WRKYs is a unique family of transcription factors (TFs) in plants, and belongs to the typical multifunctional regulator. It is involved in the regulation of multiple signaling pathways. This type of transcription factor is characterized to contain about 60 highly conservative amino acids as the WRKY domain, and usually also has the Cys2His2 or Cys2His-Cys zinc finger structure. WRKYs can directly bind to the W-box sequence ((T)(T) TGAC (C/T)) in the promoter region of the downstream target gene, and activate or inhibit the transcription of the target genes by interacting with the target protein. They may up-regulate the expression of stress-related genes through integrating signal pathways mediated by abscisic acid (ABA) and reactive oxygen species (ROS), thus playing a vital role in regulating plant response to abiotic stresses. This review summarizes the advances in research on the structure and classification, regulatory approach of WRKYs, and the molecular mechanisms of WRKYs involved in response to drought and salt stresses, and prospects future research directions, with the aim to provide a theoretical support for the genetic improvement of crop in response to abiotic stresses.
Transcription Factors/genetics* ; Abscisic Acid ; Amino Acids ; Droughts ; Stress, Physiological/genetics*

The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.
Escherichia coli/genetics* ; Glutamine ; Zeolites/chemistry* ; Amino Acids

OBJECTIVE: To retrospectively analyze the clinical phenotypes and genetic variants in two Chinese pedigrees affected with Hereditary hypofibrinemia (IFD) and explore their molecular pathogenesis. METHODS: Two probands and their pedigree members were admitted to the First Affiliated Hospital of Wenzhou Medical University on March 30, 2021 and May 27, 2021, respectively. Clinical phenotypes of the probands were collected, and blood clotting indexes of the probands and their pedigree members were determined. Variants of the FGA, FGB and FGG genes were analyzed by Sanger sequencing, and candidate variants were verified by sequence comparison. Bioinformatic software was used to analyze the conservation of the amino acids and pathogenicity of the proteins. Alteration in protein structure and intermolecular force before and after the variant was analyzed by simulating the protein model. RESULTS: Proband 1, a 18-year-old male, had significantly low plasma fibrinogen activity (Fg:C) and plasma fibrinogen antigen (Fg:Ag), respectively at 0.80 g/L and 1.00 g/L. Proband 2, a 43-year-old male, had slightly low Fg:C and Fg:Ag at 1.35 g/L and 1.30 g/L, respectively. The Fg:C and Fg:Ag of proband 1's father, proband 2's father and son were also below the normal level. Genetic testing showed that proband 1 had harbored a heterozygous missense variant of c.688T>G (p.Phe230Val) in exon 7 of the FGG gene, which was inherited from his father. Proband 2, his father and son all had harbored a heterozygous variant of c.2516A>C (p.Asn839Thr) in exon 6 of the FGA gene. Homology analysis showed that the Phe230 and Asn839 residues were highly conserved among homologous species. Bioinformatic analysis predicted that both p.Phe230Val and p.Asn839Thr were pathogenic variants. CONCLUSION Analysis of protein simulation model showed that the p.Asn839Thr variant has changed the hydrogen bo`nd between the amino acids, thus affecting the stability of the protein structure. The heterozygous missense variants of p.Phe230Val and p.Asn839Thr probably underlay the IFD in the two pedigrees.
Humans ; Male ; Amino Acids ; East Asian People ; Exons ; Pedigree ; Retrospective Studies ; Afibrinogenemia/genetics* ; Mutation, Missense ; Fibrinogen/genetics*

Objective: To investigate the relationship between amino acid variations of respiratory syncytial virus (RSV) nonstructural protein (NS) 1 and the clinical characteristics. Method: A retrospective case review was conducted. From December 2018 to January 2020, a total of 81 cases of hospitalized children who were tested only positive for RSV by RT-PCR or PCR at the Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University were included in the study. The NS1 genes of RSV subtype A and subtype B were amplified by PCR and sequenced. The amino acid sequences were analyzed. The Chi-square test and Mann-Whitney rank sum test were used to compare the clinical characteristics and type Ⅰ interferon levels of children with or without NS1 variation in the variation and non-variation groups. Results: Among 81 cases, there were 58 males and 23 females. There were 11 cases in the variation group, the age of onset was 2.0 (1.0, 11.0) months, included 4 cases of subtype A (variant sites were: 2 cases for Lys33Gln, one case for Gly2Asp, Pro67Ser, Leu137Phe, respectively) and 7 cases of subtype B (variant sites were: two cases for Val121Ile, one case for Tyr30Cys, Val65Met, Asn85Ser, Ser118Asn, Asp124Asn, respectively). These variant sites all appeared at a very low frequency 0.08 (0.04, 0.29) % in the NCBI PROTEIN database. There were 70 cases in non-variation group, the onset age was 3.5 (1.0, 7.0) months. The proportion of dyspnea in the variation group was higher than that in the non-variation group (10/11 vs. 47% (33/70), χ²=7.31, P<0.01). Conclusions: There are some variant sites in nonstructural protein NS1 of RSV. Children may be prone to have dyspnea with NS1 variations.
Child ; Male ; Female ; Humans ; Infant ; Respiratory Syncytial Virus Infections ; Amino Acids ; Retrospective Studies ; Respiratory Syncytial Virus, Human/genetics* ; Polymerase Chain Reaction

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. METHODS: The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138⁺ and CD138^- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138⁺ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. RESULTS: Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138⁺ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138⁺ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). CONCLUSIONS MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.
Humans ; Valine-tRNA Ligase ; Multiple Myeloma/genetics* ; Metabolomics ; Amino Acids ; RNA, Transfer

OBJECTIVE: To explore the genetic basis for a patient with Alport syndrome (AS) and confirm the existence of a splicing variant. METHODS: An AS patient diagnosed at the Affiliated Hospital of Inner Mongolia Medical University on January 8, 2021 for significant proteinuria and occult hematuria was selected as the study subject. Clinical data was collected. Peripheral blood samples were collected for the extraction of genomic DNA. Whole exome sequencing and Sanger sequencing were carried out to identify potential genetic variants. An in vitro experiment was also conducted to verify the abnormal mRNA splicing. Bioinformatic software was used to analyze the conservation of amino acids of the variant sites and simulate the 3D structure of the variant collagen IV protein. Immunofluorescence and immunohistochemistry were carried out on renal tissues from the patient to confirm the presence of AS kidney injury. RESULTS: The patient, a 21-year-old male, had a 24-hour urine protein of 3.53 g/24 h, which fulfilled the diagnostic criteria for proteinuria. His blood uric acid has also increased to 491 μmol/L. DNA sequencing revealed that he has harbored a c.835-9T>A splice variant of the COL4A5 gene, which was not found in either of his parents. In vitro experiment confirmed that the variant has removed 57 bp from the exon 15 of the mRNA of the COL4A5 gene. The deletion may cause loss of amino acid residues from positions 279 to 297, which in turn may affect the stability of the secondary structure of the α5 chain encoded by the COL4A5 gene. The amino acids are conserved across various species. The result of homology modeling indicated that the trimerization of Col-IV with the mutated α5 chain could be achieved, however, the 3D structure was severely distorted. The AS kidney damage was confirmed through immunofluorescence assays. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.835-9T>A variant was classified as likely pathogenic (PVS1_Moderate+PS3_Moderate+PM2_Supporting+PS2+PP3+PP4). CONCLUSION The c.835-9T>A variant of the COL4A5 gene probably underlay the AS in this patient. In vitro experiment has confirmed the abnormal splicing caused by the variant. Histopathological examination of the kidney tissue has provided in vivo evidence for its pathogenicity. Above finding has expanded the mutational spectrum of the COL4A5 gene.
Humans ; Male ; Young Adult ; Amino Acids ; China ; Collagen Type IV/genetics* ; Exons ; Nephritis, Hereditary/genetics* ; RNA Splicing

OBJECTIVE: To analyze the difference in the gene expression, amino acid and carnitine levels in the cervical secretions between the endometria of pre-receptive and receptive stages, with an aim to provide clues for identifying new molecular markers for endometrial receptivity. METHODS: Fifty nine infertile women treated at the Department of Reproductive Medicine of Linyi People's Hospital from January 6, 2020 to January 31, 2022 were selected as as the study subjects, which were matched with 3 pairs (6 cases) of infertile women preparing for embryo transfer based on factors such as age, body mass index, and length of infertility. Endometrial tissue samples were collected for gene transcription and expression analysis. Twenty five women who had become pregnant through assisted reproductive technology were selected as the control group, and 28 non-pregnant women receiving ovulation monitoring at the Outpatient Department were enrolled as the case group. Status of endometrial receptivity was determined by ultrasonography. In the former group, endometrial tissues were sampled for sequencing, and GO and KEGG database enrichment analysis of differentially expressed genes was carried out. In the latter group, cervical secretions were collected, and amino acid and carnitine levels were measured by mass spectrometry. Statistical analysis was carried out using rank sum test, t test and chi-square test with SPSS v25.0 software. RESULTS: No difference was found in the clinical data of the patients with regard to age, body mass index, infertility years, AMH, FSH, LH, E2, and type of infertility. Compared with the receptive endometrial tissues, there were 100 significantly up-regulated genes and 191 significantly down-regulated genes in the pre-receptive endometrial tissue, with the most significantly altered ones being HLA-DRB5 and MMP10. The biological processes, molecular functions and pathways enriched by more differentially expressed genes in GO and KEGG were mainly immune regulation, cell adhesion and tryptophan metabolism. Analysis of secretion metabolism also revealed a significant difference in the levels of amino acids and carnitine metabolites between the two groups (P < 0.05), in particular those of Alanine, Valine, 3-hydroxybutyrylcarnitine (C4OH) + malonylcarnitine (C3DC)/captoylcarnitine (C10). CONCLUSION A significant difference has been discovered in the levels of gene transcription and protein expression in the endometrial tissues from the pre-receptive and receptive stages. The levels of amino acids and carnitine, such as Alanine, Valine, 3-hydroxybutyryl carnitine (C4OH)+malonyl carnitine (C3DC)/caproyl carnitine (C10), may be associated with the receptive status of the endometrium, though this need to be verified with larger samples.
Pregnancy ; Humans ; Female ; Infertility, Female/genetics* ; Endometrium/metabolism* ; Amino Acids/metabolism* ; Gene Expression ; Carnitine ; Alanine/metabolism* ; Valine/metabolism*

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.
Oxidoreductases ; Biosynthetic Pathways ; Molecular Docking Simulation ; Reproducibility of Results ; Salvia miltiorrhiza/chemistry* ; Amino Acids/metabolism* ; Plant Roots/genetics*

This study aimed to demonstrate the effect of Banxia Baizhu Tianma Decoction(BBTD) on realizing withdrawal of anti-epileptic drugs and explore the relationship between BBTD and the amino acid metabolism by transcriptomic analysis in the rat model of epilepsy induced by lithium chloride-pilocarpine. The rats with epilepsy were divided into a control group(Ctrl), an epilepsy group(Ep), a BBTD & antiepileptic drug integrative group(BADIG), and an antiepileptic drug withdrawal group(ADWG). The Ctrl and Ep were given ultrapure water by gavage for 12 weeks. The BADIG was given BBTD extract and carbamazepine solution by gavage for 12 weeks. The ADWG was given carbamazepine solution and BBTD extract by gavage for the former 6 weeks, and then only given BBTD extract for the latter 6 weeks. The therapeutic effect was evaluated by behavioral observation, electroencephalogram(EEG), and hippocampal neuronal morphological changes. High-throughput sequencing was used to obtain amino acid metabolism-related differen-tial genes in the hippocampus, and the mRNA expression in the hippocampus of each group was verified by real-time quantitative polymerase chain reaction(RT-qPCR). The hub genes were screened out through protein-protein interaction(PPI) network, and Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed. Two ceRNA networks, namely circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA, were constructed for ADWG vs BADIG. The experimental results showed that compared with those in Ep, rats in ADWG were significantly improved in the behavioral observation, EEG, and hippocampal neuronal impairment. Thirty-four amino acid metabolism-related differential genes were obtained by transcriptomic analysis, and the sequencing results were confirmed by RT-qPCR. Eight hub genes were obtained through PPI network, involving several biological processes, molecular functions, and signal pathways related to amino acid metabolism. Finally, the circRNA-miRNA-mRNA ternary transcription network of 17 circRNA, 5 miRNA, and 2 mRNA, and a lncRNA-miRNA-mRNA ternary network of 10 lncRNA, 5 miRNA, and 2 mRNA were constructed in ADWG vs BADIG. In conclusion, BBTD can effectively achieve the withdrawal of antiepileptic drugs, which may be related to the transcriptomic regulation of amino acid metabolism.
Rats ; Animals ; RNA, Circular/genetics* ; Transcriptome ; RNA, Long Noncoding/genetics* ; Anticonvulsants ; MicroRNAs/genetics* ; RNA, Messenger ; Carbamazepine ; Amino Acids ; Gene Regulatory Networks

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.
Gastrodia/genetics* ; Phylogeny ; Amino Acids ; Cloning, Molecular