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Carbene transfer reactions have emerged as pivotal methodologies for the synthesis of complex molecular architectures. Heme protein-catalyzed carbene transfer reactions have shown promising results on model compounds. However, their limited substrate scope has hindered their application in natural product functionalization. Building upon the foundation of previously published work on a carbene transferase-myoglobin variant, this study employs computer-aided protein engineering to design myoglobin variants, using either docking or the deep learning-based LigandMPNN method. These variants were utilized as catalysts in carbene transfer reactions with a selection of monoterpene substrates featuring C-C double bonds, leading to seven target products. This cost-effective methodology broadens the substrate scope for heme protein-catalyzed reactions, thereby opening novel pathways for research in heme protein functionalities and offering fresh perspectives in the synthesis of bioactive molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

VQ1 and VQ10 are largely unstructured homologous proteins with a significant potential for protein-protein interactions. Yeast two-hybrid (Y2H) analysis confirmed that both proteins interact not only with themselves and each other but also with other VQ and WRKY proteins. Screening an Arabidopsis Y2H library with VQ1 as bait identified 287 interacting proteins. Validation of the screening confirmed that interactions with VQ1 also occurred with VQ10, supporting their functional homology. Although VQ1 or VQ10 proteins do not localize in plastids, 47 VQ1-targets were found to be plastidial proteins. In planta interaction with the isoprenoid biosynthetic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was confirmed by co-immunoprecipitation. DXS oligomerizes through redox-regulated intermolecular disulfide bond formation, and the interaction with VQ1 or VQ10 do not involve their unique C residues. The VQ-DXS protein interaction did not alter plastid DXS localization or its oligomerization state. Although plants with enhanced or reduced VQ1 and VQ10 expression did not exhibit significantly altered levels of isoprenoids compared to wild-type plants, they did display significantly improved or diminished photosynthesis efficiency, respectively., (© 2024. The Author(s).)

New-to-Nature biocatalysis has emerged as a promising tool in organic synthesis thanks to progress in protein engineering. Notably, hemeproteins have been evolved into robust catalysts for carbene and nitrene transfers and related sigmatropic rearrangements. In this work, we report the first example of a [2,3]-sigmatropic Sommelet-Hauser rearrangement initiated by a carbene transfer of the sperm whale myoglobin mutant L29S,H64V,V68F that was previously reported to catalyze the mechanistically similar [2,3]-sigmatropic Doyle-Kirmse rearrangement. This repurposed heme enzyme catalyzes the Sommelet-Hauser rearrangement between ethyl diazoacetate and benzyl thioethers bearing strong electron-withdrawing substituents with good yields and enantiomeric excess. Optimized catalytic conditions in the absence of any reductant led to an increased asymmetric induction with up to 59% enantiomeric excess. This myoglobin mutant is therefore one of the few catalysts for the asymmetric Sommelet-Hauser rearrangement. This work broadens the scope of abiological reactions catalyzed by iron-carbene transferases with a new example of asymmetric sigmatropic rearrangement., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Four tunicamycin class compounds, tunicamycin VII ( 1 ), tunicamycin VIII ( 2 ), corynetoxin U17a ( 3 ), and tunicamycin IX ( 4 ), were isolated from the culture broth of the marine-derived actinomycete Streptomyces sp. MBTG32. The strain was identified using the 16S rDNA sequencing technique, and the isolated strain was closely related to Streptomyces bacillaris . The structures of the isolated compounds were elucidated based on spectroscopic data and comparisons with previously reported NMR data. Compounds 1 - 4 showed potent antibacterial activities against Gram-positive bacteria, especially Staphylococcus aureus, with MIC values of 0.13-0.25 µg/mL. Through a recombinant enzyme assay and overexpression analysis, we found that the isolated compounds exerted potent inhibitory effects on S. aureus MurNAc-pentapeptide translocase (MraY), with IC 50 values of 0.08-0.21 µg/mL. The present results support that the underlying mechanism of action of tunicamycins isolated from marine-derived Streptomyces sp. is also associated with the inhibition of MraY enzyme activity in S. aureus .

Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and β-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed β-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and β-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Low-molecular-weight (LMW) thiols are produced in all living cells in different forms and concentrations. Glutathione (GSH), coenzyme A (CoA), bacillithiol (BSH), mycothiol (MSH), ergothioneine (ET) and trypanothione T(SH) 2 are the main LMW thiols in eukaryotes and prokaryotes. LMW thiols serve as electron donors for thiol-dependent enzymes in redox-mediated metabolic and signaling processes, protect cellular macromolecules from oxidative and xenobiotic stress, and participate in the reduction of oxidative modifications. The level and function of LMW thiols, their oxidized disulfides and mixed disulfide conjugates in cells and tissues is tightly controlled by dedicated oxidoreductases, such as peroxiredoxins, glutaredoxins, disulfide reductases and LMW thiol transferases. This review provides the first summary of the current knowledge of structural and functional diversity of transferases for LMW thiols, including GSH, BSH, MSH and T(SH) 2 . Their role in maintaining redox homeostasis in single-cell and multicellular organisms is discussed, focusing in particular on the conjugation of specific thiols to exogenous and endogenous electrophiles, or oxidized protein substrates. Advances in the development of new research tools, analytical methodologies, and genetic models for the analysis of known LMW thiol transferases will expand our knowledge and understanding of their function in cell growth and survival under oxidative stress, nutrient deprivation, and during the detoxification of xenobiotics and harmful metabolites. The antioxidant function of CoA has been recently discovered and the breakthrough in defining the identity and functional characteristics of CoA S-transferase(s) is soon expected., Competing Interests: Declaration of competing interest The authors do not have a conflict of interest to report., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A feeding trial of 5-week duration was performed to assess the response of broiler chicks to dietary supplementation with different doses of myco-fabricated zinc oxide nanoparticles (ZONPs) on blood indices, physiological, immunological response, antioxidant status, intestinal microbial count, and histological changes in immune organs. A total of 162 3-day-old Ross 308 broiler chicks were weighed individually and distributed equally into 3 dietary treatments with 6 replicate of 9 chicks in each in a completely randomized design. Chicks were fed ad libitum a basal ration prepared as starter, grower, and finisher supplemented with 0 (T1, control), 40 (T2), and 60 (T3) mg zinc oxide nanoparticles (ZONPs)/kg feed. Results showed that supplementing with ZONPs at both studied levels increased the relative weights of the spleen, bursa, thymus, and liver and decreased the relative weight of the kidney, gizzard, and intestine. A significant increase in the concentrations of hemoglobin (Hb), hematocrit (PCV%), red and white blood cell counts, total protein (TP), globulin (GLOB), aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and total antioxidant capacity (TAC) and a significant decrease in malonaldehyde (MDA), uric acid, and creatinine concentration were observed. Furthermore, all immunological organs showed histological alteration and increased both types of immunity in ZONPs groups with more pronounced effects in the T2 group., (© 2023. The Author(s).)

tRNA modifications play a crucial role in ensuring accurate codon recognition and optimizing translation levels. While the significance of these modifications in eukaryotic cells for maintaining cellular homeostasis and physiological functions is well-established, their physiological roles in bacterial cells, particularly in pathogenesis, remain relatively unexplored. The TusDCB protein complex, conserved in γ-proteobacteria like Escherichia coli, is involved in sulfur modification of specific tRNAs. This study focused on the role of TusDCB in the virulence of uropathogenic E. coli (UPEC), a bacterium causing urinary tract infections. The findings indicate that TusDCB is essential for optimal production of UPEC's virulence factors, including type 1 fimbriae and flagellum, impacting the bacterium's ability to aggregate in bladder epithelial cells. Deletion of tusDCB resulted in decreased virulence against urinary tract infection mice. Moreover, mutant TusDCB lacking sulfur transfer activity and tusE- and mnmA mutants revealed the indispensability of TusDCB's sulfur transfer activity for UPEC pathogenicity. The study extends its relevance to highly pathogenic, multidrug-resistant strains, where tusDCB deletion reduced virulence-associated bacterial aggregation. These insights not only deepen our understanding of the interplay between tRNA sulfur modification and bacterial pathogenesis but also highlight TusDCB as a potential therapeutic target against UPEC strains resistant to conventional antimicrobial agents., (© 2024. The Author(s).)

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar ( Populus × canescens ) lines modified in their DXS activity. Single leaves were dynamically labeled with 13 CO 2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Neryl diphosphate (C 10 ) synthase (NDPS1), a homodimeric soluble cis-prenyltransferase from tomato, contains four disulfide bonds, including two inter-subunit S-S bonds in the N-terminal region. Mutagenesis studies demonstrated that the S-S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C-terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well-ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C-terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site-directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N-terminal regions and the closed/open states of C-terminal regions may cooperate and be important for the catalytic mechanism of NDPS1., (© 2024 Wiley‐VCH GmbH.)

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The extensive exploration of antibiotic biodegradation by antibiotic-degrading bacteria in biological wastewater treatment processes has left a notable gap in understanding the behavior of these bacteria when exposed to antibiotics and the initiation of biodegradation processes. This study, therefore, delves into the adhesive behavior of Paraclostridium bifermentans, isolated from a bioreactor treating ciprofloxacin-laden wastewater, towards ciprofloxacin molecules. For the first time, this behavior is observed and characterized through quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy. The investigation further extends to identify key regulatory factors and mechanisms governing this adhesive behavior through a comparative proteomics analysis. The results reveal the dominance of extracellular proteins, particularly those involved in nucleotide binding, hydrolase, and transferase, in the adhesion process. These proteins play pivotal roles through direct chemical binding and the regulation of signaling molecule. Furthermore, QCM-D measurements provide evidence that transferase-related signaling molecules, especially tyrosine, augment the binding between ciprofloxacin and transferases, resulting in enhance ciprofloxacin removal by P. bifermentans (increased by ∼1.2-fold). This suggests a role for transferase-related signaling molecules in manipulating the adhesive behavior of P. bifermentans towards ciprofloxacin. These findings contribute to a new understanding of the prerequisites for antibiotic biodegradation and offer potential strategies for improving the application of antibiotic-degrading bacteria in the treatment of antibiotics-laden wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate (d-GAP). DXP is an essential bacteria-specific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of d-GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote LThDP activation. Here, we show that the substitution of putative network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. Substitutions predicted to disrupt the network have the effect to activate LThDP for decarboxylation, resulting in CO 2 and acetate production. In contrast, a substitution predicted to strengthen the network fails to activate LThDP and has the effect to shift DXPS toward the closed state. Network-disrupting substitutions near the carboxylate of LThDP also have a pronounced effect to shift DXPS to an open state. These results offer initial insights to explain the long-lived LThDP intermediate and its activation through disruption of an active site network, which is unique to DXPS. These findings have important implications for DXPS function in bacteria and its development as an antibacterial target.

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro . Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.

Desulfofundulus kuznetsovii is a thermophilic, spore-forming sulphate-reducing bacterium in the family Peptococcaceae. In this study, we describe a newly isolated strain of D. kuznetsovii, strain TPOSR, and compare its metabolism to the type strain D. kuznetsovii 17 T . Both strains grow on a large variety of alcohols, such as methanol, ethanol and propane-diols, coupled to the reduction of sulphate. Strain 17 T metabolizes methanol via two routes, one involving a cobalt-dependent methyl transferase and the other using a cobalt-independent alcohol dehydrogenase. However, strain TPOSR, which shares 97% average nucleotide identity with D. kuznetsovii strain 17 T , lacks several genes from the methyl transferase operon found in strain 17 T . The gene encoding the catalytically active methyl transferase subunit B is missing, indicating that strain TPOSR utilizes the alcohol dehydrogenase pathway exclusively. Both strains grew with methanol during cobalt starvation, but growth was impaired. Strain 17 T was more sensitive to cobalt deficiency, due to the repression of its methyl transferase system. Our findings shed light on the metabolic diversity of D. kuznetsovii and their metabolic differences of encoding one or two routes for the conversion of methanol., (© 2024. The Author(s).)

Acetolactate synthase inhibitors (ALS inhibitors) and glyphosate are two classes of herbicides that act by inhibiting an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. Besides amino acid synthesis inhibition, both herbicides trigger similar physiological effects in plants. The main aim of this study was to evaluate the role of glutathione metabolism, with special emphasis on glutathione S-transferases (GSTs), in the mode of action of glyphosate and ALS inhibitors in Amaranthus palmeri. For that purpose, plants belonging to a glyphosate-sensitive (GLS) and a glyphosate-resistant (GLR) population were treated with different doses of glyphosate, and plants belonging to an ALS-inhibitor sensitive (AIS) and an ALS-inhibitor resistant (AIR) population were treated with different doses of the ALS inhibitor nicosulfuron. Glutathione-related contents, GST activity, and related gene expressions (glutamate-cysteine ligase, glutathione reductase, Phi GST and Tau GST) were analysed in leaves. According to the results of the analytical determinations, there were virtually no basal differences between GLS and GLR plants or between AIS and AIR plants. Glutathione synthesis and turnover did not follow a clear pattern in response to herbicides, but GST activity and gene expression (especially Phi GSTs) increased with both herbicides in treated sensitive plants, possibly related to the rocketing H 2 O 2 accumulation. As GSTs offered the clearest results, these were further investigated with a multiple resistant (MR) population, compressing target-site resistance to both glyphosate and the ALS inhibitor pyrithiobac. As in single-resistant plants, measured parameters in the MR population were unaffected by herbicides, meaning that the increase in GST activity and expression occurs due to herbicide interactions with the target enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[ 14 C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vibrio vulnificus (vv) is a multidrug-resistant human bacterial pathogen whose prevalence is expected to increase over the years. Transketolases (TK), transferases catalyzing two reactions of the nonoxidative branch of the pentose-phosphate pathway and therefore linked to several crucial metabolic pathways, are potential targets for new drugs against this pathogen. Here, the vvTK is crystallized and its structure is solved at 2.1 Å. A crown of 6 histidyl residues is observed in the active site and expected to participate in the thiamine pyrophosphate (cofactor) activation. Docking of fructose-6-phosphate and ferricyanide used in the activity assay, suggests that both substrates can bind vvTK simultaneously. This is confirmed by steady-state kinetics showing a sequential mechanism, on the contrary to the natural transferase reaction which follows a substituted mechanism. Inhibition by the I38-49 inhibitor (2-(4-ethoxyphenyl)-1-(pyrimidin-2-yl)-1H-pyrrolo[2,3-b]pyridine) reveals for the first time a cooperative behavior of a TK and docking experiments suggest a previously undescribed binding site at the interface between the pyrophosphate and pyridinium domains., (© 2023 The Protein Society.)

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase ( HCT ) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g -1 . Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene ( CtHCT , CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the Ct HCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

Protein S -acyl transferases (PATs) catalyze S -acylation, a reversible post-translational modification critical for membrane association, trafficking, and stability of substrate proteins. Many plant proteins are potentially S -acylated but few have corresponding PATs identified. By using genomic editing, confocal imaging, pharmacological, genetic, and biochemical assays, we demonstrate that three Arabidopsis class C PATs positively regulate BR signaling through S -acylation of BRASSINOSTEROID-SIGNALING KINASE1 (BSK1). PAT19, PAT20, and PAT22 associate with the plasma membrane (PM) and the trans -Golgi network/early endosome (TGN/EE). Functional loss of all three genes results in a plethora of defects, indicative of reduced BR signaling and rescued by enhanced BR signaling. PAT19, PAT20, and PAT22 interact with BSK1 and are critical for the S -acylation of BSK1, and for BR signaling. The PM abundance of BSK1 was reduced by functional loss of PAT19 , PAT20 , and PAT22 whereas abolished by its S -acylation-deficient point mutations, suggesting a key role of S -acylation in its PM targeting. Finally, an active BR analog induces vacuolar trafficking and degradation of PAT19, PAT20, or PAT22, suggesting that the S -acylation of BSK1 by the three PATs serves as a negative feedback module in BR signaling., Competing Interests: Competing interests statement:The authors declare no competing interest.

Triclosan has been extensively used as a preservative in cosmetics and personal care products. However, its accumulation represents a real environmental threat. Thus, its phytotoxic impact needs more consideration. Our study was conducted to highlight the phytotoxic effect of triclosan on the growth, ROS homeostasis, and detoxification metabolism of two different plant species i.e., legumes (Glycine max) and grass (Avena sativa). Moreover, we investigated the potentiality of plant growth-promoting bacteria (ST-PGPB) in mitigating the phytotoxic effect of triclosan. Triclosan induced biomass (fresh and dry weights) reduction in both plants, but to a higher extent in oats. This decline was associated with a noticeable increment in the oxidative damage (e.g., MDA and H 2 O 2 ) and detoxification metabolites such as metallothionein (MTC), phytochelatins (PCs), and glutathione-S-transferase (GST). This elevation was associated with a remarkable reduction in both enzymatic and non-enzymatic antioxidants. On the other hand, the bioactive strain of ST-PGPB, Salinicoccus sp. JzA1 significantly alleviated the harmful effect of triclosan on both soybean and oat plants by enhancing their biomass, photosynthesis, as well as levels of minerals (K, Ca, P, Mn, and Zn). In parallel, a striking quenching in oxidative damage and an obvious improvement in non-enzymatic (polyphenols, tocopherols, flavonoids) and enzymatic antioxidants were observed. Furthermore, Salinicoccus sp. JzA1 augmented the detoxification metabolism by enhancing the levels of phytochelatins, metallothionein, and glutathione-S-transferase (GST) activity in a species-specific manner which is more apparent in soybean rather than in oat plants. To this end, stress mitigating impact of Salinicoccus sp. JzA1 provides a basis to improve the resilience of crop species under cosmetics and personal care products toxicity., Competing Interests: Declaration of competing interest All the authors disclosed that there is no any financial or personal relationship that may be perceived as influencing their work., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Background: Nickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure., Methods: We employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation., Result: Exposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, β-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3β/β-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3β/β-Catenin pathway and c-Myc transcription in HBECs post nickel exposure., Conclusion: Our results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/β-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chuanshu Huang reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2024. Published by Elsevier Inc.)

Zika virus (ZIKV) spread is considered a major public health threat by the World Health Organization (WHO). There are no vaccines or drugs available to control the infection of the Zika virus, therefore a highly effective medicinal molecule is urgently required. In this study, a computationally intensive investigation was performed to identify a potent natural compound that could inhibit the ZIKV NS5 methyltransferase. This research approach is based on target-based drug identification principles where the native inhibitor SAH (S-adenosylhomocysteine) of ZIKV NS5 methyltransferase was selected as a reference. High-throughput virtual screening and tanimoto similarity coefficient were applied to the natural compound library for ranking the potential candidates. The top five compounds were selected for interaction analysis, MD simulation, total binding free energy through MM/GBSA, and steered MD simulation. Among these compounds, Adenosine 5'-monophosphate monohydrate, Tubercidin, and 5-Iodotubercidin showed stable binding to the protein compared to the native compound, SAH. These three compounds also showed less fluctuations in RMSF in contrast to native compound. Additionally, the same interacting residues observed in SAH also made strong interactions with these three compounds. Adenosine 5'-monophosphate monohydrate and 5-Iodotubercidin had greater total binding free energies than the reference ligand. Moreover, the dissociation resistance of all three compounds was equivalent to that of the reference ligand. This study suggested binding properties of three-hit compounds that could be used to develop drugs against Zika virus infections.Communicated by Ramaswamy H. Sarma.

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants' cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized., Methods: Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes., Results: We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions., Conclusions: Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling., Competing Interests: Disclosures None.

Macrophage-driven chronic low-grade inflammatory response is intimately associated with pathogenesis of insulin resistance and type 2 diabetes (T2D). However, the molecular basis for skewing of pro-inflammatory macrophage is still elusive. Here, we describe the mechanism and significance of TGS1/PIMT (PRIP-Interacting protein with Methyl Transferase domain) in regulating macrophage activation and polarization and its impact on the development of insulin resistance in skeletal muscle cells. We show altered expression of TGS1 in M1 polarized cultured macrophages, bone marrow-derived (BMDM) and adipose tissue macrophages. Moreover, in High Fat Diet (HFD)-fed mice enhanced TGS1 expression is predominantly localized to the nucleus of adipose tissue macrophages suggesting its potential functional role. Gain and loss of TGS1 expression in macrophage further established its role in the secretion of pro-inflammatory mediators. Mechanistically, TGS1 controls the transcription of numerous genes linked to inflammation by forming a complex with Histone Acetyl Transferase (HAT)-containing transcriptional co-activators CBP and p300. Functionally, TGS1 mediated macrophage inflammatory response induces the development of insulin resistance in skeletal muscle cells and adipocytes. Our findings thus demonstrate an unexpected contribution of TGS1 in the regulation of macrophage mediated inflammation and insulin resistance highlighting that TGS1 antagonism could be a promising therapeutic target for the management of inflammation and insulin resistance in T2D., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Anthocyanins are a flavonoid compound known as one of the most important chromogenic substances. They play several functions, including health promotion and sustaining plants during adverse conditions. They are synthesized at the endoplasmic reticulum and sequestered in the vacuole. In this work, we generated knock-out lines of OsGSTU34, a glutathione transporter's tau gene family, with no transgene line and off-target through CRISPR/Cas9 mutagenesis and highlighted the loss of pigmentation in rice flowers, leaves, stems, shoots, and caryopsis. The anthocyanin quantification in the wild-type BLWT and mutant line BLG34-8 caryopsis showed that cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G) were almost undetectable in the mutant line. A tandem mass tag (TMT) labeling proteomic analysis was conducted to elucidate the proteomic changes in the BLWT and BLG34-8. The result revealed that 1175 proteins were altered, including 408 that were down-regulated and 767 that were upregulated. The accumulation of the OsGSTU34-related protein (Q8L576), along with several anthocyanin-related proteins, was down-regulated. The enrichment analysis showed that the down-regulated proteins were enriched in different pathways, among which the phenylpropanoid biosynthesis pathway, flavonoid biosynthesis metabolites, and anthocyanin biosynthesis pathway. Protein interaction network prediction revealed that glutathione-S-transferase (Q8L576) was connected to the proteins involved in the flavonoid and anthocyanin biosynthesis pathways, such as flavanone 3-dioxygenase 1 (Q7XM21), leucoanthocyanidin dioxygenase 1 (Q93VC3), 4-coumarate-CoA ligase 2 (Q42982), phenylalanine ammonia-lyase (P14717), chalcone synthase 1 (Q2R3A1), and 4-coumarate-CoA ligase 5 (Q6ZAC1). However, the expression of the most important anthocyanin biosynthesis gene was not altered, suggesting that only the transport mechanism was affected. Our findings highlight new insight into the anthocyanin pigmentation in black rice and provide new perspectives for future research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bovine herpesvirus 1(BoHV-1) is an important bovine pathogen that causes great economic loss to cattle farms worldwide. The virus-productive infection in bovine kidney (MDBK) cells results in ATP depletion. The mechanisms are not well understood. Mitochondrial fatty acid β-oxidation (FAO) is an important energy source in many tissues with high energy demand. Since carnitine palmitoyl-transferase 1 A (CPT1A) is the rate-limiting enzyme of FAO, we investigated the interactions between virus-productive infection and CPT1A signaling. Here, we found that virus-productive infection at the later stage significantly decreased CPT1A protein levels in all the detected cells, including MDBK, A549, and Neuro-2A cells, differentially altered the accumulation of CPT1A proteins in the nucleus and cytosol, and re-localized the protein in the nucleus. Etomoxir (ETO), an irreversible inhibitor of CPT1A, inhibited viral replication and partially interfered with the ability of BoHV-1 to alter CPT1A accumulation in the nucleus but not in the cytosol. Furthermore, ETO consistently reduced RNA levels of two viral regulatory proteins (bICP0 and bICP22) and protein expression of virion-associated proteins during productive infection, further supporting the important roles of CPT1A signaling in BoHV-1 productive infection. These data, for the first time, suggest that CPT1A is potentially involved in BoHV-1 productive infection., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Histone acetylation that controlled by two mutually antagonistic enzyme families, histone acetyl transferases (HATs) and histone deacetylases (HDACs), as one of major epigenetic mechanisms controls transcription and its abnormal regulation was implicated in various aspects of cancer. However, the comprehensive understanding of HDACs and HATs in cancer is still lacking. Systematically analysis through 33 cancer types based on next-generation sequence data reveals heterogeneous expression pattern of HDACs and HATs across different cancer types. In particular, HDAC10 and HDAC6 show significant downregulation in most cancers. Principal components analysis (PCA) of pan-cancer reveals significant difference of HDACs and HATs between normal tissues and normal tissue adjacent to the tumor. The abnormal expression of HDACs and HATs was partially due to CNV and DNA methylation in multiple types of cancer. Prognostic significance (AUC reached 0.736) of HDACs and HATs demonstrates a five-gene signature including KAT2A, HAT1, KAT5, CREBBP and SIRT1 in KIRC. Analysis of NCI-60 drug database reveals the cytotoxic effect of several drugs are associated with dysregulated expression of HDACs and HATs. Analysis of immune infiltration and immunotherapy reveals that KAT2B and HDAC9 are associated with immune infiltration and immunotherapy. Our analysis provided comprehensive understanding of the regulation and implication of HDACs and HATs in pan-cancer. These findings provide novel evidence for biological investigating potential individual HDACs and HATs in the development and therapy of cancer in the future.

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

Several related progeroid disorders are caused by defective post-translational processing of prelamin A, the precursor of the nuclear scaffold protein lamin A, encoded by LMNA . Prelamin A undergoes farnesylation and additional modifications at its C-terminus. Subsequently, the farnesylated C-terminal segment is cleaved off by the zinc metalloprotease ZMPSTE24. The premature aging disorder Hutchinson Gilford progeria syndrome (HGPS) and a related progeroid disease, mandibuloacral dysplasia (MAD-B), are caused by mutations in LMNA and ZMPSTE24 , respectively, that result in failure to process the lamin A precursor and accumulate permanently farnesylated forms of prelamin A. The farnesyl transferase inhibitor (FTI) lonafarnib is known to correct the aberrant nuclear morphology of HGPS patient cells and improves lifespan in children with HGPS. Importantly, and in contrast to a previous report, we show here that FTI treatment also improves the aberrant nuclear phenotypes in MAD-B patient cells with mutations in ZMPSTE24 (P248L or L425P). As expected, lonafarnib does not correct nuclear defects for cells with lamin A processing-proficient mutations. We also examine prelamin A processing in fibroblasts from two individuals with a prevalent laminopathy mutation LMNA -R644C. Despite the proximity of residue R644 to the prelamin A cleavage site, neither R644C patient cell line shows a prelamin A processing defect, and both have normal nuclear morphology. This work clarifies the prelamin A processing status and role of FTIs in a variety of laminopathy patient cells and supports the FDA-approved indication for the FTI Zokinvy for patients with processing-deficient progeroid laminopathies, but not for patients with processing-proficient laminopathies.

Eukaryotic dehydrodolichyl diphosphate synthases (DHDDSs), cis-prenyltransferases (cis-PTs) synthesizing precursors of dolichols to mediate glycoprotein biosynthesis require partners, for eample Nus1 in yeast and NgBR in animals, which are cis-PTs homologues without activity but to boost the DHDDSs activity. Unlike animals, plants have multiple cis-PT homologues to pair or stand alone to produce various chain-length products with less known physiological roles. We chose Cinnamomum kanehirae, a tree that contains two DHDDS-like and three NgBR-like proteins from genome analysis, and found that one DHDDS-like protein acted as a homodimeric cis-PT to make a medium-chain C55 product, while the other formed heterodimeric complexes with either one of two NgBR homologues to produce longer-chain products. Both complexes were functional to complement the growth defect of the yeast rer2 deficient strain at a higher temperature. From the roles for the polyprenol and dolichol biosynthesis and sequence motifs, their homologues in various species were compared to reveal their possible evolutionary paths., (© 2023 The Protein Society.)

Elevated intraocular pressure (IOP) in glaucoma causes retinal ganglion cell (RGC) loss and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. In this paper, we aimed to develop a novel gene therapy for glaucoma using an AAV2-based thioredoxin 2 (Trx2)-exoenzyme C3 transferase (C3) fusion protein expression vector (scAAV2-Trx2-C3). We evaluated the therapeutic effects of this vector in vitro and in vivo using dexamethasone (DEX)-induced glaucoma models. We found that scAAV2-Trx2-C3-treated HeLa cells had significantly reduced GTP-bound active RhoA and increased phosphor-cofilin Ser3 protein expression levels. scAAV2-Trx2-C3 was also shown to inhibit oxidative stress, fibronectin expression, and alpha-SMA expression in DEX-treated HeLa cells. NeuN immunostaining and TUNEL assay in mouse retinal tissues was performed to evaluate its neuroprotective effect upon RGCs, whereas changes in mouse IOP were monitored via rebound tonometer. The present study showed that scAAV2-Trx2-C3 can protect RGCs from degeneration and reduce IOP in a DEX-induced mouse model of glaucoma, while immunohistochemistry revealed that the expression of fibronectin and alpha-SMA was decreased after the transduction of scAAV2-Trx2-C3 in murine eye tissues. Our results suggest that AAV2-Trx2-C3 modulates the outflow resistance of the trabecular meshwork, protects retinal and other ocular tissues from oxidative damage, and may lead to the development of a gene therapeutic for glaucoma.

SETD7 (SET7/9, KMT7) is a lysine methyltransferase that targets master regulators of cell proliferation and differentiation. Here, the impact of inhibiting SETD7 catalytic activity on mammary epithelial cell differentiation was studied by focusing on genes associated with epithelial differentiation, lactogenesis, and lipid metabolism in HC11 and EpH4 cell lines. Setd7 mRNA and protein levels were induced upon lactogenic differentiation in both cell lines. Inhibition of SETD7 activity by the compound (R)-PFI-2 increased cell proliferation and downregulated E-cadherin, beta-catenin, lactoferrin, insulin-like growth factor binding protein 5, and beta-casein levels. In addition, inhibition of SETD7 activity affected the lipid profile and altered the mRNA expression of the phospholipid biosynthesis-related genes choline phosphotransferase 1, and ethanolamine-phosphate cytidylyltransferase. Altogether, the results suggest that inhibiting SETD7 catalytic activity impairs mammary epithelial and lactogenic differentiation., (© 2023 Federation of European Biochemical Societies.)

Spodoptera litura, a polyphagous lepidopteran pest, demonstrates a remarkable capacity to adapt to varying host plants by efficiently detoxifying phytochemicals. However, the underlying mechanism for this adaptation is not well understood. Herein, twenty eplison glutathione S-transferase genes (GSTes) were characterized and their roles in phytochemical tolerance were analyzed in S. litura. Most of the GSTe genes were mainly expressed in the larval midgut and fat body. Exposure to the phytochemicals, especially xanthotoxin, induced the expression of most GSTe genes. Molecular docking analysis revealed that xanthotoxin could form stable bonds with six xanthotoxin-responsive GSTes, with binding free energies ranging from -36.44 to -68.83 kcal mol -1 . Knockdown of these six GSTe genes increased the larval susceptibility to xanthotoxin. Furthermore, xanthotoxin exposure significantly upregulated the expression of two transcription factor genes CncC and MafK. Silencing of either CncC or MafK reduced the expression of GSTe16, which exhibited the largest change in response to xanthotoxin. Additionally, analysis of the promoter sequence of GSTe16 revealed the presence of seven CncC/Maf binding sites. Luciferase reporter assays showed that CncC and MafK enhanced the expression of GSTe16, leading to the increased xanthotoxin tolerance in S. litura. These findings provide insight into the functions and transcriptional regulatory mechanisms of GSTes, thereby enhancing our understanding of the role of GSTs in the adaptation of lepidopteran pests to phytochemicals., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance., Competing Interests: The authors declare no conflict of interest.

A decreased hemoglobin synthesis is contemplated as a pathological indication of β-thalassemia. Recent studies show that EPZ035544 from Epizyme could induce fetal hemoglobin (HbF) levels due to its proven capability to inhibit euchromatin histone lysine methyl transferase (EHMT2). Therefore, the development of EHMT2 inhibitors is considered promising in managing β-thalassemia. Our strategy to find novel compounds that are EHMT2 inhibitors relies on the virtual screening of ligands that have a structural similarity to N 2-[4-methoxy-3-(2,3,4,7-tetrahydro-1 H -azepin-5-yl) phenyl]- N 4,6-dimethyl-pyrimidine-2,4-diamine (F80) using the PubChem database. In silico docking studies using Autodock Vina were employed to screen a library of 985 compounds and evaluate their binding ability with EHMT2. The selection of hit compounds was based on the docking score and mode of interaction with the protein. The top two ranked compounds were selected for further investigations, including pharmacokinetic properties analysis and molecular dynamics simulations (MDS). Based on the obtained docking score and interaction analysis, N -(4-methoxy-3-methylphenyl)-4,6-diphenylpyrimidin-2-amine (TP1) and 2- N -[4-methoxy-3-(5-methoxy-3 H -indol-2-yl)phenyl]-4- N ,6-dimethylpyrimidine-2,4-diamine (TP2) were found to be promising candidates, and TP1 exhibited better stability in the MDS study compared to TP2. In summary, our approach helps identify potential EHMT2 inhibitors, and further validation using in vitro and in vivo experiments could certainly enable this molecule to be used as a therapeutic drug in managing β-thalassemia disease.

Diabetic nephropathy (DN) is a dreadful complication of diabetes that affects ∼50% of diabetics and is a leading cause of end-stage renal disease (ESRD). Studies have linked aberrant expression of lysine methyltransferases (KMTs) to the onset and progression of DN. SET7 is a KMT that methylates specific lysine residues of the histone and nonhistone proteins. It plays an important role in the transforming growth factor-β (TGF-β)-induced upregulation of extracellular matrix (ECM)-associated genes that are responsible for the inflammatory cascade observed in DN. Inhibiting SET7 has potential to attenuate renal disorders in animal studies. This review will focus on the role of SET7 in DN and its potential as a therapeutic target to combat DN., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cadmium (Cd) is one of the dangerous factors that have negative impacts on plants and human health. Recently, many researchers have been looking for biostimulants to use as bioprotectants that can help or ameliorate plants' tolerance against abiotic stress, including Cd. To test the dangerousness of Cd accumulated in the soil, 200 µM of the latter was applied to sorghum seeds at germination and maturation stages. At the same time, Atriplex halimus water extract (0.1%, 0.25%, 0.5%) was applied to test its efficacy on Cd alleviation in sorghum plants. The obtained results showed that the tested concentrations enhanced the tolerance of sorghum to Cd by enhancing the germination indexes parameters such as germination percentage (GP), seedling vigor index (SVI), and reducing the mean germination time (MGT) of sorghum seeds grown under cadmium stress. On the other hand, the morphological parameters (height and weight) as well as the physiological parameters (chlorophyll and carotenoid) were stimulated in treated maturated sorghum plants under Cd stress. In addition, 0.5% and 0.25% of Atriplex halimus extract (AHE) stimulated the antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase, glutathione-s-transferase, and glutathione reductase. In the same time, an increase in carbon-nitrogen enzymes was recorded in the case of AHE treatment; phosphoenol pyruvate carboxylase, glutamine synthase, glutamate dehydrogenase, and amino acid transferase were all upregulated. These results suggest that using AHE as a biostimulant could be a better strategy to enhance the tolerance of sorghum plants to Cd stress., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-β-D-Galf2Ac-(1→3)-β-D-Galp-(1→3)-α-D-Galp-(1→3)-β-D-Galf-(1→3)-β-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F., Competing Interests: Conflict of interest U. A. B. has Intellectual Property rights on some reagents used in the study. F. A. G., J. S. S., J. J. C., and M. H. N. are U. A. B. employees. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development., (© 2023 Wiley Periodicals LLC.)

Many bacterial surface proteins and carbohydrates are modified with phosphorylcholine (ChoP), which contributes to host mimicry and can also promote colonization and survival in the host. However, the ChoP biosynthetic pathways that are used in bacterial species that express ChoP have not been systematically studied. For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as Neisseria meningitidis and Neisseria gonorrhoeae. This raises a question as to the origin of the ChoP used for macromolecule biosynthesis in these species. In the current study, we used in silico analyses to identify the potential pathways involved in ChoP biosynthesis in genomes of the 26 bacterial species reported to express a ChoP-modified biomolecule. We used the four known ChoP biosynthetic pathways and a ChoP transferase as search terms to probe for their presence in these genomes. We found that the Lic-1 pathway is primarily associated with organisms producing ChoP-modified carbohydrates, such as lipooligosaccharide. Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins. Additionally, ChoP biosynthesis pathways, such as phospholipid N -methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP-modified proteins. Thus, a major finding of this study is the association of a particular ChoP biosynthetic pathway with a cognate, target ChoP-modified surface factor; i.e., protein versus carbohydrate. This survey failed to identify a known biosynthetic pathway for some species that express ChoP, indicating that a novel ChoP biosynthetic pathway(s) may remain to be identified. IMPORTANCE The modification of bacterial surface virulence factors with phosphorylcholine (ChoP) plays an important role in bacterial virulence and pathogenesis. However, the ChoP biosynthetic pathways in bacteria have not been fully understood. In this study, we used in silico analysis to identify potential ChoP biosynthetic pathways in bacteria that express ChoP-modified biomolecules and found the association between a specific ChoP biosynthesis pathway and the cognate target ChoP-modified surface factor., Competing Interests: The authors declare no conflict of interest.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65 nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Skeletal muscle plays a key role in systemic energy homeostasis besides its contractile function, but what links these functions is poorly defined. Protein Arginine Methyl Transferase 5 (PRMT5) is a well-known oncoprotein but also expressed in healthy tissues with unclear physiological functions. As adult muscles express high levels of Prmt5, we generated skeletal muscle-specific Prmt5 knockout (Prmt5 MKO ) mice. We observe reduced muscle mass, oxidative capacity, force production, and exercise performance in Prmt5 MKO mice. The motor deficiency is associated with scarce lipid droplets in myofibers due to defects in lipid biosynthesis and accelerated degradation. Specifically, PRMT5 deletion reduces dimethylation and stability of Sterol Regulatory Element-Binding Transcription Factor 1a (SREBP1a), a master regulator of de novo lipogenesis. Moreover, Prmt5 MKO impairs the repressive H4R3 symmetric dimethylation at the Pnpla2 promoter, elevating the level of its encoded protein ATGL, the rate-limiting enzyme catalyzing lipolysis. Accordingly, skeletal muscle-specific double knockout of Pnpla2 and Prmt5 normalizes muscle mass and function. Together, our findings delineate a physiological function of PRMT5 in linking lipid metabolism to contractile function of myofibers., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background/aims: Our previous work identified the dimethyladenosine transferase 1 homolog as a novel prognostic factor for detecting human gastric carcinoma with high sensitivity and specificity. The high expression of dimethyladenosine transferase 1 is closely associated with the occurrence and progression of gastric carcinoma. However, the underlying mechanism of dimethyladenosine transferase 1 for the occurrence and development of gastric carcinoma is not well elucidated yet., Materials and Methods: In our present study, the biological role of dimethyladenosine transferase 1 on cell proliferation, apoptosis, and cell cycle progression in human gastric carcinoma cells was investigated through in vitro and in vivo assays by the overexpression and knockdown of dimethyladenosine transferase 1 2-way authentication method., Results: We found that the overexpression of dimethyladenosine transferase 1 significantly promotes cell proliferation (P < .001) and inhibition of cell apoptosis (P < .01) in SGC-7901 cells. However, the in vivo experiment results of the knockdown dimethyladenosine transferase 1 using small interfering RNAs in the MKN-45 are just the opposite. Reverse-transcriptase polymerase chain reaction and western blotting analysis revealed that overexpressed dimethyladenosine transferase 1 in SGC-7901 cells significantly activated the AKT pathway compared to control cells. In contrast, we found that apoptosis genes such as Caspase-3 and Caspase-9 were downregulated in those cells. The xenograft nude mice model exhibited increased tumor growth (P < .01) and weight loss (P < .01), with the overexpression of dimethyladenosine transferase 1 homolog in the SGC-7901 cells. These results have been further confirmed through backward verification in dimethyladenosine transferase 1 knockdown cells., Conclusions: Taken together, our results indicated that the dimethyladenosine transferase 1 plays a crucial role in stimulating cancer cell proliferation and contributes to apoptosis resistance in human gastric carcinoma. Meanwhile, it provides a potential therapeutic target for gastric carcinoma treatment and is worthy of further studies.

Lettuce produces natural rubber (NR) with an average M w of > 1 million Da in laticifers, similar to NR from rubber trees. As lettuce is an annual, self-pollinating, and easily transformable plant, it is an excellent model for molecular genetic studies of NR biosynthesis. CRISPR/Cas9 mutagenesis was optimized using lettuce hairy roots, and NR-deficient lettuce was generated via bi-allelic mutations in cis-prenyltransferase (CPT). This is the first null mutant of NR deficiency in plants. In the CPT mutant, orthologous CPT counterparts from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were expressed under a laticifer-specific promoter to examine how the average M w of NR is affected. No developmental defects were observed in the NR-deficient mutants. The lettuce mutants expressing guayule and goldenrod CPT produced 1.8 and 14.5 times longer NR, respectively, than the plants of their origin. This suggests that, although goldenrod cannot synthesize a sufficiently lengthy NR, goldenrod CPT has the catalytic competence to produce high-quality NR in the cellular context of lettuce laticifers. Thus, CPT alone does not determine the length of NR. Other factors, such as substrate concentration, additional proteins, and/or the nature of protein complexes including CPT-binding proteins, influence CPT activity in determining NR length., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Mycobacterial galactan biosynthesis is critical for cell viability and growth, therefore an effort was made to study galactofuranosyl transferase 1, encoded by MRA&#95;3822 in Mycobacterium tuberculosis H37Ra (Mtb-Ra). Galactofuranosyl transferases are involved in the biosynthesis of mycobacterial cell wall galactan chain and have been shown to be essential for in-vitro growth of Mycobacterium tuberculosis. In Mtb-Ra and Mycobacterium tuberculosis H37Rv (Mtb-Rv), two galactofuranosyl transferases are present, GlfT1 acts as initiator of galactan biosynthesis and GlfT2 continues with the subsequent polymerization events. GlfT2 has been well studied however GlfT1 inhibition/down-regulation and its effect on mycobacterial survival fitness has not been evaluated. To study the Mtb-Ra survival after GlfT1 silencing, Mtb-Ra knockdown and complemented strains were developed. In this study we show that GlfT1 down-regulation leads to increased susceptibility to ethambutol. Expression of glfT1 was up-regulated in the presence of ethambutol, and also in the presence of oxidative and nitrosative stress and upon exposure to low pH. Also, reduced biofilm formation, increased accumulation of ethidium bromide, and reduced tolerance to peroxide, nitric oxide and acid stress, were observed. The present study also demonstrates that GlfT1 down-regulation leads to reduced survival of Mtb-Ra in macrophages and in mice., Competing Interests: Declaration of competing interest Authors declare that there is no competing interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2- C -methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea ., Competing Interests: The authors declare no conflict of interest.