Single cell RNA-sequencing profiling to improve the translation between human IBD and in vivo models.

Inflammatory bowel disease (IBD) is an umbrella term for two conditions (Crohn's Disease and Ulcerative Colitis) that is characterized by chronic inflammation of the gastrointestinal tract. The use of pre-clinical animal models has been invaluable for the understanding of potential disease mechanisms. However, despite promising results of numerous therapeutics in mouse colitis models, many of these therapies did not show clinical benefits in patients with IBD. Single cell RNA-sequencing (scRNA-seq) has recently revolutionized our understanding of complex interactions between the immune system, stromal cells, and epithelial cells by mapping novel cell subpopulations and their remodeling during disease. This technology has not been widely applied to pre-clinical models of IBD. ScRNA-seq profiling of murine models may provide an opportunity to increase the translatability into the clinic, and to choose the most appropriate model to test hypotheses and novel therapeutics. In this review, we have summarized some of the key findings at the single cell transcriptomic level in IBD, how specific signatures have been functionally validated in vivo, and highlighted the similarities and differences between scRNA-seq findings in human IBD and experimental mouse models. In each section of this review, we highlight the importance of utilizing this technology to find the most suitable or translational models of IBD based on the cellular therapeutic target.

A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes Treg cell activation and homeostasis.

The molecular programs involved in regulatory T (Treg) cell activation and homeostasis remain incompletely understood. Here, we show that T cell receptor (TCR) signaling in Treg cells induces the nuclear translocation of serine/threonine kinase 4 (Stk4), leading to the formation of an Stk4-NF-κB p65-Foxp3 complex that regulates Foxp3- and p65-dependent transcriptional programs. This complex was stabilized by Stk4-dependent phosphorylation of Foxp3 on serine-418. Stk4 deficiency in Treg cells, either alone or in combination with its homolog Stk3, precipitated a fatal autoimmune lymphoproliferative disease in mice characterized by decreased Treg cell p65 expression and nuclear translocation, impaired NF-κB p65-Foxp3 complex formation, and defective Treg cell activation. In an adoptive immunotherapy model, overexpression of p65 or the phosphomimetic Foxp3S418E in Stk3/4-deficient Treg cells ameliorated their immune regulatory defects. Our studies identify Stk4 as an essential TCR-responsive regulator of p65-Foxp3-dependent transcription that promotes Treg cell-mediated immune tolerance.

Vibrio cholerae high cell density quorum sensing activates the host intestinal innate immune response.

Quorum sensing fundamentally alters the interaction of Vibrio cholerae with aquatic environments, environmental hosts, and the human intestine. At high cell density, the quorum-sensing regulator HapR represses not only expression of cholera toxin and the toxin co-regulated pilus, virulence factors essential in human infection, but also synthesis of the Vibrio polysaccharide (VPS) exopolysaccharide-based matrix required for abiotic and biotic surface attachment. Here, we describe a feature of V. cholerae quorum sensing that shifts the host-pathogen interaction toward commensalism. By repressing pathogen consumptive anabolic metabolism and, in particular, tryptophan uptake, V. cholerae HapR stimulates host intestinal serotonin production. This, in turn, activates host intestinal innate immune signaling to promote host survival.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

The Efficacy of Vitamin K, A Member Of Naphthoquinones in the Treatment of Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: Redox dysregulation originating from metabolic alterations in cancer cells contributes to their proliferation, invasion, and resistance to therapy. Conversely, these features represent a specific vulnerability of malignant cells that can be selectively targeted by redox chemotherapeutics. Amongst them, Vitamin K (VitK) carries the potential against cancer stem cells, in addition to the rest of tumor mass. OBJECTIVES: To assess the possible benefits and safety of VitK for cancer treatment using a systematic review and meta-analysis with a mixed-methods approach. METHODS: We performed a systematic search on several electronic databases for studies comparing VitK treatment with and without combination to the control groups. For quantitative studies, fully or partially reported clinical outcomes such as recurrence rates, survival, overall response and adverse reactions were assessed. For qualitative studies, a narrative synthesis was accomplished. RESULTS: Our analysis suggested that the clinical outcome of efficacy, the pooled hazard ratio for progression-free survival, and the pooled relative risk for overall survival, and overall response were significantly higher in the VitK therapy group compared to the placebo group (p<0.05). We did not observe any significant difference in the occurrence of adverse events between groups. Among qualitative studies, VitK treatment targeting myelodysplastic syndrome and advanced solid tumors resulted in 24.1% and 10% of clinical response, respectively. CONCLUSION: VitK not only exerts antitumor effects against a wide range of tumor types, but it also has excellent synergism with other therapeutic agents.

Potential Mechanism of Cellular Uptake of the Excitotoxin Quinolinic Acid in Primary Human Neurons.

In Alzheimer's disease (AD), excessive amounts of quinolinic acid (QUIN) accumulate within the brain parenchyma and dystrophic neurons. QUIN also regulates glutamate uptake into neurons, which may be due to modulation of Na+-dependent excitatory amino acid transporters (EAATs). To determine the biological relationships between QUIN and glutamate dysfunction, we first quantified the functionality and kinetics of [3H]QUIN uptake in primary human neurons using liquid scintillation. We then measured changes in the protein expression of the glutamate transporter EAAT3 and EAAT1b in primary neurons treated with QUIN and the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (2,4-PDC) using western blotting and immunohistochemistry. Immunohistochemistry was further used to elucidate intracellular transport of exogenous QUIN and the lysosomal-associated membrane protein 2 (LAMP2). Structural insights into the binding between QUIN and EAAT3 were further investigated using molecular docking techniques. We report significant temperature-dependent high-affinity transport leading to neuronal uptake of [3H]QUIN with a Km of 42.2 µM, and a Vmax of 9.492 pmol/2 min/mg protein, comparable with the uptake of glutamate. We also found that QUIN increases expression of the EAAT3 monomer while decreasing the functional trimer. QUIN uptake into primary neurons was shown to involve EAAT3 as uptake was significantly attenuated following EAAT inhibition. We also demonstrated that QUIN increases the expression of aberrant EAAT1b protein in neurons further implicating QUIN-induced glutamate dysfunction. Furthermore, we demonstrated that QUIN is metabolised exclusively in lysosomes. The involvement of EAAT3 as a modulator for QUIN uptake was further confirmed using molecular docking. This study is the first to characterise a mechanism for QUIN uptake into primary human neurons involving EAAT3, opening potential targets to attenuate QUIN-induced excitotoxicity in neuroinflammatory diseases.

Methionine Availability in the Arthropod Intestine Is Elucidated through Identification of Vibrio cholerae Methionine Acquisition Systems.

While only a subset of Vibrio cholerae strains are human diarrheal pathogens, all are aquatic organisms. In this environment, they often persist in close association with arthropods. In the intestinal lumen of the model arthropod Drosophila melanogaster, methionine and methionine sulfoxide decrease susceptibility to V. cholerae infection. In addition to its structural role in proteins, methionine participates in the methionine cycle, which carries out synthetic and regulatory methylation reactions. It is, therefore, essential for the growth of both animals and bacteria. Methionine is scarce in some environments, and the facile conversion of free methionine to methionine sulfoxide in oxidizing environments interferes with its utilization. To ensure an adequate supply of methionine, the genomes of most organisms encode multiple high-affinity uptake pathways for methionine as well as multiple methionine sulfoxide reductases, which reduce free and protein-associated methionine sulfoxide to methionine. To explore the role of methionine uptake and reduction in V. cholerae colonization of the arthropod intestine, we mutagenized the two high-affinity methionine transporters and five methionine sulfoxide reductases encoded in the V. cholerae genome. We show that MsrC is the sole methionine sulfoxide reductase active on free methionine sulfoxide. Furthermore, in the absence of methionine synthesis, high-affinity methionine uptake but not reduction is essential for V. cholerae colonization of the Drosophila intestine. These findings allow us to place a lower limit of 0.05 mM and an upper limit of 0.5 mM on the methionine concentration in the Drosophila intestine.IMPORTANCE Methionine is an essential amino acid involved in both biosynthetic and regulatory processes in the bacterial cell. To ensure an adequate supply of methionine, bacteria have evolved multiple systems to synthesize, import, and recover this amino acid. To explore the importance of methionine synthesis, transport, and recovery in any environment, all of these systems must be identified and mutagenized. Here, we have mutagenized every high-affinity methionine uptake system and methionine sulfoxide reductase encoded in the genome of the diarrheal pathogen V. cholerae We use this information to determine that high-affinity methionine uptake systems are sufficient to acquire methionine in the intestine of the model arthropod Drosophila melanogaster but are not involved in virulence and that the intestinal concentration of methionine must be between 0.05 mM and 0.5 mM.

Vibrio cholerae Sheds Its Coat to Make Itself Comfortable in the Gut.

Invertebrates molt, furry mammals shed, and human skin exfoliates. In this issue of Cell Host & Microbe, Zingl et al. describe a virulence mechanism in which the bacterial pathogen Vibrio cholerae jettisons outer membrane proteins and lipids in vesicles as it enters the mammalian intestine.

Microbial Control of Intestinal Homeostasis via Enteroendocrine Cell Innate Immune Signaling.

A community of commensal microbes, known as the intestinal microbiota, resides within the gastrointestinal tract of animals and plays a role in maintenance of host metabolic homeostasis and resistance to pathogen invasion. Enteroendocrine cells, which are relatively rare in the intestinal epithelium, have evolved to sense and respond to these commensal microbes. Specifically, they express G-protein-coupled receptors and functional innate immune signaling pathways that recognize products of microbial metabolism and microbe-associated molecular patterns, respectively. Here we review recent evidence from Drosophila melanogaster that microbial cues recruit antimicrobial, mechanical, and metabolic branches of the enteroendocrine innate immune system and argue that this response may play a role not only in maintaining host metabolic homeostasis but also in intestinal resistance to invasion by bacterial, viral, and parasitic pathogens.

The Precursor to Glutathione (GSH), γ-Glutamylcysteine (GGC), Can Ameliorate Oxidative Damage and Neuroinflammation Induced by Aß40 Oligomers in Human Astrocytes.

Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH levels or impairment in the catalytic activity of the GSH biosynthetic enzyme glutamate cysteine ligase (GCL). γ-glutamylcysteine (GGC), a precursor to glutathione (GSH), can replenish depleted GSH levels under oxidative stress conditions, by circumventing the regulation of GSH biosynthesis and providing the limiting substrate. Soluble amyloid-ß (Aß) oligomers have been shown to induce oxidative stress, synaptic dysfunction and memory deficits which have been reported in Alzheimer's disease (AD). Calcium ions, which are increased with age and in AD, have been previously reported to enhance the formation of Aß40 oligomers, which have been casually associated with the pathogenesis of the underlying neurodegenerative condition. In this study, we examined the potential beneficial effects of GGC against exogenous Aß40 oligomers on biomarkers of apoptosis and cell death, oxidative stress, and neuroinflammation, in human astrocytes. Treatment with Aß40 oligomers significantly reduced the cell viability and apoptosis of astrocyte brain cultures and increased oxidative modifications of DNA, lipids, and protein, enhanced pro-inflammatory cytokine release and increased the activity of the proteolytic matrix metalloproteinase enzyme, matric metalloproteinase (MMP)-2 and reduced the activity of MMP-9 after 24 h. Co-treatment of Aß40 oligomers with GGC at 200 µM increased the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) and led to significant increases in the levels of the total antioxidant capacity (TAC) and GSH and reduced the GSSG/GSH ratio. GGC also upregulated the level of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and attenuated the changes in metalloproteinase activity in oligomeric Aß40-treated astrocytes. Our data provides renewed insight on the beneficial effects of increased GSH levels by GGC in human astrocytes, and identifies yet another potential therapeutic strategy to attenuate the cytotoxic effects of Aß oligomers in AD.

Heterologous Production and Purification of a Functional Chloroform Reductive Dehalogenase.

Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli and Bacillus megaterium. Here, we report the recombinant expression of soluble and functional TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA was demonstrated. This is the first known report of heterologous expression and purification of a respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

A bacterial chloroform reductive dehalogenase: purification and biochemical characterization.

We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103 units mg protein-1 . The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV-visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.

Corrigendum: Identification of Cerebral Metal Ion Imbalance in the Brain of Ageing Octodon degus.

[This corrects the article on p. 66 in vol. 9, PMID: 28405187.].

Identification of Cerebral Metal Ion Imbalance in the Brain of Aging Octodon degus.

The accumulation of redox-active transition metals in the brain and metal dyshomeostasis are thought to be associated with the etiology and pathogenesis of several neurodegenerative diseases, and Alzheimer's disease (AD) in particular. As well, distinct biometal imaging and role of metal uptake transporters are central to understanding AD pathogenesis and aging but remain elusive, due inappropriate detection methods. We therefore hypothesized that Octodon degus develop neuropathological abnormalities in the distribution of redox active biometals, and this effect may be due to alterations in the expression of lysosomal protein, major Fe/Cu transporters, and selected Zn transporters (ZnTs and ZIPs). Herein, we report the distribution profile of biometals in the aged brain of the endemic Chilean rodent O. degus-a natural model to investigate the role of metals on the onset and progression of AD. Using laser ablation inductively coupled plasma mass spectrometry, our quantitative images of biometals (Fe, Ca, Zn, Cu, and Al) appear significantly elevated in the aged O. degus and show an age-dependent rise. The metals Fe, Ca, Zn, and Cu were specifically enriched in the cortex and hippocampus, which are the regions where amyloid plaques, tau phosphorylation and glial alterations are most commonly reported, whilst Al was enriched in the hippocampus alone. Using whole brain extracts, age-related deregulation of metal trafficking pathways was also observed in O. degus. More specifically, we observed impaired lysosomal function, demonstrated by increased cathepsin D protein expression. An age-related reduction in the expression of subunit B2 of V-ATPase, and significant increases in amyloid beta peptide 42 (Aß42), and the metal transporter ATP13a2 were also observed. Although the protein expression levels of the zinc transporters, ZnT (1,3,4,6, and 7), and ZIP7,8 and ZIP14 increased in the brain of aged O. degus, ZnT10, decreased. Although no significant age-related change was observed for the major iron/copper regulator IRP2, we did find a significant increase in the expression of DMT1, a major transporter of divalent metal species, 5'-aminolevulinate synthase 2 (ALAS2), and the proto-oncogene, FOS. Collectively, our data indicate that transition metals may be enriched with age in the brains of O. degus, and metal dyshomeostasis in specific brain regions is age-related.

Molecular Targets of Tannic Acid in Alzheimer's Disease.

Tannic acid (TA) is a naturally occurring plant-derived polyphenol found in several herbaceous and woody plants, including legumes, sorghum, beans, bananas, persimmons, rasberries, wines and a broad selection of teas. Clinically, TA has strong antioxidant/free radical scavenging, antiinflammatory, anti-viral/bacterial, and anti-carcinogenic properties. While the aetiology of Alzheimer's disease (AD) remains unclear, this complex multifactorial neurodegenerative disorder remains the most common form of dementia, and is a growing public health concern worldwide. The neuroprotective effects of TA against AD have been shown in several in vitro and in vivo models of AD. Apart from its potent antioxidant and anti-inflammatory roles, evidence suggests that TA is also a natural inhibitor of ß-secretase (BACE1) activity and protein expression. BACE1 is the primary enzyme responsible for the production and deposition of Aß peptide. TA also destabilises neurotoxic amyloid beta (Aß) fibrils in vitro. Apart from its effects on the Aß cascade, TA can also inhibit the in vitro aggregation of tau peptide, a core component of intracellular neurofibrillary tangles (NFTs). This review summarizes the relevance of TA and TA-related vegetable extracts (tannins) in the pathogenesis of AD and its enzymatic targets. It also highlights the significance of TA as an important lead compound against AD.

Characterization of the Kynurenine Pathway in CD8+ Human Primary Monocyte-Derived Dendritic Cells.

The kynurenine (KYN) pathway (KP) is a major degradative pathway of the amino acid, L-tryptophan (TRP), that ultimately leads to the anabolism of the essential pyridine nucleotide, nicotinamide adenine dinucleotide. TRP catabolism results in the production of several important metabolites, including the major immune tolerance-inducing metabolite KYN, and the neurotoxin and excitotoxin quinolinic acid. Dendritic cells (DCs) have been shown to mediate immunoregulatory roles that mediated by TRP catabolism. However, characterization of the KP in human DCs has so far only been partly delineated. It is critical to understand which KP enzymes are expressed and which KP metabolites are produced to be able to understand their regulatory effects on the immune response. In this study, we characterized the KP in human monocyte-derived DCs (MDDCs) in comparison with the human primary macrophages using RT-PCR, high-pressure gas chromatography, mass spectrometry, and immunocytochemistry. Our results show that the KP is entirely expressed in human MDDC. Following activation of the KP using interferon gamma, MDDCs can mediate apoptosis of T h cells in vitro. Understanding the molecular mechanisms regulating KP metabolism in MDDCs may provide renewed insight for the development of novel therapeutics aimed at modulating immunological effects and peripheral tolerance.

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein).

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

Genomic, transcriptomic and proteomic analyses of Dehalobacter UNSWDHB in response to chloroform.

Organohalide respiring bacteria (ORB) are capable of utilising organohalides as electron acceptors for the generation of cellular energy and consequently play an important role in the turnover of natural and anthropogenically-derived organohalides. In this study, the response of a Dehalobacter sp. strain UNSWDHB to the addition of trichloromethane (TCM) after a 50 h period of its absence (suffocation) was evaluated from a transcriptomic and proteomic perspective. The up-regulation of TCM reductive dehalogenase genes (tmrABC) and their gene products (TmrABC) was confirmed at both transcriptional and proteomic levels. Other findings include the upregulation of various hydrogenases (membrane-associated Ni-Fe hydrogenase complexes and soluble Fe-Fe hydrogenases), formate dehydrogenases, complex I and a pyrophosphate-energized proton pump. The elevated expression of enzymes associated with carbon metabolism, including complete Wood Ljungdahl pathway, during TCM respiration raises interesting questions on possible fates of intracellular formate and its potential role in the physiology of this bacterium. Overall, the findings presented here provide a broader view on the bioenergetics and general physiology of Dehalobacter UNSWDHB cells actively respiring with TCM.

Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation.

Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e., organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDases), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses on RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.