Search Results (872)

Senecavirus A (SVA) is an emerging pathogen that poses a significant threat to the global swine industry. With the advent of SVA vaccines, there is a growing need to develop serological diagnostic methods for evaluating vaccine-induced immunity. This study successfully established an indirect enzyme-linked immunosorbent assay (iELISA) through heterologous expression of a novel VP2-VP3-VP1 tandem recombinant protein in Escherichia coli (E. coli), which was constructed by integrating B-cell epitopes from VP1, VP2, and VP3. Comparative analysis using indirect ELISA revealed that the tandem recombinant VP2-VP3-VP1 protein and VP2 exhibited superior immunoreactivity. Consequently, the iELISAs for the tandem protein and VP2 were selected for further validation. Following optimization, the cut-off for the rVP2-VP3-VP1 iELISA was set at a sample-to-positive (S/P) ratio ≥ 0.60, while that for the rVP2 iELISA was set at ≥0.53. Analysis of kinetic sera from inactivated vaccine-immunized pigs showed that the rVP2-VP3-VP1 iELISA detected seroconversion synchronously with neutralizing antibodies, earlier than anti-VP2 antibodies. Finally, a serological survey for SVA was conducted in parts of mainland China from 2023 to 2024, with the rVP2-VP3-VP1 iELISA revealing an overall seroprevalence of 20.8%. These results indicate that the established detection method can be effectively used to evaluate SVA immunity and for epidemic surveillance. Full article

Blood-sucking organisms produce various anticoagulant proteins that prevent blood clotting in their prey. Even in well-studied species like Hirudo medicinalis, many such proteins remain unidentified. We previously described a novel cysteine-rich anticoagulant (CRA), a distant homolog of antistasin. Later, we discovered another, much larger homolog in the medicinal leech. Its amino acid sequence is also highly cysteine-rich. Analysis of cysteine patterns showed four antistasin-like domain motifs, with one of them strongly disrupted. Since both antistasin and CRA contain two such domains, the new protein represents a duplicated antistasin-like structure. We cloned its cDNA, expressed the recombinant protein in Escherichia coli, purified it by metal-chelate chromatography, refolded it, and tested its anticoagulant properties. Using standard clinical assays—activated partial thromboplastin time, prothrombin time, and thrombin time—we found that the protein inhibited coagulation in all tests, though to varying degrees. These findings suggest that different antistasin-like anticoagulants in the leech enable it to block both intrinsic and extrinsic coagulation pathways, while hirudin inhibits the final step of clot formation. The combination of different anticoagulant proteins allows the leech to effectively prevent the prey’s blood from clotting during feeding. Full article

Rapid antimicrobial resistance (AMR) prediction from MALDI-TOF mass spectrometry (MS) remains challenging, particularly when training artificial intelligence (AI) models under small-sample constraints. Performance is often hampered by the high dimensionality of spectral data and the subtle nature of resistance-related signals: full-spectrum approaches risk overfitting to high-dimensional noise, whereas peak-selection strategies risk discarding structurally informative, low-intensity signals. Here, we propose ReShuffle-MS, a region-guided data augmentation framework for MS data. Each spectrum is partitioned into a Main Discriminative Region (MDR) and a Peripheral Peak Region (PPR). By recombining signals within the PPR across samples of the same class while keeping the MDR intact, ReShuffle-MS generates structure-preserving augmented samples. On a clinical dataset for Escherichia coli (E. coli) levofloxacin resistance prediction, ReShuffle-MS delivered significant and consistent performance gains. It improved the average accuracy of classical machine learning models by 3.7% and enabled a one-dimensional convolutional neural network (CNN) to achieve 83.25% accuracy and 97.28% recall. Visualization using Grad-CAM revealed a shift from sparse, peak-dependent attention toward broader and more meaningful spectral patterns. Validation on the external DRIAMS-C dataset for ceftriaxone resistance further demonstrated that the method generalizes to a distinct laboratory setting and a different antibiotic target. These findings suggest that ReShuffle-MS can enhance the robustness and clinical utility of AI-based AMR prediction from routinely acquired MALDI-TOF spectra. Full article

Red sea bream (Pagrus major) aquaculture represents one of the most economically important marine aquaculture industries in Japan and East Asia. However, viral diseases, particularly those caused by red sea bream iridovirus (RSIV), pose a serious threat to aquaculture production in this region. In this study, we applied high-hydrostatic-pressure (HHP) refolding technology to develop a recombinant vaccine targeting the RSIV major capsid protein (MCP). The recombinant MCP (RSIV-rMCP) expressed in Escherichia coli was insoluble; however, HHP treatment under alkaline (pH 10) conditions in the presence of arginine successfully solubilised the protein while preserving its structural integrity. The solubilised protein (HHP–RSIV-rMCP) induced strong RSIV-specific IgM responses and enhanced disease resistance in red sea bream. In contrast, sera from fish immunised with a commercial formalin-inactivated vaccine exhibited minimal reactivity to HHP–RSIV-rMCP but reacted significantly to formalin-treated HHP–RSIV-rMCP. These results indicate that the HHP–RSIV-rMCP vaccine induces conformation-specific IgM antibodies and that structural preservation is crucial for maintaining antigenicity. Collectively, our findings demonstrate that HHP refolding technology is an effective strategy for preparing structurally preserved antigens. Full article

Xylanases (EC 3.2.1.8) are value-added enzymes essential for biomass deconstruction and are widely used in the pulp and paper, food, feed, and biofuel sectors. This review provides a comprehensive analysis of the current state and future prospects of xylanase research and application. It begins by examining the structural diversity of xylan substrates and the corresponding classification of xylanase enzymes, their catalytic mechanisms, and methods for their functional study, such as inhibitor analysis. The discussion then covers the challenges and methods involved in the purification of xylanases from complex biological mixtures. While natural microbial sources (fungi and bacteria) remain important, the limitations of wild-type (WT) strains for industrial production are highlighted. The review assesses the most common recombinant production systems, including Escherichia coli, Bacillus subtilis, and Komagataella phaffii, comparing their advantages for high-yield enzyme production. Finally, the paper focuses on protein engineering strategies as powerful tools for enhancing key enzyme properties (thermostability, specific activity, and pH tolerance). By integrating fundamental knowledge with applied technological approaches, this review underscores the critical role of xylanases in industrial biotechnology and identifies future research directions for their optimization. Full article

Transmissible gastroenteritis (TGE), caused by the TGE virus (TGEV), is a highly contagious enteric disease characterized by vomiting, dehydration, and watery diarrhea. It mainly endangers piglets within two weeks of age, with a 100% mortality rate, inflicting severe economic losses on the global swine industry. Since enteric tropism of the virus and mucosa serves as the first line of defense against viral invasion, an oral vaccine inducing sufficient secretory immunoglobulin A (SIgA) antibodies in animals should be developed. Being a generally recognized as safe (GRAS) microorganism, Bacillus subtilis can form endospores under extreme environmental conditions, which confer resistance to the hostile gastric environment and have been widely employed as delivery vehicles for oral vaccines owing to their immunoadjuvant activity and non-specific antidiarrheal effects. In this study, the AD antigenic epitope of the TGEV S protein was selected as the immunogen. The mature peptide of the B subunit of the heat-labile enterotoxin from enterotoxigenic Escherichia coli served as a mucosal adjuvant, and B. subtilis WB800N was used as the delivery host to construct the recombinant strain pHT43-LTB-AD/WB800N. After confirming the successful expression of the target protein, oral immunization was performed using mice as a model. The results demonstrated that this recombinant strain induced robust mucosal, humoral, and cellular immunity, along with considerable levels of neutralizing antibodies. These findings indicate that recombinant B. subtilis could serve as an oral vaccine candidate to combat TGEV infections. Full article

Antibiotic persistence is a transient phenotype in which a subset of genetically susceptible bacteria survives lethal antibiotic exposure without acquiring resistance. However, survival alone does not define a persister cell—only cells that successfully recover, resume growth, and produce viable progeny complete the persister cycle. Recent studies in Escherichia coli show that persister awakening is a multistage process shaped by dormancy depth, metabolic state, and antibiotic-induced damage. Upstream induction mechanisms, including stringent-response signaling and toxin–antitoxin–mediated growth arrest, primarily determine dormancy depth but do not directly control awakening kinetics. During the lag phase, persister cells undergo coordinated recovery involving detoxification of residual antibiotics, ATP restoration, dissolution of protein aggregates, and ribosome reactivation. After exposure to fluoroquinolones, awakening additionally requires SOS-driven DNA repair via homologous recombination or transcription-coupled repair. In contrast, β-lactam–exposed persister cells rely mainly on efflux-mediated detoxification and asymmetric damage partitioning. Failure to restore proteostasis or resolve damage results in abortive recovery or cell death. Only after damage processing and metabolic reactivation can persister cells resume division and generate viable progeny. This review integrates current molecular insights into persister cell recovery in E. coli, highlighting the lag phase as the critical barrier between survival and true persistence. Full article

In vertebrates, the interleukin-1β molecule (IL-1β) is among the most important proinflammatory cytokines and plays crucial roles in shaping injury progression, immunological challenges, and local and systemic responses to infection. In the current study, a cDNA encoding the IL-1β gene in Asian seabass (Lates calcarifer) (LcIL-1β) was identified at both the nucleotide and protein levels. Its immune responses were investigated in various tissues from diseased and normal fish. Recombinant rLcIL-1β was produced in Escherichia coli. Furthermore, its ability to control two fish pathogenic bacteria, Flavobacterium covae and Streptococcus iniae, was assessed in vitro. Transcriptional expression was quantified by qRT–PCR, which revealed the highest levels in whole blood, followed by the liver, gills and midgut. Immune response analyses of the head kidney, whole blood, liver, gills, spleen and intestines of fish infected with F. covae and S. iniae at concentrations of 1 × 10³, 1 × 10⁴ and 1 × 10⁵ CFU/fish, respectively, revealed significant upregulation of LcIL-1β (p < 0.05) for 6–24 h (h) after induction. Interestingly, compared with the control treatment, the application of 1, 10 and 100 µg of rLcIL-1β greatly increased the phagocytic activity and phagocytic index of phagocytes (p < 0.05). Antibacterial function analyses of F. covae and S. iniae revealed minimal inhibitory concentrations (MICs) of 29.17 and 85.25 μg/mL, respectively. Finally, injection of S. iniae following rLcIL-1β revealed that 50 and 100 µg of the target protein demonstrated significant functional activity in safeguarding Asian seabass from these pathogenic bacteria (p < 0.05). This information revealed that LcIL-1β in Asian seabass significantly drives immune defense mechanisms against pathogenic bacteria, which is important for the development of effective disease prevention methods for Asian seabass aquaculture. Full article

Botulinum neurotoxin serotype A (BoNT/A) is the most potent known neurotoxin. While its light chain (LC) catalytic domain is a prime target for next-generation vaccines and therapeutics, the functional differences among BoNT/A subtype LCs (A1, A2, A3) remain to be definitively characterized, despite notable sequence variation. This work aimed to systematically compare the proteolytic activity and immunoprotective efficacy of recombinant BoNT/A1-LC, A2-LC, and A3-LC. Recombinant A1-LC-His, A2-LC-His, A3-LC-His, and A3-LC-Twin-Strep proteins were expressed in Escherichia coli (E. coli) and purified with affinity chromatography. Their proteolytic activity was assessed via in vitro SNAP-25 cleavage assays. The protective potency of these antigens was evaluated in a mouse model. In vitro cleavage assays revealed a substrate cleavage efficiency order of A2-LC > A1-LC > A3-LC. In vivo, both A1-LC and A2-LC immunization conferred robust, broad protection against high-dose challenges with all three toxin subtypes. In stark contrast, A3-LC provided only minimal protection against its homologous toxin and none against heterologous subtypes. Crucially, the functional deficit of A3-LC was confirmed to be an intrinsic property, as the A3-LC-TS variant, designed to exclude tag-specific interference, exhibited comparable low efficacy. According to structural research, A3-LC’s compromised function may be caused by a four-amino-acid loss. The inferior performance of A3-LC is inherent to its primary structure. This work identified A1-LC or A2-LC as the potential proteolytic activity molecule and vaccine antigen by demonstrating functional differences among BoNT/A subtype LCs. These findings provide crucial insights for developing subtype-specific countermeasures against botulism. Full article

Hydrogenases are key enzymes in microbial energy metabolism, catalyzing the reversible conversion between molecular hydrogen and protons. Among them, [NiFe]-hydrogenases are particularly attractive for biocatalytic applications due to the oxygen tolerance of several members of this class and their ability to couple hydrogen oxidation with redox cofactor regeneration. In this study, a recombinant soluble [NiFe]-hydrogenase from Cupriavidus necator H16 was successfully expressed in Escherichia coli BL21 (DE3), purified, and characterised with a focus on its applicability for NAD⁺ regeneration. Unlike previous studies that primarily used native C. necator extracts or complex maturation systems, this work provides the first quantitative demonstration that an aerobically purified recombinant soluble [NiFe]-hydrogenase expressed in E. coli can function effectively as an NAD⁺ regeneration catalyst and operate within multi-enzymatic cascade reactions under application-relevant conditions. The crude recombinant enzyme displayed a volumetric activity of 0.273 ± 0.024 U/mL and a specific activity of 0.018 ± 0.002 U/mg_cells in the hydrogen oxidation assay, while purification yielded a specific activity of 0.114 ± 0.001 U/mg with an overall recovery of 79.2%. The enzyme exhibited an optimal temperature of 35 °C and a pH optimum of 7.00. Thermal stability analysis revealed rapid deactivation at 40 °C (k_d = 0.4186 ± 0.0788 h⁻¹, t_1/2 ≈ 1.7 h) and substantially slower deactivation at 4 °C (k_d = 0.1141 ± 0.0139 h⁻¹, t_1/2 ≈ 6.1 h). Batch NADH oxidation experiments confirmed efficient cofactor turnover and high specificity towards NADH over NADPH. Finally, integration of the hydrogenase into a one-pot two-enzyme glucose oxidation system demonstrated its capacity for in situ NAD⁺ regeneration, although the reaction stopped after approximately 5 min due to acidification from gluconic acid formation, highlighting pH control as a key requirement for future process optimization. Full article

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is a devastating pine disease that is characterized by rapid transmission, high lethality, and limited control options. In our previous study, the fucosyltransferase gene (fut) which encoded fucosyltransferase (FUT) was found to be a putative virulence determinant in PWN, which regulates pathogenicity of nematodes. To investigate the functional role of the fut gene in PWN, a comprehensive analysis was conducted to understand its molecular structure and biological activity. The full-length open reading frame (ORF) of fut was amplified using reverse transcription PCR (RT-PCR) and successfully ligated into the pET-28a expression vector. Heterologous expression of the recombinant FUT was achieved in Escherichia coli Rosetta (DE3) through induction with 1.0 mM isopropyl-β-D-thiogalactoside (IPTG), followed by purification via nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Biochemical characterization revealed that the recombinant FUT exhibited optimal enzymatic activity at 30 °C and pH 8.0, respectively. Furthermore, RNA interference (RNAi) validated by RT-qPCR was used to explore the biological functions of fut in PWN, and results indicated that downregulation of the fut gene could significantly reduce the vitality, reproduction, pathogenicity, development, and lifespan of PWN. Furthermore, gallic acid as an inhibitor of FUT displayed a strong inhibitory effect on recombinant FUT activity and nematicidal activity against PWNs in vitro and could alleviate the wilt symptom of pine seedlings inoculated with PWNs at a concentration of 100 μg/mL, indicating that it has the potential to be a novel nematicide. Collectively, these results establish fut as a critical virulence determinant in PWN and highlight its potential as a molecular target for controlling pine wilt disease. Full article

Photocatalytic antimicrobial materials represent a promising class of sustainable disinfection technologies, leveraging the generation of reactive oxygen species (ROS) under light irradiation for environmental and biomedical applications. Bismuth vanadate (BiVO₄), a visible-light-responsive semiconductor, has garnered considerable interest due to its suitable bandgap and chemical stability. However, its photocatalytic performance is critically limited by rapid charge carrier recombination and a relatively weak intrinsic built-in electric field. In this study, we report a novel composite strategy to address these limitations by coupling BiVO₄ with tourmaline, a naturally abundant piezoelectric mineral exhibiting spontaneous polarization. The integration of tourmaline induces a built-in electric field that synergistically aligns with and amplifies the internal field of BiVO₄, which substantially improves charge separation and carrier transport dynamics. The resulting tourmaline/BiVO₄ heterostructure demonstrates remarkedly enhanced antibacterial activity under visible-light irradiation against both Escherichia coli and Staphylococcus aureus, significantly outperforming pristine BiVO₄. Mechanistic investigations attribute this enhancement to the polarization-induced modulation of interfacial charge dynamics, which boosts ROS generation and accelerates microbial inactivation kinetics. This work presents a generalizable strategy for the rational design of high-efficiency photocatalytic antimicrobial systems, offering potential utility in water treatment, healthcare sterilization, and environmental remediation. Full article

Cymbidium sinense is a highly valued ornamental orchid renowned for its strong floral fragrance. In this study, dihydro-β-ionone was identified as a major volatile compound in C. sinense ‘Qi Hei’. Its emission increased progressively during flower development and was predominantly released from the sepals and petals. Transcriptome analysis of flowers at three developmental stages led to the identification of four double-bond reductase genes, designated CsDBR1–CsDBR4. Spatiotemporal expression profiling demonstrated that transcript levels of CsDBRs were highest in sepals and petals, showing a significant positive correlation with dihydro-β-ionone accumulation (p < 0.05). Heterologous expression in Escherichia coli and subsequent in vitro enzymatic assays confirmed that recombinant CsDBR1, CsDBR2, and CsDBR4 proteins catalyze the conversion of β-ionone to dihydro-β-ionone, whereas CsDBR3 exhibited no detectable activity. Transient expression in Nicotiana benthamiana leaves further verified the in planta function of CsDBR1, CsDBR2, and CsDBR4, resulting in elevated production of dihydro-β-ionone upon infiltration of β-ionone. Substrate specificity assays revealed that CsDBR2 and CsDBR4 also reduced 1-octen-3-one, 3-nonen-2-one, and pulegone. Collectively, these findings demonstrate that CsDBR1, CsDBR2, and CsDBR4 are key enzymes responsible for dihydro-β-ionone biosynthesis in C. sinense, providing a genetic foundation for molecular breeding aimed at improving floral fragrance in orchids. Full article

Background/Objectives: The detection of anti-Coxiella antibodies using serological methods is essential for identifying exposed ruminants and preventing this important zoonotic disease in livestock. In recent years, numerous attempts have been made to increase diagnostic performance as well as simplify the production of serological assays. Commercially available tests often use whole-cell antigens, which can decrease specificity and require high-level biosafety facilities for manufacturing. The aim of this work was to produce three Coxiella burnetii (C. burnetii) antigens in recombinant form and assess them for the detection of anti-Coxiella antibodies in ruminants. Methods: Three recombinant C. burnetii antigens (Com-1, MceB, AdaA) were selected among immunodominant antigens and produced in a heterologous system (Escherichia coli). Following purification, the proteins were utilized to coat ELISA plates and evaluated for seroreactivity against sera from both negative and positive cattle. Results: Com-1 demonstrated the greatest agreement with the commercial test, albeit moderate. MceB exhibited nonspecific reactivity against a large number of sera, while the AdaA showed reactivity against only a few positive sera. Conclusions: Our findings are consistent with previous research, indicating that utilizing a single antigen to identify exposed animals is unfeasible with current knowledge, most likely due to the complex immunological response following C. burnetii infection in cattle. Consequently, it is critical to continue testing and identifying immunoreactive antigens in order to further investigate them and, potentially, select the most appropriate. Full article

The phylum Armatimonadota represents one of the least characterized bacterial lineages, with only three formally described species despite its widespread distribution in various environments. Deep subsurface thermal environments harbor significant microbial diversity and represent promising sources for novel enzyme discovery through metagenomic approaches. This study reports the identification, cloning, and biochemical characterization of Cel7465, a novel endoglucanase from an uncultured GBS-DC family within the order Fimbriimonadales. The enzyme was identified through metagenomic analysis of microbial mats from the Biragzang deep thermal well (North Ossetia, Russia, 48 °C) and successfully expressed in cells of Escherichia coli strain ArcticExpress (DE3). Phylogenetic analysis assigned Cel7465 to glycoside hydrolase family 5 subfamily 36. The purified recombinant enzyme exhibited optimal activity at 55 °C and pH 8.0, with high specific activity of 4347 U/mg. The enzyme demonstrated broad pH tolerance (50% activity retained from pH 4.0 to 10.0) and notable thermal stability, retaining 60% activity after one hour at 80 °C and 20% after four hours. Remarkably, the presence of Mn²⁺ ions enhanced enzyme activity more than 7-fold, while Mg²⁺ and Ca²⁺ ions provided moderate activation. Cel7465 represents the first biochemically characterized glycoside hydrolase from the order Fimbriimonadales, expanding our understanding of enzymatic capabilities within the understudied phylum Armatimonadota and demonstrating the continued potential of extreme environments as sources of novel industrial biocatalysts. Full article