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The Journal of Pharmacology and... May 2024Ulcerative colitis (UC) is an immune-mediated inflammatory disease that can lead to persistent damage and even cancer without any intervention. Conventional treatments...
Ulcerative colitis (UC) is an immune-mediated inflammatory disease that can lead to persistent damage and even cancer without any intervention. Conventional treatments can alleviate UC symptoms but are costly and even cause various side effects. Tauroursodeoxycholic acid (TUDCA), a secondary bile acid derivative, possesses anti-inflammatory and cytoprotective properties for various diseases, but its potential therapeutic benefits in UC have not been fully explored. Mice were subjected to colitis induction using 3% dextran sulfate sodium (DSS). The therapeutic effect of TUDCA was evaluated by body weight loss, disease activity index (DAI), colon length, and spleen weight ratio. Tissue pathology was assessed using H&E staining, while the levels of pro-inflammatory and anti-inflammatory cytokines in colonic tissue were quantified via enzyme-linked immunosorbent assay (ELISA). Tight junction proteins were detected by immunoblotting and intestinal permeability was assessed using fluorescein isothiocyanate (FITC)-dextran. Moreover, the gut microbiota was profiled using high-throughput sequencing of the 16S rDNA gene. TUDCA alleviated the colitis in mice, involving reduced DAI, attenuated colon and spleen enlargement, ameliorated histopathological lesions, and normalized the levels of pro-inflammatory and anti-inflammatory cytokines. Furthermore, TUDCA treatment inhibited the downregulation of intestinal barrier proteins including ZO-1 and occludin, thus reducing intestinal permeability. The analysis of gut microbiota suggested that TUDCA modulated the dysbiosis in mice with colitis, especially for the remarkable rise in TUDCA exerted a therapeutic efficacy in DSS-induced colitis by reducing intestinal inflammation, protecting intestinal barrier integrity, and restoring gut microbiota balance. This study demonstrates the potential therapeutic benefits of Tauroursodeoxycholic acid (TUDCA) in ulcerative colitis (UC). TUDCA effectively alleviated colitis symptoms in mice, including reducing inflammation, restoring intestinal barrier integrity and the dysbiosis of gut microbiota. This work highlights the promising role of TUDCA as a potentially alternative treatment, offering new insights into managing this debilitating condition.
PubMed: 38816229
DOI: 10.1124/jpet.123.002020 -
The Journal of Biological Chemistry May 2024Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene...
Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways, to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multi-module proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain-length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid (Kdo)-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137), and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.
PubMed: 38815868
DOI: 10.1016/j.jbc.2024.107420 -
The Journal of Biological Chemistry May 2024ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice...
ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
PubMed: 38815867
DOI: 10.1016/j.jbc.2024.107418 -
The Journal of Biological Chemistry May 2024GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest...
GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest sub-group of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.
PubMed: 38815865
DOI: 10.1016/j.jbc.2024.107421 -
Ultrasonics Sonochemistry May 2024The utilization of metallic nanoparticles in bio-nanofabrication holds significant potential in the field of applied research. The current study applied and compared...
UPLC-qTOF-MS phytochemical profile of Commiphora gileadensis leaf extract via integrated ultrasonic-microwave-assisted technique and synthesis of silver nanoparticles for enhanced antibacterial properties.
The utilization of metallic nanoparticles in bio-nanofabrication holds significant potential in the field of applied research. The current study applied and compared integrated ultrasonic-microwave-assisted extraction (US/MICE), ultrasonic extraction (USE), microwave-assisted extraction (MICE), and maceration (MAE) to extract total phenolic content (TPC). In addition, the study examined the antioxidant activity of Commiphora gileadensis (Cg) leaf. The results demonstrated that the TPC of US/MICE exhibited the maximum value at 59.34 ± 0.007 mg GAE/g DM. Furthermore, at a concentration of 10 μg/mL, TPC displayed a significant scavenging effect on DPPH (56.69 %), with an EC (6.48 μg/mL). Comprehensive metabolite profiling of the extract using UPLC-qTOF-MS/MS was performed to identify active agents. A total of 64 chromatographic peaks were found, out of which 60 were annotated. The most prevalent classes of metabolites found were polyphenols (including flavonoids and lignans), organic compounds and their derivatives, amides and amines, terpenes, and fatty acid derivatives. Transmission electron microscopy (TEM) revealed the aggregate size of the synthesized nanoparticles and the spherical shape of C. gileadensis-mediated silver nanoparticles (Cg-AgNPs). The nanoparticles had a particle size ranging from 7.7 to 42.9 nm. The Cg-AgNPs exhibited more inhibition zones against S. aureus and E. coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Cg-extract, AgNPs, and Cg-AgNPs were also tested. This study demonstrated the feasibility of using combined ultrasonic-microwave-assisted extraction to separate and extract chemicals from C. gileadensis on a large scale. These compounds have potential use in the pharmaceutical industry. Combining antibacterial and biocompatible properties in materials is vital for designing new materials for biomedical applications. Additionally, the results showed that the biocompatibility of the Ag-NPs using C. gileadensis extracts demonstrated outstanding antibacterial properties.
PubMed: 38815489
DOI: 10.1016/j.ultsonch.2024.106923 -
Redox Biology May 2024Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to...
Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to cysteine. As glutathione redox is important in muscle regeneration in aging, we hypothesized that xCT exerts upstream control over skeletal muscle glutathione redox, metabolism and regeneration. Bioinformatic analyses of publicly available datasets revealed that expression levels of xCT and GSH-related genes are inversely correlated with myogenic differentiation genes. Muscle satellite cells (MuSCs) isolated from Slc7a11 mice, which harbour a mutation in the Slc7a11 gene encoding xCT, required media supplementation with 2-mercaptoethanol to support cell proliferation but not myotube differentiation, despite persistently lower GSH. Slc7a11 primary myotubes were larger compared to WT myotubes, and also exhibited higher glucose uptake and cellular oxidative capacities. Immunostaining of myogenic markers (Pax7, MyoD, and myogenin) in cardiotoxin-damaged tibialis anterior muscle fibres revealed greater MuSC activation and commitment to differentiation in Slc7a11 muscle compared to WT mice, culminating in larger myofiber cross-sectional areas at 21 days post-injury. Slc7a11 mice subjected to a 5-week exercise training protocol demonstrated enhanced insulin tolerance compared to WT mice, but blunted muscle mitochondrial biogenesis and respiration in response to exercise training. Our results demonstrate that the absence of xCT inhibits cell proliferation but promotes myotube differentiation by regulating cellular metabolism and glutathione redox. Altogether, these results support the notion that myogenesis is a redox-regulated process and may help inform novel therapeutic approaches for muscle wasting and dysfunction in aging and disease.
PubMed: 38815331
DOI: 10.1016/j.redox.2024.103213 -
JCI Insight May 2024The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells...
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
PubMed: 38815134
DOI: 10.1172/jci.insight.174329 -
PloS One 2024After its emergence in China, the coronavirus SARS-CoV-2 has swept the world, leading to global health crises with millions of deaths. COVID-19 clinical manifestations...
BACKGROUND
After its emergence in China, the coronavirus SARS-CoV-2 has swept the world, leading to global health crises with millions of deaths. COVID-19 clinical manifestations differ in severity, ranging from mild symptoms to severe disease. Although perturbation of metabolism has been reported as a part of the host response to COVID-19 infection, scarce data exist that describe stage-specific changes in host metabolites during the infection and how this could stratify patients based on severity.
METHODS
Given this knowledge gap, we performed targeted metabolomics profiling and then used machine learning models and biostatistics to characterize the alteration patterns of 50 metabolites and 17 blood parameters measured in a cohort of 295 human subjects. They were categorized into healthy controls, non-severe, severe and critical groups with their outcomes. Subject's demographic and clinical data were also used in the analyses to provide more robust predictive models.
RESULTS
The non-severe and severe COVID-19 patients experienced the strongest changes in metabolite repertoire, whereas less intense changes occur during the critical phase. Panels of 15, 14, 2 and 2 key metabolites were identified as predictors for non-severe, severe, critical and dead patients, respectively. Specifically, arginine and malonyl methylmalonyl succinylcarnitine were significant biomarkers for the onset of COVID-19 infection and tauroursodeoxycholic acid were potential biomarkers for disease progression. Measuring blood parameters enhanced the predictive power of metabolic signatures during critical illness.
CONCLUSIONS
Metabolomic signatures are distinctive for each stage of COVID-19 infection. This has great translation potential as it opens new therapeutic and diagnostic prospective based on key metabolites.
Topics: Humans; COVID-19; Machine Learning; Male; Female; Biomarkers; Middle Aged; Metabolomics; Adult; Severity of Illness Index; SARS-CoV-2; Aged; Metabolome
PubMed: 38814977
DOI: 10.1371/journal.pone.0302977 -
PloS One 2024Histone variants are paralogs that replace canonical histones in nucleosomes, often imparting novel functions. However, how histone variants arise and evolve is poorly...
Histone variants are paralogs that replace canonical histones in nucleosomes, often imparting novel functions. However, how histone variants arise and evolve is poorly understood. Reconstruction of histone protein evolution is challenging due to large differences in evolutionary rates across gene lineages and sites. Here we used intron position data from 108 nematode genomes in combination with amino acid sequence data to find disparate evolutionary histories of the three H2A variants found in Caenorhabditis elegans: the ancient H2A.ZHTZ-1, the sperm-specific HTAS-1, and HIS-35, which differs from the canonical S-phase H2A by a single glycine-to-alanine C-terminal change. Although the H2A.ZHTZ-1 protein sequence is highly conserved, its gene exhibits recurrent intron gain and loss. This pattern suggests that specific intron sequences or positions may not be important to H2A.Z functionality. For HTAS-1 and HIS-35, we find variant-specific intron positions that are conserved across species. Patterns of intron position conservation indicate that the sperm-specific variant HTAS-1 arose more recently in the ancestor of a subset of Caenorhabditis species, while HIS-35 arose in the ancestor of Caenorhabditis and its sister group, including the genus Diploscapter. HIS-35 exhibits gene retention in some descendent lineages but gene loss in others, suggesting that histone variant use or functionality can be highly flexible. Surprisingly, we find the single amino acid differentiating HIS-35 from core H2A is ancestral and common across canonical Caenorhabditis H2A sequences. Thus, we speculate that the role of HIS-35 lies not in encoding a functionally distinct protein, but instead in enabling H2A expression across the cell cycle or in distinct tissues. This work illustrates how genes encoding such partially-redundant functions may be advantageous yet relatively replaceable over evolutionary timescales, consistent with the patchwork pattern of retention and loss of both genes. Our study shows the utility of intron positions for reconstructing evolutionary histories of gene families, particularly those undergoing idiosyncratic sequence evolution.
Topics: Animals; Histones; Evolution, Molecular; Introns; Caenorhabditis elegans; Amino Acid Sequence; Phylogeny; Conserved Sequence; Caenorhabditis elegans Proteins; Male
PubMed: 38814971
DOI: 10.1371/journal.pone.0300190 -
PloS One 2024Untargeted metabolomics investigations have characterized metabolic disturbances associated with various diseases in domestic cats. However, the pre-analytic stability...
Untargeted metabolomics investigations have characterized metabolic disturbances associated with various diseases in domestic cats. However, the pre-analytic stability of serum metabolites in the species is unknown. Our objective was to compare serum metabolomes from healthy cats stored at -20°C for up to 12 months to samples stored at -80°C. Serum samples from 8 adult, healthy cats were stored at -20°C for 6 months, -20°C for 12 months, or -80°C for 12 months. Untargeted liquid chromatography-mass spectrometry was used to generate serum metabolite profiles containing relative abundances of 733 serum metabolites that were compared among storage conditions. Unsupervised analysis with principal component analysis and hierarchical clustering of Euclidian distances revealed separation of samples from individual cats regardless of storage condition. Linear mixed-effects models identified 75 metabolites that differed significantly among storage conditions. Intraclass correlation analysis (ICC) classified most serum metabolites as having excellent (ICC ≥ 0.9; 33%) or moderate (ICC 0.75-0.89; 33%) stability, whereas 13% had poor stability (ICC < 0.5). Biochemicals that varied significantly among storage conditions and classified with poor stability included glutathione metabolites, amino acids, gamma-glutamyl amino acids, and polyunsaturated fatty acids. The benzoate; glycine, serine and threonine; tryptophan; chemical (xenobiotics); acetylated peptide, and primary bile acid sub pathways were enriched among highly stable metabolites, whereas the monohydroxy fatty acid, polyunsaturated fatty, and monoacylglycerol sub-pathways were enriched among unstable metabolites. Our findings suggest that serum metabolome profiles are representative of the cat of origin, regardless of storage condition. However, changes in specific serum metabolites, especially glutathione, gamma-glutamyl amino acid, and fatty acid metabolites were consistent with increased sample oxidation during storage at -20°C compared with -80°C. By investigating the pre-analytic stability of serum metabolites, this investigation provides valuable insights that could aid other investigators in planning and interpreting studies of serum metabolomes in cats.
Topics: Animals; Cats; Metabolomics; Metabolome; Male; Female; Chromatography, Liquid; Mass Spectrometry; Amino Acids
PubMed: 38814947
DOI: 10.1371/journal.pone.0303500