An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

Acyl-CoA synthetase 1 plays an important role on pollen development and male fertility in tomato.

The development of pollen is critical to male reproduction in flowering plants. Acyl-CoA synthetase (ACOS) genes play conserved functions in regulating pollen development in various plants. Our previous work found that knockout of the SlACOS1 gene in tomato might decrease fruit setting. The current study further revealed that SlACOS1 was important to pollen development and male fertility. The SlACOS1 gene was preferentially expressed in the stamen of the flower with the highest expression at the tetrad stage of anther development. Mutation of the SlACOS1 gene by the CRISPR/Cas9-editing system reduced pollen number and viability as well as fruit setting. The tapetum layer exhibited premature degradation and the pollen showed abnormal development appearing irregular, shriveled, or anucleate in Slacos1 mutants at the tetrad stage. The fatty acid metabolism in anthers was significantly impacted by mutation of the SlACOS1 gene. Furthermore, targeted fatty acids profiling using GC-MS found that contents of most fatty acids except C18:1 and C18:2 were reduced. Yeast complementation assay demonstrated that the substrate preferences of SlACOS1 were C16:0 and C18:0 fatty acids. Male fertility of Slacos1 mutant could be slightly restored by applying exogenous palmitic acid, a type of C16:0 fatty acid. Taken together, SlACOS1 played important roles on pollen development and male fertility by regulating the fatty acid metabolism and the development of tapetum and tetrad. Our findings will facilitate unraveling the mechanism of pollen development and male fertility in tomato.

[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

BAG6-A from Fragaria viridis pollen modulates gametophyte development in diploid strawberry.

Male and female gametophyte development processes are essential steps in the life cycles of all land plants. Here, we characterized a gene, FviBAG6-A, screened from the Fragaria viridis (2 n = 2x=14) pollen cDNA library and physically interacted with S-RNase. Ubiquitinated of Sa-RNase might be determined by the interaction of FviBAG6-A in the ubiquitin-proteasome system during fertilization. We found that overexpression of FviBAG6-A in Arabidopsis caused shorter silique length, and decreased silique number. Moreover, overexpression of FviBAG6-A in Fragaria vesca (2 n = 2x=14) led to a greatly reduced seed number, with nearly 80% of the seeds aborted. Analyses of paraffin sections and reactive oxygen species (ROS) content revealed that the majority of severe pollen defects were likely due to the early degradation of the tapetum and middle layer as a result of ROS accumulation and abnormal development of the uninucleate megaspore mother. Moreover, the FviBAG6-A interact with the E3 ligase SIZ1 and contribute to the SUMOylation of FviBAG6-A , which may be induced by the high level of ROS content, further promoting gametophyte abortion in strawberry transgenic lines. This study characterized the FviBAG6-A and reveals its novel function in gametophyte development.

A short-chain acyl-CoA synthetase that supports branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids (BCFAs). The branched-chain acyl-CoA precursors, utilized to initiate fatty acid synthesis, are derived from branched-chain ketoacid dehydrogenase (Bkd), a multiprotein complex that converts α-keto acids to their corresponding acyl-CoAs; however, Bkd KO strains still contain BCFAs. Here, we show that commonly used rich medias contain substantial concentrations of short-chain acids, like 2-methylbutyric and isobutyric acids, that are incorporated into membrane BCFAs. Bkd-deficient strains cannot grow in defined medium unless it is supplemented with either 2-methylbutyric or isobutyric acid. We performed a screen of candidate KO strains and identified the methylbutyryl-CoA synthetase (mbcS gene; SAUSA300_2542) as required for the incorporation of 2-methylbutyric and isobutyric acids into phosphatidylglycerol. Our mass tracing experiments show that isobutyric acid is converted to isobutyryl-CoA that flows into the even-chain acyl-acyl carrier protein intermediates in the type II fatty acid biosynthesis elongation cycle. Furthermore, purified MbcS is an ATP-dependent acyl-CoA synthetase that selectively catalyzes the activation of 2-methylbutyrate and isobutyrate. We found that butyrate and isovalerate are poor MbcS substrates and activity was not detected with acetate or short-chain dicarboxylic acids. Thus, MbcS functions to convert extracellular 2-methylbutyric and isobutyric acids to their respective acyl-CoAs that are used by 3-ketoacyl-ACP synthase III (FabH) to initiate BCFA biosynthesis.

Genome-Wide Identification and Expression Analysis of the 4-Coumarate: CoA Ligase Gene Family in Solanum tuberosum.

4-coumarate: CoA ligase (4CL) is not only involved in the biosynthetic processes of flavonoids and lignin in plants but is also closely related to plant tolerance to abiotic stress. UV irradiation can activate the expression of 4CL genes in plants, and the expression of 4CL genes changed significantly in response to different phytohormone treatments. Although the 4CL gene has been cloned in potatoes, there have been fewer related studies of the 4CL gene family on the potato genome-wide scale. In this study, a total of 10 potato 4CL genes were identified in the potato whole genome. Through multiple sequence alignment, phylogenetic analysis as well as gene structure analysis indicated that the potato 4CL gene family could be divided into two subgroups. Combined with promoter cis-acting element analysis, transcriptome data, and RT-qPCR results indicated that potato 4CL gene family was involved in potato response to white light, UV irradiation, ABA treatment, MeJA treatment, and PEG simulated drought stress. Abiotic stresses such as UV, ABA, MeJA, and PEG could promote the up-regulated expression of St4CL6 and St4CL8 but inhibits the expression of St4CL5. The above results will increase our understanding of the evolution and expression regulation of the potato 4CL gene family and provide reference value for further research on the molecular biological mechanism of 4CL participating in response to diverse environmental signals in potatoes.

Protection Against Drug-Induced Liver Injuries Through Nutraceuticals via Amelioration of Nrf-2 Signaling.

Hepatotoxicity caused by the overdose of various medications is a leading cause of drug-induced liver injury. Overdose of drugs causes hepatocellular necrosis. Nutraceuticals are reported to prevent drug-induced liver failure. The present article aims to review the protection provided by various medicinal plants against hepatotoxic drugs. Ayurveda is considered a conventional restorative arrangement in India. It is consistently used for ages and is still used today to cure drug-induced hepatotoxicity by focusing on antioxidant stress response pathways such as the nuclear factor erythroid-2 (Nrf-2) antioxidant response element signaling pathway. Nrf-2 is a key transcription factor that entangles Kelch-like ECH-associating protein 1, a protein found in the cell cytoplasm. Some antioxidant enzymes, such as gamma glycine cysteine ligase (γ-GCL) and heme oxygenase-1 (HO-1), are expressed in Nrf-2 targeted genes. Their expression, in turn, decreases the stimulation of hepatic macrophages and induces the messenger RNA (mRNA) articulation of proinflammatory factors including tumor necrosis factor α. This review will cover various medicinal plants from a mechanistic view and how they stimulate and interact with Nrf-2, the master regulator of the antioxidant response to counterbalance oxidative stress. Interestingly, therapeutic plants have become popular in the medical sector due to safer yet effective supplementation for the prevention and treatment of new human diseases. The contemporary study is expected to collect information on a variety of therapeutic traditional herbs that have been studied in the context of drug-induced liver toxicity, as nutraceuticals are the most effective treatments for oxidative stress-induced hepatotoxicity. They are less genotoxic, have a lower cost, and are readily available. Together, nutraceuticals exert protective effects against drug-induced hepatotoxicity through the inhibition of oxidative stress, inflammation, and apoptosis. Its mechanism(s) are considered to be associated with the γ-GCL/HO-1 and Nrf-2 signaling pathways.KEY TEACHING POINTSThe liver is the most significant vital organ that carries out metabolic activities of the body such as the synthesis of glycogen, the formation of triglycerides and cholesterol, as well as the formation of bile.Acute liver failure is caused by the consumption of certain drugs; drug-induced liver injury is the major condition.The chemopreventive activity of nutraceuticals may be related to oxidative stress reduction and attenuation of biosynthetic processes involved in hepatic injury via amelioration of the nuclear factor erythroid-2 (Nrf-2) signaling pathway.Nrf-2 is a key transcription factor that is found in the cell cytoplasm resulting in the expression of various genes such as gamma glycine cysteine ligase and heme oxygenase-1.Nutraceutical-rich phytochemicals possess high antioxidant activity, which helps in the prevention of hepatic injury.

Inhibition on XBP1s-driven lipogenesis by Qushi Huayu Decoction contributes to amelioration of hepatic steatosis induced by fructose.

ETHNOPHARMACOLOGICAL RELEVANCE: Qushi Huayu Decoction (QHD) is a traditional Chinese medicine formula consisting of five herbs, which has been used for non-alcoholic fatty liver disease (NAFLD) treatment in clinic for decades in China and validated in several NAFLD animal models. The hepatic de novo lipogenesis (DNL) is enhanced greatly to contribute to steatosis in NAFLD. The spliced form of X-box binding protein 1 (XBP1s) initiates DNL independently of sterol regulatory element-binding protein (SREBP) and carbohydrate-responsive element-binding protein (ChREBP). AIM OF THE STUDY: To disclose the mechanism of inhibition on hepatic DNL by QHD and the responsible compounds. METHODS: The effects of QHD on hepatic DNL were evaluated in mice induced by high-fructose diet (HFru). The effects of the serum-absorbed compounds of QHD on XBP1s were evaluated in HepG2 cells induced by tunicamycin. Hepatic histology, triglyceride (TG) and nonesterified fatty acids were observed. Hepatic apolipoprotein B100 and very low-density lipoprotein were measured to reflect lipid out-transport. The mRNA expression of XBP1s and its target genes were detected by real-time polymerase chain reaction. The protein expression of TG synthetases and DNL enzymes, and inositol requirement enzyme 1 alpha (IRE1α), phosphorylated IRE1α and XBP1s were detected in liver tissue and HepG2 cells by western-blot. The binding activity of SREBP1, protein expression of ChREBP and XBP1s were detected in the nuclear extracts of liver tissue. RESULTS: Dynamical observing suggested feeding with HFru for 2 weeks was sufficient to induce hepatic lipogenesis and XBP1s. QHD ameliorated liver steatosis without enhancing out-transport of lipids, accompanied with more inhibitory effects on DNL enzymes than TG synthetases. QHD inhibits the nuclear XBP1s without affecting ChREBP and SREBP1. In QHD, chlorogenic acid, geniposide and polydatin inhibit lipogenesis initiated by XPB1s. CONCLUSION: QHD probably decreases hepatic DNL by inhibiting XBP1s independent of SREBP1 and ChREBP. Chlorogenic acid, geniposide and polydatin are the potential responsible compounds.

Gallic acid impairs fructose-driven de novo lipogenesis and ameliorates hepatic steatosis via AMPK-dependent suppression of SREBP-1/ACC/FASN cascade.

Accumulating evidence suggests that de novo lipogenesis is a typical characteristic facilitating nonalcoholic fatty liver disease (NAFLD) progression. Gallic acid (GA) is a naturally occurring phenolic acid with metabolic disease-related clinical significance and preclinical benefits. This study aimed to evaluate the anti-steatotic potentials of GA in a fructose-induced NAFLD mouse model featuring a hepatic lipogenic phenotype. The results revealed that GA alleviated hepatic steatosis, oxidative stress, and inflammatory response in fructose-fed mice. Mechanistically, GA treatment restored AMP-activated protein kinase α (AMPKα) phosphorylation, resulting in downregulations of pro-lipogenic factors, including sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthetase (FASN), and acetyl-CoA carboxylase (ACC), in hepatocytes of mice and in vitro. Furthermore, computational docking analysis indicated that GA could directly interact with AMPKα/ß subunits to stabilize its activation. These results suggest that GA ameliorates fructose-induced hepatosteatosis by restraining hepatic lipogenesis via AMPK-dependent suppression of the SREBP-1/ACC/FASN cascade. Altogether, this study demonstrates that GA supplement may be a promising therapeutic strategy in NAFLD, especially in the subset with enhanced hepatic lipogenesis.

[Elucidation and Application of Novel Action of Therapeutic Agents for Diabetic Neuropathy].

Epalrestat is the only aldose reductase inhibitor that is currently available for diabetic peripheral neuropathy. Oxidative stress impairs endothelial cells, thereby leading to numerous pathological conditions. Increasing antioxidative ability is important to prevent cellular toxicity induced by reactive oxygen species. Epalrestat increases antioxidant defense factors such as glutathione and γ-glutamylcysteine ligase in vascular endothelial cells through activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This increases suppression of oxidative stress-induced cellular toxicity. Cadmium is an industrial and environmental pollutant that targets the vascular endothelium. The vascular system is critically affected by cadmium toxicity. Therapeutic treatment against cadmium toxicity is chelation therapy that promotes metal excretion; however, cadmium chelators can cause renal toxicity. Therefore, safe and efficient therapeutic agents are required. Epalrestat suppresses cadmium-induced cytotoxicity in vascular endothelial cells through activation of Nrf2. In addition, epalrestat affects the intracellular levels of cadmium, cadmium transporter Zrt-Irt-like protein 8 (ZIP8), and metallothionein (MT). The upregulation of ZIP8 and MT may be involved in the suppression of cadmium-induced cytotoxicity by epalrestat. Drug repurposing is a new strategy for drug discovery in which the pharmacological action of existing medicines whose safety and pharmacokinetics have already been confirmed clinically and whose use has been approved is examined comprehensively at the molecular level. The results can be applied to the development of existing drugs for use as medicines for the treatment of other diseases. This review provides useful findings for future expansion of indications as research leading to drug repurposing of epalrestat.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

The selenophosphate synthetase family: A review.

Selenophosphate synthetases use selenium and ATP to synthesize selenophosphate. This is required for biological utilization of selenium, most notably for the synthesis of the non-canonical amino acid selenocysteine (Sec). Therefore, selenophosphate synthetases underlie all functions of selenoproteins, which include redox homeostasis, protein quality control, hormone regulation, metabolism, and many others. This protein family comprises two groups, SelD/SPS2 and SPS1. The SelD/SPS2 group represent true selenophosphate synthetases, enzymes central to selenium metabolism which are present in all Sec-utilizing organisms across the tree of life. Notably, many SelD/SPS2 proteins contain Sec as catalytic residue in their N-terminal flexible selenium-binding loop, while others replace it with cysteine (Cys). The SPS1 group comprises proteins originated through gene duplications of SelD/SPS2 in metazoa in which the Sec/Cys-dependent catalysis was disrupted. SPS1 proteins do not synthesize selenophosphate and are not required for Sec synthesis. They have essential regulatory functions related to redox homeostasis and pyridoxal phosphate, which affect signaling pathways for growth and differentiation. In this review, we summarize the knowledge about the selenophosphate synthetase family acquired through decades of research, encompassing their structure, mechanism, function, and evolution.

Antimicrobial metabolite of Cordyceps tenuipes targeting MurE ligase and histidine kinase via in silico study.

Cordyceps spp. are widely healthy foods around the world with several traditional uses and bio-functionalities. The chemical characterization of ethyl acetate-soluble extract of the entomopathogenic fungus Cordyceps tenuipes NBRC 111,630 afforded two new metabolites with 1,6-dioxaspiro[4.4]nonane motif, tenuipesone A (1) and tenuipesone B (2), along with four well-known metabolites (3-6). The elucidation of the chemical structures was carried out via extensive spectroscopic experiments including FTIR, HRMS, 1D-NMR, and 2D-NMR. The probable biosynthetic pathway of 1 and 2 was hypothesized. From the six isolates, beauvericin (6) exhibited antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with respective MIC of 6.25 and 12.5 µM. Docking results exhibited that beauvericin (6) has significant binding affinities against MurE and HK proteins with ΔG = - 8.021 and - 8.585 kcal/mol, respectively. KEY POINTS: • Six compounds, including two new, were isolated from the entomopathogenic fungus Cordyceps tenuipes. • Plausible biosynthetic pathway of compounds 1, 2, 4, and 5 was hypothesized. • Beauvericin (6) exhibited significant antimicrobial activity against Bacillus subtilis and Staphylococcus aureus alongside binding affinities against MurE and HK proteins in MOE study.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model: A study based on proteomics.

BACKGROUND: Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease. Acupuncture and moxibustion is proved effective in treating UC, but the mechanism has not been clarified. Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC, which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC. AIM: To investigate the mechanism of electroacupuncture (EA) and herb-partitioned moxibustion (HM) on UC rats by using proteomics technology. METHODS: Male Sprague-Dawley rats were randomly divided into the normal (N) group, the dextran sulfate sodium (DSS)-induced UC model (M) group, the HM group, and the EA group. UC rat model was prepared with 3% DSS, and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Isotope-labeled relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues, followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins (DEPs) between groups. Then western blot was used for verification of selected DEPs. RESULTS: The macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group (P < 0.01). Compared with the N group, a total of 202 DEPs were identified in the M group, including 111 up-regulated proteins and 91 down-regulated proteins, of which 25 and 15 proteins were reversed after HM and EA interventions, respectively. The DEPs were involved in various biological processes such as biological regulation, immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity, intestinal immune network for immunoglobulin A (IgA) production, and FcγR-mediated phagocytosis. The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups, EA and M groups both included immune-associated and oxidative phosphorylation. Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions, and the expression of oxidative phosphorylation pathway-related proteins, including ATP synthase subunit g (ATP5L), ATP synthase beta subunit precursor (Atp5f), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) were down-regulated after HM and EA interventions. Subsequent verification of selected DEPs (Synaptic vesicle glycoprotein 2A; nuclear cap binding protein subunit 1; carbamoyl phosphate synthetase 1; Cox4i1; ATP synthase subunit b, Atp5f1; doublecortin like kinase 3) by western blot confirmed the reliability of the iTRAQ data, HM and EA interventions can significantly down-regulate the expression of oxidative phosphorylation-associated proteins (Cox4i1, Atp5f1) (P < 0.01). CONCLUSION: EA and HM could regulate the expression of ATP5L, Atp5f1, Cox4i1 that associated with oxidative phosphorylation, then might regulate immune-related pathways of intestinal immune network for IgA production, FcγR-mediated phagocytosis, thereby alleviating colonic inflammation of DSS-induced UC rats.

Mori fructus aqueous extracts attenuate carbon tetrachloride-induced renal injury via the Nrf2 pathway and intestinal flora.

Mori fructus aqueous extracts (MFAEs) have been used as a traditional Chinese medicine for thousands of years with the function of strengthening the liver and tonifying the kidney. However, its inner mechanism to alleviative renal injury is unclear. To investigate the attenuation of MFAEs on nephrotoxicity and uncover its potential molecular mechanism, we established a nephrotoxicity model induced by carbon tetrachloride (CCl4). The mice were randomly divided into control group, CCl4 model group (10% CCl4), CCl4 + low and high MFAEs groups (10% CCl4 + 100 mg/kg and 200 mg/kg MFAEs). We found that MFAEs decreased the kidney index of mice, restored the pathological changes of renal structure induced by CCl4, reduced cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (Kim-1) blood urea nitrogen and creatinine contents in serum, promoted the nuclear transportation of Nrf2 (nuclear factor erythroid derived 2 like 2), elevated the expression of HO-1 (heme oxygenase 1), GPX4 (glutathione peroxidase 4), SLC7A11 (solute carrier family 7 member 11), ZO-1 (zonula occludens-1) and Occludin, suppressed the expression of Keap1 (kelch-like ECH-associated protein 1), HMGB1 (High Mobility Group Protein 1), ACSL4 (acyl-CoA synthetase long chain family member 4) and TXNIP (thioredoxin interacting protein), upregulated the flora of Akkermansia, Anaerotruncus, Clostridium_sensu_stricto, Ihubacter, Alcaligenes, Dysosmobacter, and downregulated the flora of Clostridium_XlVa, Helicobacter, Paramuribaculum. Overlapped with Disbiome database, Clostridium_XlVa, Akkermansia and Anaerotruncus may be the potential genera treated with renal injury. It indicated that MFAEs could ameliorate kidney injury caused by CCl4 via Nrf2 signaling.

Shenmai Injection Attenuates Myocardial Ischemia/Reperfusion Injury by Targeting Nrf2/GPX4 Signalling-Mediated Ferroptosis.

OBJECTIVE: To examine the effect of Shenmai Injection (SMJ) on ferroptosis during myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism. METHODS: A total of 120 SPF-grade adult male SD rats, weighing 220-250 g were randomly divided into different groups according to a random number table. Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of 120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration of Fe2+ in heart tissues and the levels of creatine kinase-MB (CK-MB), troponin I (cTnl), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured using assay kits, and the expressions of Nrf2, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were examined by Western blot. RESULTS: Compared with the sham group, I/R significantly injured heart tissues, as evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum CK-MB, cTnI and MDA levels, and myocardial Fe2+ concentrations; and the decreases in SOD activity (P<0.05). These results were accompanied by ultrastructural alterations to the mitochondria, increased expression of ACSL4 and inhibited the activation of Nrf2/GPX4 signalling (P<0.05). Compared with I/R group, pretreatment with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury, Fe2+ concentrations and ACSL4 expression and attenuated mitochondrial impairment, while 14 mg/kg erastin exacerbated myocardial I/R injury (P<0.05). In addition, cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385, as evidenced by the increased myocardial infarct size, CK-MB, cTnI, MDA and Fe2+ concentrations, and the decreased SOD activity (P<0.05). CONCLUSIONS: Ferroptosis is involved in myocardial I/R injury. Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a strategy for the prevention and treatment of ischemic heart diseases.

Identification, Characterization and Expression Profiling of the RS Gene Family during the Withering Process of White Tea in the Tea Plant (Camellia sinensis) Reveal the Transcriptional Regulation of CsRS8.

Raffinose synthetase (RS) is a key enzyme in the process of raffinose (Raf) synthesis and is involved in plant development and stress responses through regulating Raf content. As a sweetener, Raf makes an important contribution to the sweet taste of white tea. However, studies on the identification, analysis and transcriptional regulation of CsRSs (Camellia sinensis RS genes) are still lacking. In this study, nine CsRSs were identified from the tea plant (Camellia sinensis) genome database. The CsRSs were classified into five groups in the phylogenetic tree. Expression level analysis showed that the CsRSs varied in different parts of the tea plant. Transcriptome data showed that CsRSs could respond to persistent drought and cold acclimation. Except for CsRS5 and CsRS9, the expression pattern of all CsRSs increased at 12 h and decreased at 30 h during the withering process of white tea, consistent with the change trend of the Raf content. Furthermore, combining yeast one-hybrid assays with expression analysis, we found that CsDBB could potentially regulate the expression of CsRS8. Our results provide a new perspective for further research into the characterization of CsRS genes and the formation of the white tea flavour.

Mechanistic analysis of carbon-carbon bond formation by deoxypodophyllotoxin synthase.

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.