Transferase Activity Research Articles

Itaconate mechanism of action and dissimilation in Mycobacterium tuberculosis

Itaconate, an abundant metabolite produced by macrophages upon interferon-γ stimulation, possesses both antibacterial and immunomodulatory properties. Despite its crucial role in immunity and antimicrobial control, its mechanism of action and dissimilation are poorly understood. Here, we demonstrate that infection of mice with Mycobacterium tuberculosis increases itaconate levels in lung tissues. We also show that exposure to itaconate inhibits M. tuberculosis growth in vitro, in macrophages, and mice. We report that exposure to sodium itaconate (ITA) interferes with the central carbon metabolism of M. tuberculosis. In addition to the inhibition of isocitrate lyase (ICL), we demonstrate that itaconate inhibits aldolase and inosine monophosphate (IMP) dehydrogenase in a concentration-dependent manner. Previous studies have shown that Rv2498c from M. tuberculosis is the bona fide (S)-citramalyl-CoA lyase, but the remaining components of the pathway remain elusive. Here, we report that Rv2503c and Rv3272 possess itaconate:succinyl-CoA transferase activity, and Rv2499c and Rv3389c possess itaconyl-CoA hydratase activity. Relative to the parental and complemented strains, the ΔRv3389c strain of M. tuberculosis was attenuated for growth in itaconate-containing medium, in macrophages, mice, and guinea pigs. The attenuated phenotype of ΔRv3389c strain of M. tuberculosis is associated with a defect in the itaconate dissimilation and propionyl-CoA detoxification pathway. This study thus reveals that multiple metabolic enzymes are targeted by itaconate in M. tuberculosis. Furthermore, we have assigned the two remaining enzymes responsible for the degradation of itaconic acid into pyruvate and acetyl-CoA. Finally, we also demonstrate the importance of enzymes involved in the itaconate dissimilation pathway for M. tuberculosis pathogenesis.

Mononuclear Aluminum-Fluoride Ions, AlFx(+/-)-Study of Plausible Frameworks of Complexes with Biomolecules and Their In Vitro Toxicity.

The importance of fluorine and aluminum in all aspects of daily life has led to an enormous increase in human exposure to these elements in their various forms. It is therefore important to understand the routes of exposure and to investigate and understand the potential toxicity. Of particular concern are aluminum-fluoride complexes (AlFx), which are able to mimic the natural isostructural phosphate group and influence the activity of numerous essential phosphoryl transferases. Our review of salts of ionic AlFx species, which plausibly form the framework of complexes with biomolecules, revealed that the octahedral configuration of aluminum in the active site of the enzyme is preferred over the trigonal-bipyramidal structure. The effects of varying concentrations of fluoride, aluminum and AlFx-from micromolar to millimolar levels-on the viability and apoptosis rate of THP-1 monocytes were investigated using phosphate buffer solution as a culture media to simulate physiological conditions. Our results suggest that aluminum can reduce the direct toxicity of fluoride through the formation of AlFx. In view of the results found, further in vitro studies are required to clarify the toxicity mechanisms of these species.

A facile assay for zDHHC palmitoyl transferase activation elucidates effects of mutation and modification.

At least 10% of proteins constituting the human proteome are subject to S-acylation by a long-chain fatty acid, thioesterified to a Cys thiol side chain. Fatty S-acylation (prototypically, S-palmitoylation) operates across eukaryotic phylogeny and cell type. S-palmitoylation is carried out in mammalian cells by a family of 23-24 dedicated zDHHC palmitoyl transferase enzymes, and mutation of zDHHCs is associated with a number of human pathophysiologies. Activation of the zDHHCs by auto-S-palmitoylation, the transthioesterification of the active site Cys by fatty acyl-CoA, is the necessary first step in zDHHC-mediated protein S-palmitoylation. Most prior in vitro assessments of zDHHC activation have utilized purified zDHHCs, a time- and effort-intensive approach, which removes zDHHCs from their native membrane environment. We describe here a facile assay for zDHHC activation in native membranes. We overexpressed HA-tagged wild-type or mutant zDHHCs in cultured HEK293 cells and prepared a whole membrane fraction, which was incubated with fluorescent palmitoyl CoA (NBD-palmitoyl-CoA) followed by SDS-PAGE, fluorescence imaging and western blotting for HA. We show by mutational analysis that, as assayed, zDHHC auto-S-palmitoylation by NBD-palmitoyl-CoA is limited to the active site Cys. Application of the assay revealed differential effects on zDHHC activation of posttranslational zDHHC modification, and of zDHHC mutations associated with human disease, in particular cancer. Our assay provides a facile means of assessing zDHHC activation and thus of differentiating the effects of zDHHC mutation and post-translational modification on zDHHC activation versus secondary effects on zDHHC functionality resulting from altered zDHHC interaction with substrate palmitoyl-proteins.

Altering the intracellular trafficking of Necator americanus GST-1 antigen yields novel hookworm mRNA vaccine candidates.

The antigen Na-GST-1, expressed by the hookworm Necator americanus, plays crucial biochemical roles in parasite survival. This study explores the development of mRNA vaccine candidates based on Na-GST-1, building on the success of recombinant Na-GST-1 (rNa-GST-1) protein, currently assessed as a subunit vaccine candidate, which has shown promise in preclinical and clinical studies. By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, we developed three variants of Na-GST-1 as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. After one immunization in mice, mRNA vaccines induced an earlier onset of antigen-specific antibodies compared to rNa-GST-1. Following two immunizations, mRNA vaccines induced similar or superior levels of antigen-specific antibodies compared to rNa-GST-1. Secretory Na-GST-1 was comparable to rNa-GST1 in producing neutralizing antibodies against Na-GST-1's thiol transferase activity, while native Na-GST-1 induced a more robust CD8+ T cell response due to its intracellular accumulation. Although PM Na-GST-1 elicited one of highest titers of antigen-specific antibody and a diverse set of memory T-cell populations, it resulted in a lower ratio of neutralizing antibodies after IgG purification compared to the other vaccine candidates. These findings emphasize the importance of antigen localization in tailoring immune responses and suggest that extracellular antigens are more effective for inducing humoral responses, whereas cytosolic antigen accumulation enhances MHC-1 peptide presentation. Future studies will determine if these in vitro and immunogenicity findings translate to in vivo efficacy. Altogether, mRNA vaccines offer numerous possibilities in the development of multivalent vaccines with single or multiple antigens.

Oxidative Homeostasis in Follicular Fluid and Embryo Quality-A Pilot Study.

The objective of this study was to measure the different redox biomarker levels within the follicular fluid (FF) and evaluate correlations with embryo quality using the one follicle-one oocyte/embryo approach. The prospective study included 54 women (average age 34.6 ± 3.0 years). Out of the 235 mature metaphase II cells that underwent intracytoplasmic sperm injection, fertilization was achieved in 177 cells, producing 92 Grade I embryos, 26 Grade II embryos, 39 Grade III embryos, and 20 Grade IV embryos. The activities of antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione transferase were significantly higher in the group consisting of lower-quality (Grades II-IV) embryos in comparison with top-quality (Grade I) embryos (p = 0.011; p = 0.021; p = 0.008, respectively). The concentration of oxidative stress markers, malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and thiol groups was significantly increased in the group with lower-quality embryos (Grades II-IV) compared to top-quality embryos (0.027; 0.018; 0.021, respectively). Furthermore, a significant positive correlation between each oxidative marker and the activities of antioxidant enzymes was observed (p < 0.001). According to our findings, the best embryos and, consequently, better in vitro fertilization outcomes are linked to low levels of oxidative stress and low antioxidant enzyme activity.

Decoding gene expression dynamics during seed development in sesame (Sesamum indicum L.) through RNA-Seq analysis.

Sesame (Sesamum indicum L., 2n = 2× = 26) from the Pedaliaceae family is primarily grown for its high oil content, rich in unsaturated fatty acids like linoleic acid (LA) and alpha-linolenic acid (ALA). However, the molecular mechanisms of sesame oil accumulation remain poorly understood. This study analyzed transcriptomes at two seed development stages: Young Stage (YS, pods 1.5-2.5 cm) and Mature Stage (MS, brown pods >2.5 cm), to explore regulatory mechanisms and identify key genes involved in lipid biosynthesis. From 25,173 genes, 18,820 with expression values >10 CPM in at least 70 % of replicates were included in differential expression (DE) analysis. Active expression (LFC > 0) was observed in 9372 and 9448 genes at YS and MS, respectively. DEGs were annotated, revealing roles in various biological processes, (e.g., mRNA metabolic process, reproduction-related developmental processes, seed development), molecular functions (e.g., aminoacyltransferase activity, ubiquitin-like protein and ubiquitin-protein transferase activities), and cellular components (e.g., peroxisome, microbody, lipid droplet). KEGG analysis highlighted genes involved in fatty acid synthesis (e.g., fabG, fabZ), TAG biosynthesis (DGAT1, GPAT), and alpha-linolenic acid metabolism (AOS, LCAT3). Key genes upregulated at MS included SIN_1025205 (protein transport), SIN_1006853 (acetylajmalan esterase), and SIN_1003267 (gamma-cadinene synthase). The study generated a valuable transcriptome dataset and gene list for seed development and lipid biosynthesis, which will be validated through functional studies. An interactive webpage is provided for data exploration.

Mondia whitei fruit extract abates cadmium chloride-induced nephrotoxicity via the suppression of oxidative stress in rats

Cadmium (Cd) is an industrial heavy metal that causes toxic effects to several body organs including the kidney. Medicinal plants are used in managing various diseases caused by environmental toxicants. This study aimed to evaluate the protective ability of Mondia whitei fruit extract (MWE) against nephrotoxicity induced by CdCl₂ exposure in Wistar rats. Twenty-five male Wistar rats of 90 to 130 g body weight (b.w) were used in this study. The animals were indiscriminately assigned to five groups of five animals per group. The control group were rats in group I, whereas group II to V rats were orally administered CdCl₂ (5 mg/kg body weight (b.w)) for 5 days. Simultaneously, the rats in group III to V were co-treated with 70 mg/kg b.w of silymarin, 250 and 500 mg/kg b.w of MWE, respectively. The CdCl2 significantly (p &lt; 0.05) increased the levels of serum renal biomarkers such as urea and creatinine, total protein and albumin. Furthermore, a substantial (p &lt; 0.05) elevation in the nitric oxide (NO) and malondialdehyde (MDA) levels, and a concomitant (p &lt; 0.05) reduction in the kidney activities of superoxide dismutase (SOD) and glutathione transferase (GST), and the level of reduced glutathione (GSH) were observed in the CdCl2-intoxicated rats. Alterations in the kidney's histology were also noted. Nonetheless, treatment with MWE markedly returned to normal the levels of serum kidney parameters and tissue NO and MDA. In addition, the MWE significantly (p &lt; 0.05) elevated tissue antioxidant levels and restored the kidney histology. The result of this study suggest that MWE could potentially serve as an alternative therapy in managing CdCl2-induced nephrotoxicity.

Characterization of Helicobacter pylori immunoreactive proteins NusB, isoprenyl transferase, and hypothetical protein via immunoproteomics and molecular modeling approaches.

The microaerophilic Gram-negative bacterium H. pylori is associated with various gastric complications and affects nearly half of the global population. Current sero-diagnostic methods for H. pylori diagnosis are often insensitive or lack specificity. This study aimed to detect H. pylori immunoreactive proteins to improve diagnostic tools. H. pylori isolates from biopsy samples were characterized using biochemical and molecular techniques. An immunoproteomics approach involving immunoprecipitation and mass spectrometry identified three immunoreactive proteins: Transcription antitermination protein NusB, Isoprenyl transferase, and a hypothetical protein associated with a transposase gene. Bioinformatics analysis revealed that these proteins are involved in RNA binding, termination of DNA-templated transcription, cell and energy metabolism, transferase activity, regulation, and ribosomal biosynthesis pathways. CD4 T cell and Class-I immunogenicity predictions highlighted NusB's strong potential to stimulate an immune response. Immune simulations demonstrated robust antibody production, particularly in response to NusB. Additionally, molecular docking studies with phenolic compounds (Gnetol, Isohomovanillic acid, Licoisoflavone A, and Chrysosplenol D) against the three proteins, followed by molecular dynamics (MD) simulations, confirmed the stability and favorable interactions of these protein-phenolic compound complexes. This integrative approach, combining immunoproteomics, bioinformatics, molecular docking, and MD simulations, underscores the potential of these immunoreactive proteins for vaccine development and improved diagnostic methods.

Transcriptomic analysis and epigenetic regulators in human oocytes at different stages of oocyte meiotic maturation.

Human oocytes are highly specialized cells with the capacity to store and regulate mRNAs during oocyte maturation, in preparation for post-fertilization steps. Here we performed single-oocyte transcriptomic analysis of human oocytes in three meitoic maturation stages - Germinal Vesicle (GV; n=6), Metaphase I (MI; n=6) and Metaphase II (MII; n=7). Single-oocyte transcriptomic analysis revealed that the total number of expressed genes progressively decreased from GV to MII stages, with 9660 genes being transcribed in GV, 8734 in MI and 5889 in MII. The same tendency was observed for the number of uniquely expressed genes, with 1328 uniquely expressed genes in GV, 401 in MI and 72 in MII. GO analysis of the uniquely expressed genes showed distinct terms in GV oocytes such as transferase activity, organonitrogen compound metabolic process and ncRNA processing. Analysis of Differentially Expressed Genes (DEGs) between the three maturation stages revealed 1165 DEGs between GV and MII oocytes, with 635 being upregulated and 528 downregulated, 42 DEGs between GV and MI, with 38 being upregulated and 4 downregulated, and no significant changes in gene expression between MI and MII oocytes. Comprehensive analysis of epigenetic regulators showed high expression of several histone-modifying enzymes, namely deacetylases, acetylases, lysine demethylases and methyltransferases, and DNA methylation regulators, namely the maintenance methyltransferase DNMT1 and its co-regulators DPPA3 and UHRF1. Some of these epigenetic regulators were differentially expressed between maturation stages, namely SIRT3, SIRT6, KDM3AP1, KMT2E, DNMT1, DPPA3 and the MEST and RASGRF1 imprinted genes. Our study contributes with important information on the transcriptional landscape of human oocytes in different stages of meiotic maturation, providing important insights into candidate biomarkers of human oocyte quality.

Interactions between the gut microbiome and DNA methylation patterns in blood and visceral adipose tissue in subjects with different metabolic characteristics

BackgroundThe gut microbiome has been reported to induce epigenetic modifications in the host, which may be involved in the pathophysiology of metabolic diseases.ObjectiveTo evaluate the potential interactions between the gut microbiome and DNA methylome in subjects with different metabolic characteristics.MethodsSixty-four participants with different metabolic characteristics (i.e., participants without obesity -healthy controls-, and participants with obesity and normal insulin sensitivity/insulin resistance/ type 2 diabetes-T2DM-) were included in this study. A machine learning approach was performed in order to identify distinctive patterns in three omics (gut microbiome, blood DNA methylome, and visceral adipose tissue-VAT- DNA methylome) according to the different study groups.ResultsRobust distinctive distribution patterns of the three different omics were found between healthy controls and patients with obesity; participants with and without T2DM, and patients with obesity with and without insulin resistance. Importantly, strong correlations between the gut microbiome (including Odoribacteriaceae and Christensenllaceae families) and both blood and VAT DNA methylome were found. Moreover, in the entire study population, three main bacterial genera (Sutterella, Collinsella and Eubacterium) were related to the epigenetic regulation of different genes involved in distinct processes related to cellular metabolism and metabolic diseases, including small ubiquitin-related modifier (SUMO) transferase activity or lipid binding.ConclusionWe show that distinctive interactions between the gut microbiome and DNA methylome may occur in subjects with different metabolic characteristics. Further research is needed to elucidate the potential role of these interactions in the pathophysiology of obesity and related comorbidities.

Effect of DNA-binding Proteins on Terminal Deoxynucleotidyl Transferase Activity in Systems with Homopolymer Substrates

Effect of DNA-binding Proteins on Terminal Deoxynucleotidyl Transferase Activity in Systems with Homopolymer Substrates

Self-assembling nanoparticles for delivery of miR-603 and miR-221 in glioblastoma as a new strategy to overcome resistance to temozolomide

Glioblastoma (GBM) is a highly aggressive brain cancer with poor clinical outcome. Unfortunately, chemotherapy with temozolomide (TMZ) has a limited efficacy due to resistance mainly attributed to O6-methylguanine methyl transferase (MGMT) activity. Recently, miR-603 and miR-221 have been identified to target MGMT, thus improving the efficacy of temozolomide (TMZ) in the treatment of GBM. Previously, self-assembling nanoparticles (SANPs) have been proposed to deliver miRNAs into the brain. Here, SANP co-encapsulating miRNA-603 (miR-603) and miRNA-221 (miR-221) have been developed to enhance the efficacy of TMZ in the treatment of GBM by preventing the occurrence of chemoresistance. Preliminarily, SANPs encapsulating miRNAs were optimized in terms of lipid composition to assure physical stability and no hemolytic activity. Subsequently, SANPs with the lowest cytotoxicity and excellent internalization efficiency of miRNAs were selected through MTT assay and real-time PCR, respectively. To evaluate a potential synergistic effect between TMZ and miRNAs, MTT and clonogenic assays were performed. In our biological model, miRNA delivery via SANPs in combination with TMZ treatment strongly reduced cell viability and tumorigenic potential. Finally, in vivo assays were carried out on orthotopic xenograft mouse models. The treatment with SANPs encapsulating both miRNAs in combination with TMZ greatly decreased tumour growth, and even more significantly increased animal survival. In conclusion, this strategy provides the rationale for the development of new therapeutic approaches based on SANP technology to deliver miRNAs that play a key role in suppressing tumour.

Determination of Haptoglobin, Serum Amyloid A, Some Other Acute Phase Proteins, and Biochemical Parameters in Cattle with Hydatid Cysts

The aim of this study is to determine some acute phase proteins and biochemical parameter levels in cattle infected with the hydatid cysts. In the study, a total of 30, Brown Swiss cattle including 15 infected with the hydatid cysts in the study group and 15 in the control group were used. Haptoglobin, serum amyloid A (SAA), ceruloplasmin, interleukin (IL)-6, total protein, albumin, aspartate amino transferase (AST), gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) activity, urea, creatinine, iron (Fe) levels were determined colorimetrically. When the animals infected with hydatid cysts and control group were compared, it was determined that haptoglobin, SAA, IL-6, AST, ALP, GGT activity, urea, creatinine levels and acute phase protein index values increased, while albumin levels decreased. In addition, it was determined that the ceruloplasmin and total protein levels increased, globulin, albumin/globulin and Fe levels decreased, but they were statistically insignificant. In conclusion, it was determined that there were significant changes in the biochemical parameters and increased haptoglobin and SAA synthesis in the animals infected with the hydatid cysts, and it is thought that these parameters may contribute to the pathogenesis and diagnosis of the disease.

Association Between Hyperlipidaemia and Selected Cholestatic Markers in 74 Dogs with Suspect Acute Pancreatitis.

The association between hyperlipidaemia and acute pancreatitis is unknown in dogs. This study aimed to investigate the association between hyperlipidaemia and other markers of cholestasis in dogs with suspect acute pancreatitis. Case records of dogs with suspect acute pancreatitis were retrospectively reviewed. Dogs that had pre-existing disorders or drug therapies associated with hyperlipidaemia, hypocholesterolaemia, or incomplete biochemical data were excluded. In total, 74 dogs met the inclusion criteria. There were 33 (44.6%) dogs with hypercholesterolaemia (HC) and 41 (55.4%) without (NC). Increased triglyceride concentrations were significantly (p = 0.005) more common in HC dogs (n = 13, 39.4%) compared with NC dogs (n = 4, 9.8%), but no value exceeded 5 mmol/L. The ALP activity was significantly higher in the HC group compared to NC group (932 (461-7271) and 380 (135-1312) IU/L, respectively, p = 0.001). There was a moderate positive correlation between cholesterol concentration and ALP activity (r = 0.498, p < 0.001) and a weak positive correlation between cholesterol concentration and gamma-glutamyl transferase activity (r = 0.296, p = 0.011). Cholesterol concentration was correlated with ALP and GGT activities suggesting an association between cholestasis and hypercholesterolaemia in dogs with acute pancreatitis.

Abstract 4138633: Infusion of human apolipoprotein A-I (CSL112) promotes several aspects of reverse cholesterol transport

Background: High-density lipoprotein (HDL)-cholesterol is inversely correlated with cardiovascular risk, but increasing its circulating concentration is insufficient to prevent adverse cardiovascular outcomes. Instead, the emerging paradigm is on increasing the function of HDL and its major protein constituent apolipoprotein A-I (apoA-I), to increase reverse cholesterol transport. Objective: To investigate the effect of apoA-I [human] (CSL112) infusion on HDL protein composition, and provide further insights into the mechanism of action of CSL112 administered post-acute myocardial infarction (AMI). Methods: A mass spectrometry (MS)-based proteomic approach was used to evaluate changes in HDL protein composition in patients (n=50) from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study who received either placebo or CSL112 post AMI. HDL was immuno-isolated from patient plasma using anti-apoA-I antibodies. Cholesterol esterification rate (CER) was measured to determine lecithin-cholesterol acyl transferase (LCAT) activity. Cholesterol efflux capacity (CEC) and hepatocyte uptake were assessed using patient serum in ex vivo cell-based assays. Results: CSL112 induced extensive rearrangement of HDL proteins at 4 hours post-infusion. Levels of apolipoproteins A2, B, C, and E as well as the acute phase proteins serum amyloid A1 and A4 were significantly reduced. By contrast, apoA-I, apoM, and LCAT significantly increased. Elevated apoA-I and LCAT levels on HDL were associated with an increase in CEC, plasma HDL-C levels, and CER in CSL112-treated patients. Furthermore, enhanced CEC strongly correlated with cholesterol uptake by hepatic cells (r=0.95 p&lt;0.001). Conclusion: CSL112 altered HDL composition and increased HDL functionality by promoting multiple steps of the reverse cholesterol transport pathway.

Disperse Red 1 azo dye: Consequences of low-dose/low-concentration exposures in mice and zebrafish

Color Index Disperse Red 1 (DR1), an azo dye widely used in the textile industry and released into aquatic environments, is genotoxic in somatic cells, but little is known concerning its effects on the reproductive system or the early stages of embryonic development. We have assessed the effects on the spermatozoa of male mice following oral exposure to the dye, at low doses, for 14 days. Measured endpoints were DNA damage (comet assay), miRNA-34c levels, and sperm number, morphology, and motility. Exposure caused decreased miRNA-34c levels. We have also examined dye effects on zebrafish embryos and larvae, which included developmental impairment, altered glutathione transferase activity, and effects on reactive oxygen species and lipid peroxidation levels.

Molecular Cloning, Characterization, and Application of a Novel Multifunctional Isoamylase (MIsA) from Myxococcus sp. Strain V11.

A novel multifunctional isoamylase, MIsA from Myxococcus sp. strain V11, was expressed in Escherichia coli BL21(DE3). Sequence alignment revealed that MIsA is a typical isoamylase that belongs to glycoside hydrolase family 13 (GH 13). MIsA can hydrolyze the α-1,6-branches of amylopectin and pullulan, as well as the α-1,4-glucosidic bond in amylose. Additionally, MIsA demonstrates 4-α-D-glucan transferase activity, enabling the transfer of α-1,4-glucan oligosaccharides between molecules, particularly with linear maltooligosaccharides. The Km, Kcat, and Vmax values of the MIsA for amylopectin were 1.22 mM, 40.42 µmol·min-1·mg-1, and 4046.31 mM·min-1. The yields of amylopectin and amylose hydrolyzed into oligosaccharides were 10.16% and 11.70%, respectively. The hydrolysis efficiencies were 55%, 35%, and 30% for amylopectin, soluble starch, and amylose, respectively. In the composite enzyme hydrolysis of amylose, the yield of maltotetraose increased by 1.81-fold and 2.73-fold compared with that of MIsA and MTHase (MCK8499120) alone, respectively.

Analysis of Alternative Splicing Events and Identification of Key Genes in Brassica napus Leaves Infected by Leptosphaeria biglobosa

Alternative splicing (AS) is a prevalent post-transcriptional regulatory mechanism in eukaryotes and plays a crucial role in plant disease resistance. Here, we used the Illumina Novaseq sequencing platform to conduct transcriptome sequencing on canola (Brassica napus) leaves infected with the blackleg pathogen (Leptosphaeria biglobosa strain nm−1) at 0 h, 72 h, 120 h, and 168 h post-inoculation to investigate the mechanism of AS coordination with transcriptional regulation in canola’s response to blackleg disease. The rMATS software (4.1.0) was employed to analyze different AS events in samples taken at 72 h, 120 h, and 168 h. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to elucidate the biological functions of differentially spliced genes at various time points, while Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify key modules and hub genes. As a result, our analysis reveals 16908 AS events across three time points, with 221 being differently spliced. Intron retention (RI) was the most common AS event, accounting for approximately 55% of all events, while alternative 5′ splice site events were least common, comprising only 2%. Furthermore, a total of 213 significantly differentially spliced genes were identified, which were enriched in functions related to protein kinase activity, transferase activity, and pathways such as MAPK signaling pathway—plant and plant hormone signal transduction. WGCNA identified three key modules and ten hub genes, including calcium-binding transcription activator 1, LRR class receptor serine/threonine protein kinase FEI 2, PLATZ transcription factor family proteins, serine/threonine protein kinase PRP4, and E3 ubiquitin ligase SUD1, all of which are associated with canola resistance to L. biglobosa. Thus, this study provides a theoretical basis for identifying disease-resistance genes involved in AS and for exploring the functions of AS gene isoforms in canola.

SIRT2-mediated deacetylation of glutathione transferase alleviates oxidative damage and increases the heat tolerance of Pleurotus ostreatus

High-temperature stress (HS) severely threatens agricultural production. Pleurotus ostreatus is cultivated in many parts of the world, and its growth is strongly affected by HS. We previously reported that metabolic rearrangement occurred in HS, but the gene expression levels of several key enzymes remained unchanged. Therefore, in this study, we investigated the contribution of posttranslational modifications of proteins to HS resistance in P. ostreatus. We found that the level of acetylation of P. ostreatus decreased under short-term HS treatment and increased as the duration of HS treatment increased. Acetylation omics revealed that almost all metabolic enzymes were acetylated. We found that deacetylation under HS can improve the growth recovery ability of mycelia, the activity of matrix-degrading enzyme, and the contents of antioxidants, such as nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH), but can decreased H2O2 levels. In vitro acetylation experiments and point mutations revealed that the deacetylase SIRT2 increased the activity of glutathione transferases (GSTs) by deacetylating GST1 66K, GST2 206K, and GST2 233K. Together, SIRT2 is activated by short-term HS and improves its antioxidant activity by deacetylating GSTs, thereby improving the resistance of P. ostreatus to HS. In this study, we identified new non-histone substrate proteins and new lysine acetylation sites of SIRT2 under HS. We also discovered the role of non-histone acetylation in the adaptation of organisms to HS.

Modulation of Carnitine Palmitoyl Transferase 1b Expression and Activity in Muscle Pathophysiology in Osteoarthritis and Osteoporosis

In the pathophysiology of osteoarthritis and osteoporosis, articular cartilage and bone represent the target tissues, respectively, but muscle is also involved. Since many changes in energy metabolism occur in muscle with aging, the aim of the present work was to investigate the involvement of carnitine palmitoyl transferase 1b (Cpt1b) in the muscle pathophysiology of the two diseases. Healthy subjects (CTR, n = 5), osteoarthritic (OA, n = 10), and osteoporotic (OP, n = 10) patients were enrolled. Gene expression analysis conducted on muscle and myoblasts showed up-regulation of CPT1B in OA patients; this result was confirmed by immunohistochemical and immunofluorescence analyses and enzyme activity assay, which showed increased Cpt1b activity in OA muscle. In addition, CPT1B expression resulted down-regulated in cultured OP myoblasts. Given the potential involvement of Cpt1b in the modulation of oxidative stress, we investigated ROS levels, which were found to be lower in OA myoblasts, and gene expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (Nox4), which resulted up-regulated in OA cells. Finally, the immunofluorescence of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) showed a decreased expression in OP myoblasts, with respect to CTR and OA. Contextually, through an ultrastructural analysis conducted by Transmission Electron Microscopy (TEM), the presence of aberrant mitochondria was observed in OP muscle. This study highlights the potential role of Cpt1b in the regulation of muscle homeostasis in both osteoarthritis and osteoporosis, allowing for the expansion of the current knowledge of what are the molecular biological pathways involved in the regulation of muscle physiology in both diseases.