Microbiota Dysbiosis and Gut Barrier Dysfunction Associated with Non-Alcoholic Fatty Liver Disease Are Modulated by a Specific Metabolic Cofactors' Combination.

The gut is a selective barrier that not only allows the translocation of nutrients from food, but also microbe-derived metabolites to the systemic circulation that flows through the liver. Microbiota dysbiosis occurs when energy imbalances appear due to an unhealthy diet and a sedentary lifestyle. Dysbiosis has a critical impact on increasing intestinal permeability and epithelial barrier deterioration, contributing to bacterial and antigen translocation to the liver, triggering non-alcoholic fatty liver disease (NAFLD) progression. In this study, the potential therapeutic/beneficial effects of a combination of metabolic cofactors (a multi-ingredient; MI) (betaine, N-acetylcysteine, L-carnitine, and nicotinamide riboside) against NAFLD were evaluated. In addition, we investigated the effects of this metabolic cofactors' combination as a modulator of other players of the gut-liver axis during the disease, including gut barrier dysfunction and microbiota dysbiosis. Diet-induced NAFLD mice were distributed into two groups, treated with the vehicle (NAFLD group) or with a combination of metabolic cofactors (NAFLD-MI group), and small intestines were harvested from all animals for histological, molecular, and omics analysis. The MI treatment ameliorated gut morphological changes, decreased gut barrier permeability, and reduced gene expression of some proinflammatory cytokines. Moreover, epithelial cell proliferation and the number of goblet cells were increased after MI supplementation. In addition, supplementation with the MI combination promoted changes in the intestinal microbiota composition and diversity, as well as modulating short-chain fatty acids (SCFAs) concentrations in feces. Taken together, this specific combination of metabolic cofactors can reverse gut barrier disruption and microbiota dysbiosis contributing to the amelioration of NAFLD progression by modulating key players of the gut-liver axis.

NIN Acts as a Network Hub Controlling a Growth Module Required for Rhizobial Infection.

The symbiotic infection of root cells by nitrogen-fixing rhizobia during nodulation requires the transcription factor Nodule Inception (NIN). Our root hair transcriptomic study extends NIN's regulon to include Rhizobium Polar Growth and genes involved in cell wall modification, gibberellin biosynthesis, and a comprehensive group of nutrient (N, P, and S) uptake and assimilation genes, suggesting that NIN's recruitment to nodulation was based on its role as a growth module, a role shared with other NIN-Like Proteins. The expression of jasmonic acid genes in nin suggests the involvement of NIN in the resolution of growth versus defense outcomes. We find that the regulation of the growth module component Nodulation Pectate Lyase by NIN, and its function in rhizobial infection, are conserved in hologalegina legumes, highlighting its recruitment as a major event in the evolution of nodulation. We find that Nodulation Pectate Lyase is secreted to the infection chamber and the lumen of the infection thread. Gene network analysis using the transcription factor mutants for ERF Required for Nodulation1 and Nuclear Factor-Y Subunit A1 confirms hierarchical control of NIN over Nuclear Factor-Y Subunit A1 and shows that ERF Required for Nodulation1 acts independently to control infection. We conclude that while NIN shares functions with other NIN-Like Proteins, the conscription of key infection genes to NIN's control has made it a central regulatory hub for rhizobial infection.

MtLAX2, a Functional Homologue of the Arabidopsis Auxin Influx Transporter AUX1, Is Required for Nodule Organogenesis.

Most legume plants can form nodules, specialized lateral organs that form on roots, and house nitrogen-fixing bacteria collectively called rhizobia. The uptake of the phytohormone auxin into cells is known to be crucial for development of lateral roots. To test the role of auxin influx in nodulation we used the auxin influx inhibitors 1-naphthoxyacetic acid (1-NOA) and 2-NOA, which we found reduced nodulation of Medicago truncatula. This suggested the possible involvement of the AUX/LAX family of auxin influx transporters in nodulation. Gene expression studies identified MtLAX2, a paralogue of Arabidopsis (Arabidopsis thaliana) AUX1, as being induced at early stages of nodule development. MtLAX2 is expressed in nodule primordia, the vasculature of developing nodules, and at the apex of mature nodules. The MtLAX2 promoter contains several auxin response elements, and treatment with indole-acetic acid strongly induces MtLAX2 expression in roots. mtlax2 mutants displayed root phenotypes similar to Arabidopsis aux1 mutants, including altered root gravitropism, fewer lateral roots, shorter root hairs, and auxin resistance. In addition, the activity of the synthetic DR5-GUS auxin reporter was strongly reduced in mtlax2 roots. Following inoculation with rhizobia, mtlax2 roots developed fewer nodules, had decreased DR5-GUS activity associated with infection sites, and had decreased expression of the early auxin responsive gene ARF16a Our data indicate that MtLAX2 is a functional analog of Arabidopsis AUX1 and is required for the accumulation of auxin during nodule formation in tissues underlying sites of rhizobial infection.

Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies.

INTRODUCTION: Placenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are conditions postulated to originate from initial failure of placentation, leading to clinical sequelae indicative of endothelial dysfunction. Vascular smooth muscle aberrations have also been implicated in the pathogenesis of both disorders via smooth muscle contractility and relaxation mediated by Myosin Light Chain Phosphatase (MLCP) and the oppositional contractile action of Myosin Light Chain Kinase. PPP1R12A is a constituent part of the MLCP complex responsible for dephosphorylation of myosin fibrils. We hypothesize that alternative splicing of micro-exons result in isoforms lacking the functional leucine zipper (LZ) domain which may give those cells expressing these alternative transcripts a tendency towards contraction and vasoconstriction. METHODS: Expression was determined by qRT-PCR. Epigenetic profiling consisted of bisulphite-based DNA methylation analysis and ChIP for underlying histone modifications. RESULTS: We identified several novel transcripts with alternative micro-exon inclusion that would produce LZ- PPP1R12A protein. qRT-PCR revealed some isoforms, including the PPP1R12A canonical transcript, are differentially expressed in placenta biopsies from PE and IUGR samples compared to uncomplicated pregnancies. DISCUSSION: We propose that upregulation of PPP1R12A expression in complicated pregnancies may be due to enhanced promoter activity leading to increased transcription as a response to physiological stress in the placenta, which we show is independent of promoter DNA methylation.

Metabolic Regulation of Macrophages by SIRT1 Determines Activation During Cholestatic Liver Disease in Mice.

BACKGROUND & AIMS: Inflammation is the hallmark of chronic liver disease. Metabolism is a key determinant to regulate the activation of immune cells. Here, we define the role of sirtuin 1 (SIRT1), a main metabolic regulator, in controlling the activation of macrophages during cholestatic liver disease and in response to endotoxin. METHODS: We have used mice overexpressing SIRT1, which we treated with intraperitoneal lipopolysaccharides or induced cholestasis by bile duct ligation. Bone marrow-derived macrophages were used for mechanistic in vitro studies. Finally, PEPC-Boy mice were used for adoptive transfer experiments to elucidate the impact of SIRT1-overexpressing macrophages in contributing to cholestatic liver disease. RESULTS: We found that SIRT1 overexpression promotes increased liver inflammation and liver injury after lipopolysaccharide/GalN and bile duct ligation; this was associated with an increased activation of the inflammasome in macrophages. Mechanistically, SIRT1 overexpression associated with the activation of the mammalian target of rapamycin (mTOR) pathway that led to increased activation of macrophages, which showed metabolic rewiring with increased glycolysis and broken tricarboxylic acid cycle in response to endotoxin in vitro. Activation of the SIRT1/mTOR axis in macrophages associated with the activation of the inflammasome and the attenuation of autophagy. Ultimately, in an in vivo model of cholestatic disease, the transplantation of SIRT1-overexpressing myeloid cells contributed to liver injury and fibrosis. CONCLUSIONS: Our study provides novel mechanistic insights into the regulation of macrophages during cholestatic disease and the response to endotoxin, in which the SIRT1/mTOR crosstalk regulates macrophage activation controlling the inflammasome, autophagy and metabolic rewiring.