Prokaryotic Expression Research Articles

Identification of IspD as a novel target for tuberculosis treatment using compound M6

IntroductionTuberculosis (TB) is a serious infectious disease that endangers human health, and TB becomes more difficult in eradiation due to its multidrug resistance (MDR). The objective of this research was to identify novel targets for treating TB.MethodsA 2-fold serial dilution method was used to determine minimal inhibitory concentrations (MIC) of compound M6 against Mycobacterium smegmatis (M. smegmatis). Compound M6 was subjected to reverse molecular docking with seven Mycobacterium tuberculosis proteins, and the best binding protein with the highest LibDock score was evaluated. The target protein with the highest score was purified through prokaryotic expression. Isolated target proteins were investigated for the enzyme activities and for the kinetic effect of compound M6 by absorbance detection. Subsequently, the CRISPR/Cas9 technology was employed to inhibit target gene expression for detecting MIC changes. Finally, potential targets were evaluated for the effect of the compound M6 in bacteria.ResultsThe MIC values of compound M6 against M. smegmatis were 32 μg/mL. The results from reverse molecular docking show that IspD has the highest LibDock score of 142.50, followed by Rv0674, IspF, and Dxr, with docking scores of 110.762, 71.6955, and 57.7446, respectively. IspD is a key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway of MTB. The aKi and Ki values of M6 for the substrate MEP are 609.58 μM and 81.33 μM. For CTP, the aKi and Ki values are 657.89 μM and 40.07 μM. With tetracycline inducing CRISPR/Cas9 to suppress the expression of IspD, the MIC value of M6 against IspD went down significantly from 32 μg/mL to 4 μg/mL.ConclusionIspD is a novel target of the compound M6 for treating TB.

Strain-Specific Infection of Phage AP1 to Rice Bacterial Brown Stripe Pathogen Acidovorax oryzae

Bacteriophage (phage) AP1 has been reported to effectively lyse Acidovorax oryzae, the causative agent of bacterial brown stripe in rice. However, phage AP1 exhibits strain-specific lysis patterns. In order to enhance the potential of phages for biological control of rice bacterial brown stripe, this study investigated the possible mechanism of strain-specific infection by characterizing phage AP1 and its susceptible (RS-2) and resistant (RS-1) strains. Based on the current classification standards and available database information, phage AP1 was classified into the class Caudoviricetes, and it is a kind of podophage. Comparative analysis of the susceptible and resistant strains showed no significant differences in growth kinetics, motility, biofilm formation, or effector Hcp production. Interestingly, the resistant strain demonstrated enhanced virulence compared to the susceptible strain. Prokaryotic expression studies indicated that six putative structural proteins of phage AP1 exhibited varying degrees of binding affinity (1.90–9.15%) to lipopolysaccharide (LPS). However, pull-down assays and bacterial two-hybrid analyses revealed that only gp66 can interact with four host proteins, which were identified as glycosyltransferase, RcnB, ClpB, and ImpB through immunoprecipitation and mass spectrometry analyses. The role of LPS in the specific infection mechanism of phage AP1 was further elucidated through the construction of knockout mutant strains and complementary strains targeting a unique gene cluster (wbzB, wbzC, wbzE, and wbzF) involved in LPS precursor biosynthesis. These findings provide novel insights into the mechanisms of phage-host specificity, which are crucial for the effective application of phage AP1 in controlling rice bacterial brown stripe.

Purification and Identification of the Nematicidal Activity of S1 Family Trypsin-Like Serine Protease (PRA1) from Trichoderma longibrachiatum T6 Through Prokaryotic Expression and Biological Function Assays

Background/Objectives: Heterodera avenae is a highly significant plant-parasitic nematode, causing severe economic losses to global crop production each year. Trichoderma species have been found to parasitize nematodes and control them by producing enzymes that degrade eggshells. The T. longibrachiatum T6 (T6) strain has been demonstrated the parasitic and lethal effects on H. avenae cysts and eggs, associated with the increased serine protease activity and trypsin-like serine protease gene (PRA1) expression. Methods: Our present study aimed to purify the recombinant PRA1 protease through a prokaryotic expression system and identify its nematicidal activity. Results: The recombinant PRA1 protease was identified as S1 family trypsin-like serine protease, with a molecular weight of 43.16 kDa. The purified soluble protease exhibited the optimal activity at 35 °C and pH 8.0, and also demonstrating higher hydrolytic ability toward casein and skimmed milk. Meanwhile, the Ca2+ and Mg2+ enhanced its activity, while the inhibitor PMSF significantly reduced it. The contents of H. avenae eggs leaked out after treatment with the recombinant PRA1 protease, with egg hatching inhibition and relative hatching inhibition rates at 70.60% and 66.58%, respectively. In contrast, there was no sign of content dissolution, and embryos developed normally in the control group. Conclusions: Our present study revealed that the PRA1 protease of T6 strain has a lethal effect on H. avenae eggs, which providing a theoretical basis for developing biocontrol agents to control nematodes.

Prokaryotic Expression of Coat Protein Gene of Grapevine Berry Inner Necrosis Virus and Preparation of Its Polyclonal Antibody

Grapevine berry inner necrosis virus (GINV) and grapevine Pinot gris virus (GPGV) are prevalent viral diseases affecting viticulture, posing significant threats in grape-producing regions of China. Previous studies have emphasized the harmful effects of grape viruses on the grape industry all over the world. However, few reports have focused specifically on GINV. In wild grapevines, GINV infection frequently leads to grapevine fanleaf degeneration disease (GFDD). GINV often co-occurs with other grape viruses, exacerbating its harmful effects on the grapevine industry in China. In this study, we collected grapevine samples from Taizhou city, Jiangsu Province, where GINV infection was confirmed. Based on the GINV coat protein (CP) gene, we developed a high-throughput and high-sensitivity direct antigen-coated ELISA and Dot blot assay for field diagnosis of GINV CP in grape samples. The CP gene was cloned from GINV-infected grape samples, and the GINV CP was expressed using the pET30(a) vector. Specific polyclonal antiserum CPGINV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPGINV antiserum, we developed a rapid, precise, and scalable diagnostic method for GINV in the grapevine industry. The established ELISA and Dot blot assays successfully detected GINV-infected grapevine samples. The occurrence of GINV is relatively common in China, which poses a risk of transmission and threatens the healthy development of the grape industry. Therefore, this study prepared CPGINV antiserum and established an efficient, rapid, sensitive, accurate, and high-throughput diagnostic method, providing a foundational approach for the prevention and control of vitis viral diseases.

DAAO Mutant Sites among Different Mice Strains and Their Effects on Enzyme Activity.

Previous studies reported that D-amino acid oxidase (DAAO) activity was closely associated with neuropathic pain, cognitive characteristics of schizophrenia and so on. To determine DAAO mutant sites in different strains of mice and their effects on enzyme activity, we successfully constructed a prokaryotic expression system for heterologous expression of DAAO in vitro. There were total five nucleotide mutations distributed in exons 2, 8, 9, 10 of C57 mice. Three mutations located on exons 8 and 9 were synonymous mutations and had no variation on the encoded amino acid. The remaining two mutations in exons 2 (V64A) and 10 (R295H) were non-synonymous mutations, which might affect enzymatic activity and protein structure of mDAAO. Based on the determination of the kinetic constants and IC50 of mDAAO mutants in vitro, the differences in amino acid levels at these two sites (V64A, R295H) increased the affinity of C57 DAAO with substrate and enhanced its catalytic efficiency. Besides, the IC50 value of C57 DAAO was less than that of Balb/c and other DAAO mutants (SUN: reducted by about 11.9%; CBIO: reducted by about 26.5%), which meant that the affinity of C57 DAAO with CBIO was higher.

Combining GWAS and RNA-Seq approaches identifies the FtADH1 gene for drought resistance in Tartary buckwheat

Drought is one of the major environmental constraints that significantly affects seedling emergence, yield, and quality of Tartary buckwheat, thereby hindering the development of its industry. However, the molecular mechanisms underlying drought tolerance genes in Tartary buckwheat remain largely unexplored. Alcohol dehydrogenase (ADH), one of the essential plant proteins, plays a crucial role in growth, development, and stress responses, but its specific role in drought resistance is still unclear. In this study, we identified an ADH gene FtADH1, using a membership function value of drought tolerance (MFVD) combined with a genome-wide association study (GWAS) and transcriptomic profiles that confers drought tolerance in Tartary buckwheat. Our findings demonstrated that the overexpression of FtADH1 in Arabidopsis and Tartary buckwheat hairy roots enhances drought tolerance by promoting root elongation and mitigating elevated levels of reactive oxygen species (ROS). Our findings demonstrated that FtADH1 can enhanced tolerance to drought stresses in both Tartary buckwheat and Arabidopsis. This study identifies the FtADH1 as a new player in affecting ROS level and the stress response of Tartary buckwheat by regulating protective enzyme activities at a high level to scavenge ROS and modulating root growth under drought stress. Further, we identified proteins interacting with FtADH1 through a prokaryotic expression pull-down assay combined with mass spectrometry, revealing that FtADH1 specifically interacts with the S-adenosyl-L-methionine (SAM) synthetase protein, FtSAMS1. Overexpression of FtSAMS1 was found to enhance ADH enzymatic activity, leading to increased SAM content in overexpressing Tartary buckwheat hairy roots under water-deficit conditions. Additionally, FtSAMS1 overexpression induced a drought-resistant phenotype in Arabidopsis and Tartary buckwheat hairy roots under drought stress, revealing the biological function of FtADH1. Evolutionary analysis indicates that ADH1 in Fagopyrum species has undergone significant evolutionary events, including duplication and purifying selection, which may contribute to functional diversification and adaptive advantages such as drought resistance in cultivated buckwheat. In summary, this study proposes that FtADH1 is a key contributor to drought tolerance, and its interaction with FtSAMS1 holds potential for the development of drought-resistant varieties in Tartary buckwheat and its relative species.

Metabolome and transcriptome association study reveals biosynthesis of specialized benzylisoquinoline alkaloids in Phellodendron amurense

ObjectiveBenzylisoquinoline alkaloids (BIAs) have pharmacological functions and clinical use. BIAs are mainly distributed in plant species across the order Ranunculales and the genus Phellodendron from Sapindales. The BIA biosynthesis has been intensively investigated in Ranunculales species. However, the accumulation mechanism of BIAs in Phellodendron is largely unknown. The aim of this study is to unravel the biosynthetic pathways of BIAs in Phellodendron amurens. MethodsThe transcriptome and metabolome data from 18 different tissues of P. amurense were meticulously sequenced and subsequently subjected to a thorough analysis. Weighted gene co-expression network analysis (WGCNA), a powerful systems biology approach that facilitates the construction and subsequent analysis of co-expression networks, was utilized to identify candidate genes involved in BIAs biosynthesis. Following this, recombinant plasmids containing candidate genes were expressed in Escherichia coli, a widely used prokaryotic expression system. The purpose of this genetic engineering endeavor was to express the candidate genes within the bacteria, thereby enabling the assessment of the resultant enzyme activity. ResultsThe synonymous substitutions per synonymous site for paralogs indicated that at least one whole genome duplication event has occurred. The potential BIA biosynthetic pathway of P. amurense was proposed, and two PR10/Bet v1 members, 14 CYP450s, and 33 methyltransferases were selected as related to BIA biosynthesis. One PR10/Bet v1 was identified as norcoclaurine synthase, which could catalyze dopamine and 4-hydroxyphenylacetaldehyde into (S)-norcoclaurine. ConclusionOur studies provide important insights into the biosynthesis and evolution of BIAs in non-Ranunculales species.

Expression of Immunodominant gI Protein of Duck Enteritis Virus and its Evaluation for ELISA

Background: Duck plague (DP), also known as duck viral enteritis (DVE) caused by Anatid alpha herpesvirus-1, is the major infectious viral disease reported in India very often with significant economic losses due to high morbidity and mortality. The genome is approximately 158,091 bp with a genomic arrangement pattern (UL-IRS-US-TRS) that corresponds to type-D herpesvirus. The incubation period of the disease in domestic ducks ranges from 3 to 7 days and the mortality varies from 5-100%. It has been reported that the strategies to monitor and control DVE were often frustrating because the disease tends to establish a latent infection in waterfowl and vaccination failure. The present study was taken as an attempt to develop indirect ELISA using developed recombinant gI protein to diagnose DEV in field condition. Methods: In this study, we selected a membrane glycoprotein protein I (US7) gene, that plays an important role in virion sorting, cell-cell spread and binding of IgG Fc receptor. The gI protein was cloned with pET-32a (+) and expressed in a prokaryotic (E. coli) expression system. Further, recombinant protein was purified by nickel column affinity chromatography and refolded by dialysis. The obtained concentrated protein was then evaluated for its antigenicity and reliability in an Indirect-Enzyme-linked immunosorbent assay (ELISA) for DEV antibody detection in duck sera. Result: A panel of positive, negative and vaccinal serum was evaluated which indicated the potential use of the protein in the development of a serological assay for sero- surveillance of duck plague infection.

Preparation of divalent camelid single-domain antibody and its application in immunoassays for Salmonella detection in food.

Salmonella-related foodborne infections are commonly caused by the serovars of S. Typhimurium, which can be detected using antibody-based immunoassays. The monovalent variable domain of thecamelid heavy chain antibody (VHH) performs excellently in constructing multivalent VHH variants, which generally exhibit higher affinities with antigens and consequently enhance the assay sensitivity. In this study, the divalent variants of VHHs (diVHHs) targeting S. Typhimurium were generated by encoding the monovalent VHH genes assembled in tandem with a flexible linker peptide (G4S)2. Soluble diVHHs were produced in a prokaryotic expression system and purified with a yield of 4.22 mg/L. Benefiting from their stability and antigen-binding abilities towards tested Salmonella serovars, diVHH-based immunoassays were developed. The diVHH-based sandwich immunoassay, using diVHH as capture antibody, exhibited a detection limit of 1.04×102 CFU/mL and enabled as low as 10 CFU/mL S. Typhimurium to be detectedafter 6 h of enrichment in lettuce. Furthermore, this assay can be applied to spiked lettuce, chicken, and pork samples, showing acceptable recoveries ranging from 83 to 106%. This study presented feasible strategies for VHH multivalence and established a superior sensitivity VHH-based immunoassay for monitoring and analyzing Salmonella contamination in food.

Prokaryotic expression and purification of inorganic nitrogen transporters in wheat

The nitrate transporter (NRT) and ammonium transporter (AMT) are crucial transmembrane proteins involved in the absorption, transport, and distribution of inorganic nitrogen in wheat. Obtaining NRT/AMT and preparing corresponding antibodies are conducive to probing into their tissue localization and comprehending the nitrogen utilization process in wheat. In this study, four genes (TaNPF4.5, TaNPF8.3, TaNRT3.1, TaAMT1.2) with high expression levels were chosen and cloned from 405 genes of the TaNRT/TaNPF family and 23 genes of the TaAMT family identified previously. The transmembrane domains of the four transporters were predicted by HMMER to determine the putative expression segments, followed by prokaryotic expression and purification. Under the induction with 1 mmol/L IPTG at 37 ℃, the non-transmembrane segments of TaNPF4.5, TaNPF8.3, and TaNRT3.1 reached the highest expression levels (as inclusion bodies) after 4 h, while TaAMT1.2 was expressed at the highest level (as a soluble protein) after 3 h. TaNPF4.5, TaNPF8.3, and TaNRT3.1 were purified by a pH gradient. The purity of TaNPF4.5 and TaNPF8.3 reached about 87% and 85% at pH 2.0 and pH 3.0, respectively, both of which were suitable for antibody preparation. However, the purity of TaNRT3.1 did not reach 85%. TaAMT1.2 was purified by an imidazole gradient, reaching the purity of about 95% at 20 mmol/L imidazole, and the antibody was prepared successfully. The expression, purification, and antibody preparation of TaAMT1.2 not only provides insights into the expression, purification, and antibody preparation of membrane proteins including TaNPF4.5 and TaNPF8.3 but also lays a foundation for studying the expression and localization of membrane proteins in wheat.

Prokaryotic expression of wheat TaABI5-D3 gene and polyclonal antibody preparation

Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3, one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a-TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.

Prokaryotic expression, purification, and activity of the inositol polyphosphate 5-phosphatase Gs5PTase8 from wild soybean

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP3 [inositol(1, 4, 5)trisphosphate], IP4 [inositol(1, 3, 4, 5)tetrakisphosphate], PI(4, 5)P2 [phosphatidylinositol(4, 5) bisphosphate] and PI(3, 4, 5)P3 [phosphatidylinositol(3, 4, 5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

Sequence characteristics, expression and subcellular localization of PtCYP721A57 gene from cytochrome P450 family in Polygala tenuifolia willd.

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins.

Screening of IgE-Binding Epitopes of Peach Allergenic Protein Pru p 7 Based on an Immune Microarray Assay.

Pru p 7 (also named Peamaclein) is a member of the gibberellin-regulated protein family, which is the latest foodborne allergenic protein identified in peach. In this paper, the prokaryotic expression and identification of Pru p 7 were performed, and the protein properties, structure, and homology were analyzed. In addition, a preliminary screening of B-cell linear epitopes of Pru p 7 was performed by the bioinformatics software prediction method, and three epitopes were identified using slot-blot immune microarray assay combined with an immune score matrix (P-1, AA16-21, AGYQER; P-2, AA40-46, TYGNKDE; P-3, AA52-59, DLKNSKGN). Moreover, the electrostatic potential of these epitopes was analyzed, and the stability after ultrahigh pressure treatment was also verified. Finally, the amino acids that play key immune roles in the epitopes were obtained by amino acid mutations. These results may contribute to the further understanding of Pru p 7 and the prevention of peach allergy.

Chemical and Transcriptomic Analyses Provide New Insights into Key Genes for Ginsenoside Biosynthesis in the Rhizome of Panax japonicus C. A. Meyer.

Panax japonicus C. A. Meyer is renowned for its significant therapeutic effects and is commonly used worldwide. Its active ingredients, triterpenoid saponins, show variation in content among different tissues. The tissue-specific distribution of saponins is potentially related to the expression of vital genes in the biosynthesis pathway. In this study, the contents of five saponins (ginsenoside Ro, chikusetsusaponin IV, chikusetsusaponin IVa, ginsenoside Rg1, and ginsenoside Rb1) in three different tissues were determined by HPLC. Transcriptome sequencing analysis identified differentially expressed genes (DEGs) involved in triterpenoid saponin biosynthesis, highlighting significant correlations between saponin contents and the expression levels of 10 cytochrome p450 monooxygenase (CYP) and 3 UDP-glycosyltransferase (UGT) genes. Cloning, sequencing, and prokaryotic expression of UGT genes confirmed the molecular weights of UGT proteins. Gene sequence alignment and phylogenetic analysis provided preliminary insights into UGT gene functions. Meanwhile, the function of one UGT gene was characterized in the yeast. These findings advance our understanding of the triterpenoid saponin biosynthesis in P. japonicus and support future research in traditional Chinese medicine (TCM) and synthetic biology.

Recombinant avian-derived antiviral proteins cIFITM1, cIFITM3, and cViperin as effective adjuvants in inactivated H9N2 subtype avian influenza vaccines

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control.

Preliminary evaluation of the protective effect of rEi-SAG19 on Eimeria intestinalis infection in rabbits

Eimeria intestinalis is one of the most pathogenic coccidia species in rabbits. Anticoccidial treaments are the main measures to control rabbit coccidiosis now, but there are drug resistance and residues concerns. Therefore, vaccine has been used as an alternative strategy. The surface antigens (SAGs) of apicomplexan protozoa play a role in adhesion and invasion of host intestinal cells, and are considered to be potential candidate antigens for vaccines. In this study, transcriptional analysis of 5 Ei-SAGs genes at four developmental stages was conducted, then the Ei-SAG19 gene were screened out for prokaryotic expression and the reactogenicity of recombinant SAG19 (rEi-SAG19) was investigated by immunoblotting. To assessment the protective effects of rEi-SAG19, rabbits (n = 40) were randomly divided into four groups (Blank control, PBS-infected, Trx-His-S-Quil-A-infected and rEi-SAG19 immunized groups), the rEi-SAG19 immunized group was subcutaneously immunized with 100 μg rEi-SAG19 in the neck with an interval of two weeks, and challenged with 5×104 homologous oocysts two weeks after the second immunization. Two weeks after the challenge, all rabbits were sacrificed. After that, the level of serum specific IgG antibody was detected weekly and the level of cytokines in serum before the challenge were determined. At the end of the experiment, the weight gain, oocyst reduction rate, lesion score and anticoccidial index (ACI) were calculated. The results showed that rEi-SAG19 has a good reactogenicity. The relative weight gain rate, oocyst reduction rate and ACI of the rabbits in rEi-SAG19 immunized group were 80.51%, 72.6%, and 165.1, respectively, which has a moderate protective effect. The level of serum specific IgG antibody and IL-4 rised significantly (P < 0.05), but the levels of IL-2, IFN-γ and IL-10 had no significant difference (P > 0.05). Our results indicated that rEi-SAG19 could provides moderate protective effect against E. intestinalis infection in rabbits (ACI = 165.1). Therefore, rEi-SAG19 could be used as a vaccine candidate antigen for E. intestinalis.

A new Bowman-Birk type protease inhibitor regulated by MeJA pathway in maize exhibits anti-feedant activity against the Ostrinia furnacalis.

Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.

PHD-BAH Domain in ASH1L Could Recognize H3K4 Methylation and Regulate the Malignant Behavior of Cholangiocarcinoma.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

Structural and biochemical basis for the pathogenic mutations of methylmalonate semialdehyde dehydrogenase ALDH6A1

BackgroundMethylmalonate semialdehyde dehydrogenase (ALDH6A1), encoded by the ALDH6A1 gene, is essential for the metabolic degradation of valine and thymine. Genetic alterations in ALDH6A1 lead to methylmalonate semialdehyde dehydrogenase deficiency (MMSDD), a rare disease with only five reported disease mutations, focusing on its molecular foundation but lacking in-depth mechanistic investigations. Therefore, the structural and biochemical properties of the ALDH6A1 mutants have not yet been thoroughly examined. MethodsWild-type (WT) and mutant ALDH6A1 were constructed as plasmids and purified after prokaryotic expression to obtain conformationally homogeneous and pure protein. The structures of ALDH6A1 mutants (P62S, Y172H & R535C, S262Y, P421S, and G446R) were solved using cryo-electron microscopy. Based on the results of ALDH6A1 WT, enzyme activity and thermal stability experiments of their mutants were performed to explore the ALDH6A1’s biochemical characteristics. ResultsThis study presents a structural analysis of the ALDH6A1 mutants, P62S, Y172H & R535C, S262Y, P421S, and G446R at resolutions of 3.70, 2.92, 3.12, 3.47, and 3.00Å, respectively. However, the electronic density of ALDH6A1 P421S is poor, and it is difficult to fit into this density. Furthermore, the root-mean-square deviation (r.m.s.d.) of ALDH6A1 WT with these mutants was significant. This study revealed a tetrameric structure with closely interacting monomers, except for ALDH6A1 P62S, which forms a dimer, and is consistent with the principles of the aldehyde dehydrogenase family. Moreover, these disease mutations also affect enzyme activity and thermal stability. ConclusionsOur findings shed light on disease mutations that contribute to the properties of ALDH6A1 and lead to the genesis of MMSDD from structural and biochemical perspectives, which holds promise as a potential theoretical basis for this rare disease.