate, and acetate, providing the microbiota a suitable and thrive driven environment. Feed, age, the use of feed additives and pathogenic infections are the main factors that have an effect on the microbial community within the GIT. Changes in these factors may have potential effects on the gut microbiota in the chicken intestine which in turn may have an influence on health essentially affecting growth, feed intake, and feed conversion ratio. This review will highlight limited research studies that investigated the possible role of amino acids in the gut microbiota composition of poultry.

Continuous Low-Intensity Ultrasound Improves Cartilage Repair in Rabbit Model of Subchondral Injury.

Subchondral drilling (SD), a bone marrow stimulation technique, is used to repair cartilage lesions that lack regenerative potential. Cartilage repair outcomes upon SD are typically fibrocartilaginous in nature with inferior functionality. The lack of cues to foster the chondrogenic differentiation of egressed mesenchymal stromal cells upon SD can be attributed for the poor outcomes. Continuous low-intensity ultrasound (cLIUS) at 3.8 MHz is proposed as a treatment modality for improving cartilage repair outcomes upon marrow stimulation. Bilateral defects were created by SD on the femoral medial condyle of female New Zealand white rabbits (n = 12), and the left joint received cLIUS treatment (3.8 MHz, 3.5 Vpp, 8 min/application/day) and the contralateral right joint served as the control. On day 7 postsurgery, synovial fluid was aspirated, and the cytokine levels were assessed by Quantibody™ assay. Rabbits were euthanized at 8 weeks and outcomes were assessed macroscopically and histologically. Defect areas in the right joints exhibited boundaries, incomplete fill, irregular cartilage surfaces, loss of glycosaminoglycan (GAG), and absence of chondrocytes. In contrast, the repaired defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte morphology, COL2A1 staining, and columnar organization. Synovial fluid collected from cLIUS-treated left knee joints had lower levels of IL1, TNFα, and IFNγ when compared to untreated right knee joints, alluding to the potential of cLIUS to mitigate early inflammation. Further at 8 weeks, left knee joints (n = 12) consistently scored higher on the O'Driscoll scale, with a higher percent hyaline cartilage score. No adverse impact on bone or change in the joint space was noted. Upon a single exposure of cLIUS to TNFα-treated cells, nuclear localization of pNFκB and SOX9 was visualized by double immunofluorescence and the expression of markers associated with the NFκB pathway was assayed by quantitative real-time polymerase chain reaction. cLIUS extends its chondroprotective effects by titrating pNFκB levels, preventing its nuclear translocation, while maintaining the expression of SOX9, the collagen II transcription factor. Our combined results demonstrate that healing of chondral defects treated with marrow stimulation by SD can be accelerated by employing cLIUS regimen that possesses chondroinductive and chondroprotective properties. Impact statement Repair of cartilage represents an unsolved biomedical burden. In vitro, continuous low-intensity ultrasound (cLIUS) has been demonstrated to possess chondroinductive and chondroprotective potential. To our best knowledge, the use of cLIUS to improve cartilage repair outcomes upon marrow stimulation, in vivo, has not been reported and our work reported here fills that gap. Our results demonstrated enhanced cartilage repair outcomes under cLIUS (3.8 MHz) in a rabbit model of subchondral injury by subchondral drilling. Enhanced repair stemmed from mesenchymal stem cell differentiation in vivo and the subsequent synthesis of articular cartilage-specific matrix.

Metabolic Impact of MKP-2 Upregulation in Obesity Promotes Insulin Resistance and Fatty Liver Disease.

The mechanisms connecting obesity with type 2 diabetes, insulin resistance, nonalcoholic fatty liver disease, and cardiovascular diseases remain incompletely understood. The function of MAPK phosphatase-2 (MKP-2), a type 1 dual-specific phosphatase (DUSP) in whole-body metabolism, and how this contributes to the development of diet-induced obesity, type 2 diabetes (T2D), and insulin resistance is largely unknown. We investigated the physiological contribution of MKP-2 in whole-body metabolism and whether MKP-2 is altered in obesity and human fatty liver disease using MKP-2 knockout mice models and human liver tissue derived from fatty liver disease patients. We demonstrate that, for the first time, MKP-2 expression was upregulated in liver tissue in humans with obesity and fatty liver disease and in insulin-responsive tissues in mice with obesity. MKP-2-deficient mice have enhanced p38 MAPK, JNK, and ERK activities in insulin-responsive tissues compared with wild-type mice. MKP-2 deficiency in mice protects against diet-induced obesity and hepatic steatosis and was accompanied by improved glucose homeostasis and insulin sensitivity. Mkp-2-/- mice are resistant to diet-induced obesity owing to reduced food intake and associated lower respiratory exchange ratio. This was associated with enhanced circulating insulin-like growth factor-1 (IGF-1) and stromal cell-derived factor 1 (SDF-1) levels in Mkp-2-/- mice. PTEN, a negative regulator of Akt, was downregulated in livers of Mkp-2-/- mice, resulting in enhanced Akt activity consistent with increased insulin sensitivity. These studies identify a novel role for MKP-2 in the regulation of systemic metabolism and pathophysiology of obesity-induced insulin resistance and fatty liver disease.

Characterization and Roles of Membrane Lipids in Fatty Liver Disease.

Obesity has reached global epidemic proportions and it affects the development of insulin resistance, type 2 diabetes, fatty liver disease and other metabolic diseases. Membrane lipids are important structural and signaling components of the cell membrane. Recent studies highlight their importance in lipid homeostasis and are implicated in the pathogenesis of fatty liver disease. Here, we discuss the numerous membrane lipid species and their metabolites including, phospholipids, sphingolipids and cholesterol, and how dysregulation of their composition and physiology contribute to the development of fatty liver disease. The development of new genetic and pharmacological mouse models has shed light on the role of lipid species on various mechanisms/pathways; these lipids impact many aspects of the pathophysiology of fatty liver disease and could potentially be targeted for the treatment of fatty liver disease.

Evaluation of Selected Bacteria and Yeast for Probiotic Potential in Poultry Production.

Performance and efficiency of feed utilization in poultry is highly influenced by gut health, which is dependent on intestinal microbial balance. Probiotics are live microbial feed supplements or viable microorganisms that beneficially affect the host animal by improving its gastrointestinal tract (GIT) microbial balance. However, their mode of action and suitable GIT environment favoring their colonization of the GIT is obscure. The probiotic properties of Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces boulardii were evaluated. These microbes were tested in vitro against gastrointestinal conditions for survivability and their ability to attach to the intestinal mucosa. The ability of the microbes to tolerate and survive varying pH levels and bile concentrations was assessed. The microbes were challenged with a pH of 2 to 7 for 5 h and bile concentrations of 1 to 3% for 6 hrs. The microbes were sampled hourly to evaluate growth or decline in colony-forming units (CFU). B. longum, L. Plantarum, and S. boulardii exhibited significantly higher CFU (p < 0.05) at a pH range of 5 to 7, 4 to 7, and 2 to 7, respectively, when compared with other pH levels. L. plantarum had much higher colony-forming units per mL within each pH level, except at pH 2 where S. boulardii was the only microbe to survive over time. While L. plantarum and S. boulardii were able to tolerate the various bile concentrations, B. longum and L. plantarum showed remarkable ability to attach to the intestinal mucosa and to inhibit pathogenic microbes.

Continuous Low-Intensity Ultrasound Preserves Chondrogenesis of Mesenchymal Stromal Cells in the Presence of Cytokines by Inhibiting NFκB Activation.

Proinflammatory joint environment, coupled with impeded chondrogenic differentiation of mesenchymal stromal cells (MSCs), led to inferior cartilage repair outcomes. Nuclear translocation of phosphorylated-NFκB downregulates SOX9 and hinders the chondrogenesis of MSCs. Strategies that minimize the deleterious effects of NFκB, while promoting MSC chondrogenesis, are of interest. This study establishes the ability of continuous low-intensity ultrasound (cLIUS) to preserve MSC chondrogenesis in a proinflammatory environment. MSCs were seeded in alginate:collagen hydrogels and cultured for 21 days in an ultrasound-assisted bioreactor (5.0 MHz, 2.5 Vpp; 4 applications/day) in the presence of IL1ß and evaluated by qRT-PCR and immunofluorescence. The differential expression of markers associated with the NFκB pathway was assessed upon a single exposure of cLIUS and assayed by Western blotting, qRT-PCR, and immunofluorescence. Mitochondrial potential was evaluated by tetramethylrhodamine methyl ester (TMRM) assay. The chondroinductive potential of cLIUS was noted by the increased expression of SOX9 and COLII. cLIUS extended its chondroprotective effects by stabilizing the NFκB complex in the cytoplasm via engaging the IκBα feedback mechanism, thus preventing its nuclear translocation. cLIUS acted as a mitochondrial protective agent by restoring the mitochondrial potential and the mitochondrial mRNA expression in a proinflammatory environment. Altogether, our results demonstrated the potential of cLIUS for cartilage repair and regeneration under proinflammatory conditions.

Effects of Lactobacillus reuteri and Streptomyces coelicolor on Growth Performance of Broiler Chickens.

There are well documented complications associated with the continuous use of antibiotics in the poultry industry. Over the past few decades, probiotics have emerged as viable alternatives to antibiotics; however, most of these candidate probiotic microorganisms have not been fully evaluated for their effectiveness as potential probiotics for poultry. Recent evaluation of a metagenome of broiler chickens in our laboratory revealed a prevalence of Lactobacillus reuteri (L. reuteri) and Actinobacteria class of bacteria in their gastrointestinal tract. In this study Lactobacillus reuteri and Streptomyces coelicolor (S. coelicolor) were selected as probiotic bacteria, encapsulated, and added into broiler feed at a concentration of 100 mg/kg of feed. In an 8-week study, 240 one day-old chicks were randomly assigned to four dietary treatments. Three dietary treatments contained two probiotic bacteria in three different proportions (L. reuteri and S. coelicolor individually at 100 ppm, and mixture of L. reuteri and S. coelicolor at 50 ppm each). The fourth treatment had no probiotic bacteria and it functioned as the control diet. L. reuteri and S. coelicolor were added to the feed by using wheat middlings as a carrier at a concentration of 100 ppm (100 mg/kg). Chickens fed diets containing L. reuteri and S. coelicolor mixture showed 2% improvement in body weight gain, 7% decrease in feed consumption, and 6-7% decrease in feed conversion ratios. This research suggests that L. reuteri and S. coelicolor have the potential to constitute probiotics in chickens combined or separately, depending on the desired selection of performance index.

Two novel SNPs in genes involved in immune response and their association with mandibular residual ridge resorption.

"Residual ridge resorption" (RRR) is a multifactorial condition involving bone resorption of the residual ridge. We investigated 10 single nucleotide polymorphisms (SNPs) in seven genes with the aim of identifying the genetic factors associated with RRR susceptibility. The study group included 96 RRR patients and 96 controls. Age at first edentulism, duration of edentulism, and bone height were recorded. Saliva was collected from the subjects for DNA extraction. Genotype analysis was performed on the 'SequenomMassARRAYiPLEX'. The genotype and allele frequencies calculated in patients and controls were compared. We found that rs1800896 in the IL10 gene and rs5743289 in NOD2 gene showed significant association with RRR. Within the RRR group, genotypes for each SNP were separated, and we observed that the age at first edentulism and bone height showed variations in the different genotypes of the ten studied SNPs. This study showed an association between SNPs in IL10 and NOD2 genes. It also revealed that the genotypes of the different SNPs influence bone resorption and health. Additionally, age at first edentulism and bone height were much lower in some genotypes. This study demonstrates the need for larger multicenter trials to confirm these findings. Finally, we suggest that the results of this study may be utilized for developing novel genetic diagnostic tests and for identifying Saudi individuals who may be more susceptible to RRR development following dental extraction.

A comparative analysis of microbial profile of Guinea fowl and chicken using metagenomic approach.

Probiotics are live microbial feed supplements that promote growth and health to the host by minimizing non-essential and pathogenic microorganisms in the host's gastrointestinal tract (GIT). The campaign to minimize excessive use of antibiotics in poultry production has necessitated development of probiotics with broad application in multiple poultry species. Design of such probiotics requires understanding of the diversity or similarity in microbial profiles among avian species of economic importance. Therefore, the objective of this research was to establish and compare the microbial profiles of the GIT of Guinea fowl and chicken and to establish the microbial diversity or similarity between the two avian species. A metagenomic approach consisting of the amplification and sequence analysis of the hypervariable regions V1-V9 of the 16S rRNA gene was used to identify the GIT microbes. Collectively, we detected more than 150 microbial families. The total number of microbial species detected in the chicken GIT was higher than that found in the Guinea Fowl GIT. Our studies also revealed phylogenetic diversity among the microbial species found in chicken and guinea fowl. The phylum Firmicutes was most abundant in both avian species whereas Phylum Actinobacteria was most abundant in chickens than Guinea fowls. The diversity of the microbial profiles found in broiler chickens and Guinea fowls suggest that the design of effective avian probiotics would require species specificity.