Abstract:Eumelanin, a natural biological pigment formed by the polymerization of 5,6-dihydroxyindole and its carboxylic acid derivatives, is ubiquitous in animals and microorganisms. Its highly conjugated molecular structure confers multiple functional properties, including broad-spectrum light absorption, antioxidant activity, free radical scavenging, and metal ion chelation, making it a promising material for applications in biomedicine, functional coatings, and environmental remediation. The conventional production of eumelanin relies largely on extraction from animal tissue or chemical synthesis, which are constrained by limited raw materials and uncontrollable product structures. Recently, microbial cell factory-based biosynthesis of eumelanin has attracted increasing attention due to its environmental sustainability and structural controllability. This review comprehensively summarizes the biosynthetic pathways, strategies for metabolic engineering and genetic engineering, analysis and characterization methods for eumelanin, and discusses future perspectives for the application and development of microbial eumelanin production.

Abstract:Ulcerative colitis (UC) is a chronic non-specific intestinal inflammatory disease with complex pathogenesis. Recent studies have identified a pivotal role of the gut microbiota in the etiology of the disease. Beneficial bacteria, such as Lactobacillus and Bifidobacterium, have been shown to regulate the balance of the gut microbiota, repair the mucosal barrier, and alleviate inflammation. In contrast, excessive proliferation or secretion of toxins by harmful bacteria, such as Escherichia, can damage the integrity of the mucosa, induce inflammation, and accelerate the progression of UC. The active ingredients of traditional Chinese medicine, such as Scutellariae radix polysaccharides, pulchinenosides, and ginsenoside Rg1, may offer a promising avenue for the treatment of UC by modulating the structure of the gut microbiota and enhancing the barrier function and mucosal integrity. The present article reviews the latest research progress in the regulation mechanism of the gut microbiota in UC and the traditional Chinese medicine intervention, with a view to providing new strategies and theoretical support for clinical treatment.

Abstract:Medicinal plants and microorganisms engage in complex, dynamic interactions that profoundly influence plant growth and development, the biosynthesis of secondary metabolites, resistance to pathogens, and adaptation to environmental stressors. Therefore, elucidating the chemical signaling molecules that mediate these interactions is of fundamental importance. The review systematically summarizes the types and functional mechanisms of chemical signals involved in medicinal plant-microorganism interactions. It highlights the roles of both plant- and microorganism-derived signaling molecules in shaping the rhizosphere microbial community assembly, regulating plant growth, and modulating the accumulation of bioactive secondary metabolites. In addition, we discuss the functional divergence of bidirectional signaling in both symbiotic and competitive interactions. Although many mechanism aspects of these chemical signals remain to be fully elucidated, current knowledge provides a solid framework for understanding their regulatory roles. With a “signal-microbiome-function” framework, it is possible to manipulate plant chemical signaling or screen functional microbial strains to reconstruct beneficial rhizosphere microbiomes. Such strategies hold potential for improving soil-plant-microorganism interactions, promoting plant growth, enhance the accumulation of medicinal compounds, and strengthen resistance to pathogens. Overall, these insights are of considerable significance for improving the quality of Chinese medicinal materials and promoting the sustainable and ecological cultivation of medicinal plants.

Abstract:The continuous evolution and drug-resistant mutations of influenza virus have placed higher demands on the study of its pathogenic mechanism, as well as the development of antiviral drugs and vaccines. As a key tool for real-time monitoring of viral infection, replication and transmission, viral visualization reporter systems have evolved into various strategies, including high-resolution microscopic imaging, exogenous nanomaterial labeling, recombinant reporter viruses, and virus infection-inducible reporter systems. This manuscript systematically reviewed the research progress of influenza virus visualization technologies, elaborated the principles, core breakthroughs, and application scenarios of each technique, analyzed the common limitations and specific problems existing in the current technical system, and prospected its development direction combined with cutting-edge research, providing a reference for technical selection and innovative applications in influenza virus visualization research.

Abstract:The increasing emission of sulfur-containing compounds has become a critical factor restricting ecological and environmental security and the sustainable resource utilization. Biological desulfurization technology, relying on the sulfur metabolic pathways of desulfurization microorganisms, enables the efficient conversion of sulfur-containing pollutants and the recovery of sulfur resources under mild conditions, offering both environmental benefits and economic potential. This review systematically summarized the classification and functional characteristics of desulfurization microorganisms, with emphasis on the 4S pathway, the Kodama pathway, and related metabolic modes of organic sulfur-desulfurizing bacteria, as well as the sulfate reduction and sulfur oxidation mechanisms of inorganic sulfur-desulfurizing bacteria. On this basis, the advances in engineering applications in the fields of industrial gas purification, sludge and wastewater treatment, livestock and poultry manure management, domestic waste and agriculture were summarized, and the key environmental factors and carbon source regulation mechanisms affecting desulfurization efficiency were analyzed. Furthermore, the main bottlenecks currently faced by biological desulfurization technology were summarized from the aspects of strain performance, reaction kinetics, mass transfer enhancement, and process stability, and research directions for system enhancement through functional microbial community construction, reactor optimization, and intelligent regulation were proposed. This review aims to provide theoretical references and technical insights for the deepening of biological desulfurization mechanisms and efficient engineering applications.

Abstract:As a key group of the gut microbiota that helps maintain intestinal homeostasis, probiotics have attracted significant attention in life science and medical research. Akkermansia muciniphila (AKK), a strictly anaerobic Gram-negative bacterium, has swiftly become a central topic in gut microbiota research owing to its unique metabolic properties and considerable probiotic potential. This review systematically summarizes the discovery, physiological characteristics, and isolation methodologies and screening of AKK, while placing a particular emphasis on its role in preventing and managing a spectrum of human and animal diseases (including tumors, neurodegenerative disorders, metabolic syndromes, and inflammatory bowel diseases), thereby offering novel perspectives and potential targets for disease intervention. Furthermore, this review elucidates the core mechanisms underlying the probiotic functions of AKK. Specifically, AKK exerts beneficial effects on host health and therapeutic effects on diseases primarily through three key pathways: modulating intestinal microecological balance and barrier integrity, regulating systemic metabolic processes and immune responses, and mediating intricate crosstalk between the intestine and multiple extra-intestinal systems. To assess the translational efficiency of AKK, we summarize the current application status of its functional-related proteins and preparations in health, analyze clinical application scenarios and potential, and discuss existing bottlenecks and challenges in industrial application. This review provides a comprehensive theoretical reference and directional guidance for future basic research, application development, and industrialization of AKK.

Abstract:Trichoderma reesei is recognized as the microorganism with the most potent protein secretion capability known to date. It is extensively utilized for cellulase production and has garnered significant attention in heterologous protein synthesis. Although notable progress has been achieved in the screening of expression elements, chassis engineering, and fermentation process optimization, critical challenges persist, and the large-scale industrial application of heterologous protein expression has not yet been fully realized. This paper reviews the strategies and recent research advances regarding the high-efficiency expression of heterologous proteins in T. reesei. Furthermore, this paper discusses the limiting factors for its industrial-scale application and proposes novel recommendations and strategies to enhance heterologous protein yields.

Abstract:Objective To address the scarcity of neutral sites in Corynebacterium glutamicum ATCC 13032 caused by high G+C content and short intergenic sequences, we established a computer-aided screening platform to identify and characterize efficient neutral sites suitable for heterologous gene integration, aiming to provide key component support for constructing high-performance cell factories.Methods A computational screening platform CgNSFinder was constructed to screen candidate neutral sites by integrating heuristic rules such as genomic annotation, neighborhood characteristics, length, and G+C content. The candidate sites were systematically characterized in terms of integration efficiency, cell adaptability, expression intensity, and stability through reporter gene mKate knock-in experiments. We integrated the amylase gene, lycopene synthesis gene cluster, and key L-lysine synthesis genes into the screened sites to verify their heterologous expression ability and application efficiency.Results A total of 96 candidate neutral sites were screened out, from which 24 efficient sites were confirmed by experimental characterization. Among them, 19 sites had an integration efficiency greater than 50%, and 22 sites had a fluorescence expression intensity within the range of 2 400-3 500 a.u. The NS3 site showed the best performance, and the lycopene titer reached 48.4 mg/L after multi-copy integration. The L-lysine-producing strain D301 constructed based on the screened sites achieved a titer of 49.51 g/L, a yield of 14.25 g/g, and a productivity of 1.53 g/(L·h) in a 5-L fermenter.Conclusion The established neutral site screening method is efficient and feasible. The screened neutral sites are characterized by high integration efficiency, low host interference, and stable expression. This study provides an important tool for metabolic engineering of C. glutamicum.

Abstract:Objective We investigated the characteristics of rhizosphere bacterial communities of healthy and root rot-infected strawberry plants across different plots, as well as their correlations with soil physicochemical properties, aiming to discover and isolate beneficial rhizosphere bacteria with significant inhibitory effects against strawberry root rot pathogens to support disease management.Methods We utilized high-throughput sequencing combined with bioinformatics analysis to elucidate the differential characteristics and influencing factors of rhizosphere bacterial communities between healthy and diseased strawberry plants. Then, we employed the dilution plate method and dual culture assay to isolate antagonistic bacteria. Subsequently, we determined the taxonomic status of these beneficial strains by combining morphological observation with multi-gene phylogenetic analysis.Results The bacterial community diversity varied across different cultivation bases. Compared with that of healthy plants, the rhizosphere soil samples of diseased strawberry plants from the Xinzhuang and Kangshou bases showed decreased bacterial InvSimpson, Chao1, and Shannon evenness indices (P>0.05). Conversely, the rhizosphere soil samples of diseased strawberry plants from the Taolin base showed increased InvSimpson and Shannon evenness indices (P<0.05). Beta diversity analysis revealed that root rot significantly altered the microbial community structure. The complexity and stability of the rhizosphere bacterial co-occurrence network decreased in the diseased samples. Redundancy analysis indicated that soil total organic carbon, available phosphorus, and total potassium were the primary physicochemical factors shaping the bacterial community structure in strawberry rhizosphere. We isolated two bacterial strains, N2-10 and N2-18, that exhibited significant antagonistic activity from the healthy strawberry rhizosphere. The two strains demonstrated inhibition rates ranging from 55.6% to 62.2% and 54.5% to 66.7%, respectively, against the tested strawberry root rot pathogens. We identified strains N2-10 and N2-18 as Bacillus cereus and Bacillus velezensis, respectively.Conclusion We clarify how root rot affects strawberry rhizosphere bacterial communities and identify two beneficial rhizosphere bacterial strains with strong biocontrol potential. These findings provide a theoretical basis and valuable strain resources for elucidating the mechanisms of strawberry root rot and developing microbial-based green control technologies.

Abstract:Objective To elucidate the primary bioactive component in Psoralea corylifolia seed extract that is responsible for inhibiting Pseudomonas amygdali pv. lachrymans (Pal) and to characterize its antibacterial mechanism.Methods The effect of isopsoralen on the growth curve of Pal and its minimum inhibitory concentration (MIC) were determined by the liquid culture assay. The antibacterial mechanism was investigated by transmission electron microscopy (TEM) combined with physiological and biochemical analyses.Results The MIC of isopsoralen against Pal was 400 mg/L. Isopsoralen disrupted the integrity of the bacterial cell wall and cell membrane, leading to leakage of intracellular contents. It interfered with cellular energy metabolism by inhibiting the activity of respiratory chain dehydrogenases. In addition, it impaired bacterial responses to environmental changes and colonization in the host by suppressing swarming motility, swimming motility, and biofilm formation.Conclusion Isopsoralen exerts its inhibitory activity against Pal through the synergistic actions of structural damage, energy disruption, and virulence suppression, ultimately abolishing the pathogen’s ability to infect the host. These findings provide a theoretical basis for the application of P. corylifolia seed extract as a botanical bactericide in the green management of cucumber bacterial angular leaf spot.

Abstract:Objective To investigate the effects of pea-oat mixed cropping at different ratios on soil nutrients, enzyme activities, and carbon-fixing microbial communities in the alpine region of Qinghai Province.Methods This study designed five treatments: monoculture of oat (T0), monoculture of pea (Q0), and pea-oat mixed cropping at three ratios (QT1, QT2, and QT3). Soil enzyme activities and physicochemical indicators were measured, and the community structure, diversity, and relationships with environmental factors of carbon-fixing bacteria were analyzed based on high-throughput sequencing data of cbbL.Results Compared with monoculture, mixed cropping significantly enhanced soil enzyme activities and nutrient content, with QT3 (the pea:oat ratio of 2:1) showing the most pronounced effects. Under QT3, the activities of sucrase, cellulase, protease, and other enzymes were the highest, and the content of soil organic carbon, total nitrogen, ammonium nitrogen, and other nutrients was also significantly increased. Mixed cropping optimized the community structure of soil carbon-fixing bacteria. Specifically, QT3 significantly increased the alpha diversity (with the highest Chao1 and Shannon indices) and enriched functional groups such as Cyanobacteria. Environmental factor analysis indicated that soil ammonium nitrogen, total nitrogen, and organic carbon were the main drivers of changes in the carbon-fixing bacterial community. Furthermore, microbial co-occurrence network analysis revealed that mixed cropping, especially QT3, significantly enhanced network complexity, connectivity, and modularity, which indicated higher structural stability and functional collaboration potential of the microbial community.Conclusion Pea-oat mixed cropping, particularly at a ratio of 2:1, significantly improved soil ecological functions in alpine regions by enhancing soil enzyme activities, increasing nutrient availability, and optimizing the structure and functional network of carbon-fixing microbial communities. This provides an effective approach for enhancing soil carbon sequestration potential and promoting sustainable agricultural development.

Abstract:Ergosteryl-β-glucosidase (Egh1) catalyzes the hydrolysis of ergosteryl-β-glucoside for producing free ergosterol and glucose. This enzyme plays a crucial role in maintaining intracellular membrane lipid homeostasis and regulating the morphological stability of organelles, including vacuoles. However, the biological functions of this enzyme class in plant pathogenic fungi remain poorly understood.Objective To elucidate the role of MoEGH1, an Egh1 homolog in Magnaporthe oryzae, in appressorium development, sterol metabolism, autophagy regulation, and pathogenicity.Methods MoEGH1, homologous to yeast EGH1, was identified by sequence homology analysis. A deletion mutant ΔMoegh1 was constructed through targeted gene replacement. The biological functions of MoEgh1 were systematically examined through phenotypic characterization, genetic complementation, autophagic flux assays, and assessments of appressorium formation and turgor pressure.Results The deletion of MoEGH1 markedly impaired the growth, development, and pathogenicity of M. oryzae. ΔMoegh1 displayed significantly reduced radial growth and conidiation, an increased proportion of malformed conidia, a substantial decrease in appressorial turgor pressure, and defective appressorium formation. These developmental defects led to an almost complete loss of pathogenicity. Furthermore, compared with the wild-type strain, ΔMoegh1 demonstrated heightened sensitivity to rapamycin and sterol-targeting antifungal agents, including natamycin, amphotericin B, and itraconazole. Additionally, the mutant displayed abnormally elevated activity of the target of rapamycin (TOR) signaling pathway concomitant with reduced levels of autophagy.Conclusion MoEgh1 is essential for vegetative growth, conidiation, and appressorium formation in M. oryzae. MoEgh1 regulates the autophagy process and sterol homeostasis by modulating TOR activity, thereby playing a critical role in the pathogenicity of this fungus.

Abstract:Objective To elucidate the role of arginine methyltransferase in the growth, development, and pathogenicity of Botrytis cinerea.Methods Bioinformatics approaches were used to perform sequence alignment, phylogenetic analysis, and conserved domain prediction of the arginine methyltransferase HMT2 from eight fungal species, including B. cinerea, Saccharomyces cerevisiae, Fusarium graminearum, and Magnaporthe oryzae. The gene encoding this enzyme in B. cinerea was identified as BcHMT2. The BcHMT2-deleted mutant was constructed via homologous recombination, followed by phenotypic characterization and pathogenicity assays.Results The BcHMT2-deleted mutant exhibited a significantly reduced growth rate, significantly weakend cell wall-degrading enzyme activity, and decreased sclerotial production and conidiation. Meanwhile, the hyphal cells became smaller and the conidial morphology was abnormal. In addition, the mutant displayed markedly attenuated pathogenicity on tomato fruits and tobacco leaves.Conclusion This study reveals the regulatory role of BcHMT2 in the growth, development, and pathogenicity of B. cinerea, providing novel insights into the research on the prevention and control of gray mold.

Abstract:Objective To investigate the alleviating effects and mechanisms of Lacticaseibacillus paracasei CCFM1538 on inflammatory pain in mice.Methods A mouse model of inflammatory pain was induced by complete Freund’s adjuvant, and blank, model, dexamethasone, culture medium, fermentation supernatant, and high-dose/low-dose bacterial groups were set up. The ability of CCFM1538 to alleviate inflammatory pain was evaluated based on paw thickness, thermal/mechanical withdrawal threshold, HE staining of paw skin, inflammatory cytokines, glial cell immunofluorescence, and oxidative stress indicators.Results The fermentation supernatant of CCFM1538 significantly alleviated mouse paw swelling, increased the mechanical withdrawal threshold after modeling, reduced paw inflammatory cell infiltration, and decreased the expression of pro-inflammatory cytokines (IL-6 and CCL2). High-dose CCFM1538 cells significantly reduced the expression of pro-inflammatory cytokines (IL-1β and CCL2), decreased serum malondialdehyde levels, and inhibited abnormal activation of spinal cord glial cells. In addition, both significantly increased the thermal withdrawal threshold and serum superoxide dismutase activity in mice after modeling.Conclusion The fermentation supernatant and high-dose cells of CCFM1538 can alleviate inflammatory pain by reducing inflammation, alleviating oxidative stress, and regulating spinal glial cell activation. This study provides experimental evidence for the development of related functional foods.

Abstract:Objective To investigate the mechanism by which Camphora kanahirae leaves alleviate alcoholic liver disease through the regulation of the gut microbiota-short-chain fatty acid-intestinal barrier pathway.Methods Sixty mice were randomly allocated into six groups [normal, model, silymarin (100 mg/kg), and low-, medium-, and high-dose (100, 200, and 400 mg/kg, respectively) C. kanahirae leaves], with 10 mice in each group. After 14 consecutive days of administration, a mouse model of alcoholic liver disease was established. The liver index, alanine aminotransferase (ALT) and aspartate transferase (AST) levels, colon histopathological changes, short-chain fatty acid content, and alterations in gut microbiota structure were measured.Results Compared with the model group, medium- and high-dose C. kanahirae leaves reduced ALT and AST levels (P<0.05). Pathological evaluation showed that C. kanahirae leaves at all doses alleviated alcohol-induced colon mucosal damage and inflammatory cell infiltration to varying degrees. Alcohol damage resulted in decreases in levels of short-chain fatty acids, such as acetic acid, propionic acid, butyric acid, and valeric acid (P<0.05). C. kanahirae leaves promoted the proliferation of beneficial bacteria, increased short-chain fatty acid levels, and enhanced intestinal barrier function. Furthermore, 16S rRNA gene analysis of the gut microbiota indicated that alcohol intake led to dysbiosis, and C. kanahirae leaves effectively reversed this microbial imbalance, restoring the composition and diversity of the gut microbiota.Conclusion C. kanahirae leaves exert hepatoprotective effects by regulating the gut microbiota balance, promoting short-chain fatty acid production, and maintaining intestinal barrier integrity.

Abstract:Early life is a critical window for the establishment of the infant gut microbiome and the shaping of its metabolic functions, and gestational age and feeding mode are considered key determinants of this process.Objective To compare the fecal gut microbiome and metabolome between preterm and full-term infants under breastfeeding and non-breastfeeding conditions, thus exploring the effects of feeding mode and gestational age on early-life gut microbial ecology and metabolic features.Methods Infants were assigned into four groups: breastfeeding preterm infants (BPI), non-breastfeeding preterm infants (NBPI), breastfeeding full-term infants (BTI), and non-breastfeeding full-term infants (NBTI), with 10 infants per group. The 16S rRNA gene sequencing and untargeted metabolomics analysis were performed. Alpha/beta diversity analyses, differential abundance testing, and linear discriminant analysis effect size (LEfSe) were performed to identify key microbial taxa. Partial least squares-discriminant analysis (PLS-DA), volcano plots, and KEGG pathway enrichment were employed to determine differential metabolites and functional pathways, followed by microbiome-metabolite association network analysis.Results The 16S rRNA gene sequencing showed that the gut microbiome in the BTI group was dominated by Actinomycetota, Bifidobacterium (45.98%), and Bifidobacterium breve, forming a typical “breastfeeding-type” structure. The BPI group showed enrichment of Pseudomonadota and Streptococcus, with attenuated dominance of Bifidobacterium. Among non-breastfed infants, the NBTI group was dominated by Enterococcus (59.20%), whereas the NBPI group showed a fluctuating gut microbiome. Untargeted metabolomics further revealed functional differences consistent with these compositional patterns. KEGG enrichment indicated that differential metabolites were mainly involved in amino acid metabolism, lipid metabolism, bile acid-related pathways, and carbohydrate digestion and absorption. At the level of key metabolites, the BPI group showed significant enrichment of metabolites related to antioxidant and immune support, such as glutathione and vitamin D sulfate conjugates; the BTI group enriched long-chain polyunsaturated fatty acids and indole-derived metabolites; and non-breastfeeding groups generally exhibited accumulation of fermentable carbohydrates and specific bile acid derivatives. Correlation analysis confirmed a strong positive association between Bifidobacterium and indole-related metabolite outputs in breastfeeding groups, whereas microbiome–metabolite networks were looser in non-breastfeeding groups.Conclusion Both gestational age and feeding mode are related to differences in the early gut microbiome structure and related metabolic characteristics of infants, with the association between feeding mode and metabolic profile differences being more prominent. Gestational age may affect the establishment pattern of dominant microbial taxa in the context of breastfeeding. As a pioneering exploratory study, this study preliminarily reveals the specificity of gut microbiome structure and functional metabolism in different populations of infants, providing a basis for subsequent prospective cohort validation, mechanism research, and early nutritional intervention optimization.

Abstract:Objective To investigate the efficacy of postbiotics prepared with Bifidobacterium animalis CCFM1155, a strain previously identified for its ability to enhance nuclear factor erythroid 2-related factor 2 (Nrf2) secretion in human immortalized epidermal cells, in alleviating skin aging.Methods A mouse model of skin aging was established by daily subcutaneous injection of D-galactose (500 mg/kg) for eight weeks (n=6 per group). The model mice were then orally administered either heat-inactivated cells (1155B) or concentrated fermentation supernatant (1155T) of CCFM1155 cells at a dose equivalent to 5×10⁹ CFU of live bacteria.Results The 1155B was the primary postbiotic component exerting anti-skin aging effects, whereas the 1155T group showed no significant effect. Compared with the model group, the 1155B treatment ameliorated skin aging phenotypes by improving the skin tissue structure, increasing the epidermal thickness (P<0.05) and dermal collagen fiber density (P<0.01), and decreasing the expression of the senescence marker protein p21 and the activity of senescence-associated β-galactosidase (P<0.001). In addition, the 1155B treatment enhanced the antioxidant defense system of the skin by elevating the activities of total superoxide dismutase (P<0.05) and catalase (P<0.01), as well as the content of reduced glutathione (P<0.01), while lowering the malondialdehyde level (P<0.01). Mechanism studies revealed that the 1155B upregulated both the mRNA (P<0.05) and protein (P<0.001) levels of Nrf2 in the skin. Consequently, it promoted the transcription of key downstream antioxidant molecules, including heme oxygenase-1, peroxiredoxin, thioredoxin 1, and the glutamate-cysteine ligase catalytic subunit, thereby activating the downstream signaling pathway of Nrf2.Conclusion The inactivated cells of B. animalis CCFM1155 alleviates skin aging by activating the Nrf2 signaling pathway. These findings offer a novel perspective on postbiotic regulation of the Nrf2 pathway and lay a theoretical foundation for developing safe and effective anti-skin aging products.

Abstract:Objective To investigate the effects of diets with different concentrate to roughage ratios on the growth performance, rumen fermentation parameters, and serum metabolites of Yajiang snow cattle.Methods Twenty-seven healthy individuals of Yajiang snow cattle with similar body weights were selected and randomized into three groups (n=9). Cattle in the three groups were fed diets with the concentrate to roughage ratios being 40:60 (C40 group), 50:50 (C50 group), and 60:40 (C60 group). The pre-experimental period lasted for 7 days, and the experimental period lasted for 45 days. The growth performance, nutrient digestibility, volatile fatty acid content and microbiota structure in the rumen fluid, and serum metabolite content of the Yajiang snow cattle were measured.Results With the increase in the dietary concentrate to roughage ratio, the average daily gain and average daily feed intake of Yajiang snow cattle increased, while the apparent digestibility of crude protein and acid detergent fiber decreased (all P<0.05). As the dietary concentrate to roughage ratio increased, the pH value, acetate content, and acetate/propionate ratio in the rumen fluid decreased, whereas the propionate content increased (all P<0.05). Different concentrate to roughage ratios altered the microbiota structure in the rumen fluid of Yajiang snow cattle (P<0.05 or P<0.01). Specifically, the C60 group had lower relative abundance of Bacteroidota, Verrucomicrobia, Ruminococcus, Clostridium, Butyrivibrio, Pseudobutyrivibrio, and Bibersteinia but higher relative abundance of Bacillota, Prevotella, Succiniclasticum, and Selenomonas than the C40 group. Furthermore, different concentrate to roughage ratios changed the serum metabolite content of Yajiang snow cattle (P<0.05 or P<0.01). Specifically, the C40 group had significantly higher content of 5,6-dihydrouridine, 2-hydroxybutyric acid, and nitrilotriacetic acid but significantly lower content of 6-hydroxy-2-methylindole, N-acetylneuraminic acid, 2,6-dihydroxybenzoic acid, and taurodeoxycholic acid in the serum than the C50 and C60 groups.Conclusion Under the conditions of this experiment, different dietary concentrate to roughage ratios had significant effects on the production performance, rumen fermentation parameters, and serum metabolites of Yajiang snow cattle. Compared with the high roughage diet, the high concentrate diet significantly improved the production performance and promoted the rumen fermentation, while reducing the diversity of the rumen microbiota in Yajiang snow cattle.

Abstract:Objective To investigate the specific role and molecular mechanism of the host protein protein disulfide isomerase A4 (PDIA4) during bovine viral diarrhea virus (BVDV) infection, thus providing a theoretical basis for elucidating the role of this protein in viral replication and developing novel prevention and control strategies.Methods Western blotting and qPCR were employed to analyze PDIA4 expression in Madin-Darby bovine kidney (MDBK) cells following BVDV infection. The expression levels of autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62) were measured by Western blotting and laser confocal microscopy. MDBK cell lines with pdia4 knockdown and overexpression were constructed. The effects of pdia4 on LC3 and p62 expression were examined, and the autophagic flux was evaluated by a tandem GFP-mRFP-LC3 reporter system. Cells were subjected to starvation or treated with bafilomycin A1 (BafA1), followed by BVDV infection. Viral replication was assessed by measuring BVDV mRNA levels through qPCR and double-stranded RNA (dsRNA) levels through immunofluorescence staining.Results BVDV infection significantly upregulated the endogenous expression of PDIA4. The LC3 level increased progressively with infection time, whereas the p62 level showed an initial increase followed by a decrease. The cell lines with pdia4 knockdown and overexpression were successfully established. pdia4 knockdown significantly increased both LC3 and p62 levels, whereas pdia4 overexpression increased the LC3 level and decreased the p62 level. Under starvation or BafA1 treatment, pdia4 knockdown inhibited BVDV replication, whereas pdia4 overexpression promoted BVDV replication.Conclusion BVDV infection upregulates host PDIA4 expression. PDIA4 promotes BVDV replication by activating the autophagic flux through regulation of LC3 and p62. These findings provide new insights into the pathogenic mechanism of BVDV and offer a theoretical basis for developing targeted antiviral strategies.

Abstract:Porcine deltacoronavirus (PDCoV), a major porcine intestinal coronavirus, has caused enormous economic losses to the pig industry. Up to now, there is no commercial vaccine available.Objective To obtain the epidemic strain of PDCoV and study its biological characteristics and pathogenicity, thereby providing biological materials for the development of an effective vaccine against PDCoV as soon as possible.Methods LLC-PK1 cells were used to isolate the virus from PDCoV-positive samples from a pig farm in Gansu Province. The virus was identified by observation of cytopathic effect (CPE), indirect immunofluorescence assay (IFA), and whole genome sequencing.Results After blind passage of the PDCoV-positive LLC-PK1 cells to P6, the cells became larger and rounded, and then shrunk and shed into single or clustered dense particles, typical features of CPE. The IFA results confirmed successful isolation of the PDCoV strain CHN/GSYD/2023. The whole genome sequencing and phylogenetic analysis showed that CHN/GSYD/2023 isolated in this study was more closely related to the strain from China and was located on a different clade from the strain CHN/XJYN/2016 preserved in our laboratory. After 5-day-old piglets were infected with CHN/GSYD/2023, 100% (5/5) of piglets became diseased. Autopsy and histopathology of piglets showed thinning small intestinal wall, a large amount of effusion in the intestinal cavity, ileum intestinal villus necrosis and other pathological damage.Conclusion A PDCoV strain CHN/GSYD/2023 was successfully isolated from PDCoV-positive samples in a pig farm in Gansu. The strain can be stably proliferated and passaged in LLC-PK1 cells, and it is highly pathogenic to suckling piglets.

Abstract:Objective To investigate the genomic phylogeny as well as the in vivo and in vitro infection and replication characteristics of the tick-borne parainfluenza virus type 5 (strain PIV5-JC12) recently isolated from Yunnan Province.Methods The strain PIV5-JC12 was identified through cytopathic effect (CPE) observation, negative staining electron microscopy, and an indirect immunofluorescence assay (IFA) based on the P protein. We determined its optimal culture temperature by comparing viral proliferation efficiency at 33 ℃ and 37 ℃ in Vero cells. A phylogenetic tree based on the whole genome was constructed via the maximum likelihood method to elucidate the molecular evolutionary characteristics of this strain. After infection of six representative cell lines of different origins (Vero, MDCK, HeLa, Huh7.5, MRC-5, and BHK-21), CPE observation, RT-qPCR, and tissue culture infectious dose 50% (TCID₅₀) assays were employed to evaluate the virus replication kinetics. The in vivo pathogenicity of PIV5-JC12 was evaluated in Kunming (KM) and C57BL/6J mice. Mice were infected intranasally with high and low doses (2×10⁷ TCID₅₀ and 2×10⁶ TCID₅₀) and monitored for body weight and survival rate. The viral loads in various tissue and organ samples were measured at the time points of 1, 4, 7, and 10 days post-infection (dpi), and histopathological changes were examined.Results PIV5-JC12 induced CPEs, as manifested by the rounding and detachment of Vero cells. Irregular spherical particles with diameters of 50-200 nm were observed, and the specific P protein was identified by immunofluorescence. The tick-borne strain PIV5-JC12 showed the highest sequence identity with the tiger-borne strain PIV5 (OQ236149.1). Viral replication and proliferation in Vero cells were more efficient at 37 ℃ than at 33 ℃. PIV5-JC12 infected all the six cell lines of human, murine, simian, and canine origins. However, the CPEs varied among the cell lines, being more pronounced in Vero, MDCK and BHK-21 cells. Higher RNA replication efficiency was observed in Vero, MDCK, and HeLa cells. In vivo infection studies revealed differential susceptibility of KM and C57BL/6J mice to PIV5-JC12. The body weight loss (5%) was only observed in the high-dose infected C57BL/6J mice at 10 dpi. Lung viral loads in both mouse lines reached 10⁴-10⁵ copies/g, while no infectious virus was detected in the lung tissue at any of the time points examined. Histopathological staining analysis of lung tissue at the early stage (4 dpi) and late stage (10 dpi) of infection showed no significant pathological damage.Conclusion PIV5-JC12 isolated from tick samples from Yunnan Province shows broad cellular tropism and low pathogenicity in mice. The findings provide a basis for research on the pathogenic characteristics of PIV5 and its potential application as a vaccine vector.

Abstract:Objective To investigate the ability of enterovirus A71 (EV-A71) to traverse the blood-brain barrier (BBB) and infect human cerebral organoids, as well as the impact of EV-A71 on BBB integrity following retrograde invasion into the central nervous system via neural routes, and to characterize the infection profiles of EV-A71 in the BBB and human cerebral organoids and the associated inflammatory responses.Methods An in vitro BBB model was established by seeding human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes into a Transwell system. Human cerebral organoids were generated from human induced pluripotent stem cells (hiPSCs), and a BBB-human cerebral organoid co-culture model was subsequently established. EV-A71 at three titers (10², 10⁴, and 10⁶ TCID₅₀) was used to infect each cell type of the BBB to evaluate viral replication kinetics and cytopathic effects (CPE) across different cell types. Subsequently, the BBB-human cerebral organoid co-culture model was infected with 10⁴ TCID₅₀ EV-A71 using two distinct routes: (1) an apical inoculation strategy, in which EV-A71 was directly applied to the human brain microvascular endothelial cell layer; and (2) a retrograde infection strategy, in which human cerebral organoids were first infected and subsequently co-cultured with the BBB model. Viral RNA copy numbers in culture supernatants and cells were quantified by RT-qPCR. BBB integrity was assessed by measuring transendothelial electrical resistance, and inflammatory responses were evaluated by determining the expression levels of inflammatory cytokines in human cerebral organoids using RT-qPCR.Results EV-A71 efficiently replicated in human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes, inducing pronounced CPE. Among these cell types, astrocytes were the most susceptible to EV-A71 infection, showing the most rapid progression of cytopathic changes, followed by brain vascular pericytes. Human brain microvascular endothelial cells were relatively less susceptible, requiring higher viral titers to establish effective infection. Under apical inoculation conditions, EV-A71 replicated within the BBB, compromised BBB integrity, traversed the barrier to infect human cerebral organoids, and significantly activated inflammatory responses. In the retrograde infection model, EV-A71 replicated within human cerebral organoids, preferentially targeted neurons and astrocytes, triggered cellular inflammatory responses, and subsequently invaded the BBB in a retrograde manner, resulting in compromised structural integrity of the BBB.Conclusion EV-A71 can traverse the BBB to infect human cerebral organoids and activate inflammatory responses, and can also infect human cerebral organoids first and subsequently disrupt BBB integrity in a retrograde manner. This study systematically characterizes the infection dynamics of EV-A71 in an in vitro BBB-human cerebral organoid model, provides a robust experimental model for in-depth analysis of the mechanisms underlying EV-A71 invasion of the central nervous system, and lays a research foundation for future investigations into viral pathogenesis and antiviral therapeutic development.

Abstract:Pseudomonas aeruginosa is a widely distributed Gram-negative opportunistic pathogen that poses a serious threat to immunocompromised individuals, often leading to severe clinical infections. Sodium houttuyfonate (SH), the main active derivative of the Chinese medicinal herb Houttuynia cordata, has been widely used for anti-infection therapy. Recent studies suggest that the efficacy of SH against antibiotic-resistant pathogens extends beyond direct antibacterial effects to include delicate regulation of the host immune system.Objective To investigate the role of SH in promoting M1 polarization of alveolar macrophages against P. aeruginosa infection.Methods A mouse model of acute pneumonia caused by P. aeruginosa was established via intratracheal intubation. Hematoxylin and eosin (HE) staining was used to evaluate the histopathological changes in the lung tissue. Bacterial load in the lung tissue was determined by the plate counting method, and the expression of Toll-like receptor 4 (TLR-4) in the lung tissue was analyzed by immunohistochemistry (IHC). In the cell experiment, MH-S alveolar macrophages were co-cultured with SH for 6 h to induce M1 polarization. Polarization markers CD86 and CD206 were analyzed by immunofluorescence and flow cytometry, and phagocytic capacity was evaluated with P. aeruginosa at 1×10⁵ CFU/mL. The mRNA levels and inflammation levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) were measured by RT-qPCR and ELISA, respectively. The expression of proteins in the TLR-4/MyD88/NF-κB signaling pathway was analyzed by Western blotting. To decipher the mechanism, we constructed TLR-4 overexpression and knockdown cell models to confirm SH regulation of this pathway and related inflammatory factors via TLR-4.Results The in vivo experiment results indicated that SH significantly reduced P. aeruginosa proliferation in the mouse lung tissue. Both HE staining and IHC results demonstrated that pulmonary inflammation in the treatment group was alleviated compared with the model group, suggesting that SH may enhance host immune clearance and thereby mitigate acute pulmonary infection. The cell experiment showed that SH promoted early M1 polarization of MH-S cells and enhanced their phagocytosis of P. aeruginosa. Western blotting further confirmed activation of the TLR-4/MyD88/NF-κB pathway with upregulated protein expression after SH treatment.Conclusion SH can activate the TLR-4/MyD88/NF-κB pathway to induce M1 polarization of alveolar macrophages during early infection, thereby exerting significant phagocytic and anti-infective effects.

Abstract:Yeast β-glucan is a well-established immunomodulatory agent. Studies have demonstrated that compared with vegetative yeast cells, β-glucan exposed on the surface of mutant yeast spores exhibits enhanced immunostimulatory effects.Objective To screen and identify yeast mutants with enhanced ability to activate the mammalian immune system.Methods Two yeast mutants (osw1Δ and mum3Δ) exposing β-glucan on the spore wall were constructed, and their immunostimulatory activity was evaluated via the macrophage stimulation assay and a mouse model. The role of the Dectin-1 signaling pathway was verified through small interfering RNA-mediated gene knockdown experiments.Results The spore lysates of osw1Δ and mum3Δ induced the production of inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in macrophages. Their immune activation was mainly dependent on the Dectin-1-mediated signaling pathway. In vivo studies showed that after oral administration of the osw1Δ spore lysate, the levels of IL-6 and TNF-α in the mouse serum increased by 1.8-fold and 1.7-fold, respectively, compared with those after oral administration of the wild-type spore lysate. Moreover, the osw1Δ spore lysate exerted a significant tumor-suppressive effect.Conclusion The spores of osw1Δ exhibit strong immune-activating effects. This mutant could be a candidate for the development of novel immunomodulators.

Abstract:Objective The heat-labile enterotoxin (LT) is widely recognized as a potent mucosal immunoadjuvant. However, the mechanisms underlying its interaction with host epithelial cells remain incompletely understood, which makes it difficult to separate its toxicity from its adjuvant activity. This limitation has severely hampered its clinical application. Therefore, this study aims to elucidate the differential regulatory effects of LT and its A (LTA) and B (LTB) subunits on epithelial cells, as well as the mechanism underlying the initiation of their initial adjuvant activity.Methods The biologically active LTB, LTA, and its mutant LTA(R192G) were prepared via prokaryotic expression. Using the human small intestinal epithelial cell line FHs 74 Int and a mouse jejunal ex vivo intestinal segment model, we examined cell viability and apoptosis, analyzed inflammatory cytokine expression, and investigated changes in the NF-κB and NLRP3 inflammasome pathways.Results The three prepared proteins all possessed biological activity and could be effectively internalized by cells. LTB primarily induced early apoptosis, significantly up-regulated the expression of IL-6, IL-8, IL-1β, and TNF-α, and activated the NF-κB pathway. LTA triggered significant pyroptosis, specifically up-regulated the expression of IL-18 and IL-1β, and activated the NLRP3 inflammasome pathway. The mutant LTA(R192G) exerted weakened effects but could still activate the NLRP3 pathway.Conclusion The adjuvant activity of LT stems from its subunits activating immune responses in epithelial cells through different mechanisms: LTB primarily promotes pro-inflammatory cytokine production via the NF-κB pathway, while LTA mainly relies on the NLRP3 pathway to induce IL-1β/IL-18 secretion and pyroptosis. This discovery not only reveals the initial molecular events of LT-initiated mucosal immunity, but more importantly, lays a solid theoretical foundation for developing novel, safe, and efficient mucosal vaccine adjuvants through targeted modification of LTA and LTB subunits.

Abstract:Objective Ergothioneine (EGT), a sulfur-rich derivative of histidine, is utilized in the food, pharmaceutical, and cosmetic industries. However, large-scale production of EGT faces challenges due to the high costs and inefficiency of conventional chemical synthesis and extraction techniques. This study aims to engineer Saccharomyces cerevisiae to provide a microbial platform for EGT biosynthesis.Methods The biosynthetic pathway for EGT was reconstructed in S. cerevisiae by heterologously expressing Egt1 from Neurospora crassa and Egt2 from Claviceps purpurea. To overcome the metabolic bottlenecks related to precursor supply, we optimized the upstream pathways for histidine, cysteine, methionine, and S-adenosylmethionine to enhance the flux toward EGT synthesis. Fermentation performance of the engineered strain was assessed in both shake flasks and a 5-L bioreactor.Results The engineered S. cerevisiae strain produced 312.8 mg/L of EGT in shake flask fermentation. In a 5-L bioreactor, the strain achieved the EGT titer of 1 312.2 mg/L after 168 h, with the productivity of 7.8 mg/(L·h).Conclusion This study presents a metabolic engineering strategy for producing EGT in S. cerevisiae. The approach not only significantly improves EGT biosynthesis but also serves as a reference for microbial production of other compounds.

Abstract:Raspberry ketone (RK) is a high-value natural aromatic compound widely used in food, flavors and fragrances, and daily chemical products. Traditional plant extraction methods are constrained by raw material availability and high separation costs, while chemical synthesis suffers from high environmental burden and difficulties in meeting market demand for natural-source products. Biocatalytic synthesis represents the core direction for its green manufacturing. However, existing systems remain limited by bottlenecks such as insufficient enzyme activity and substrate tolerance, poor stability of cofactor regeneration systems, and product titers insufficient for industrial needs.Objective To construct an efficient, stable, and scalable biocatalytic synthetic system for RK, overcoming existing technical bottlenecks.Methods A flavin-independent ene-reductase from Arabidopsis thaliana, AtQOR, was screened and coupled with formate dehydrogenase from Lactobacillus buchneri, LbFDH, to construct an NADPH self-recycling dual-enzyme cascade system. By optimizing vector copy number and gene expression order, an engineered strain co-expressing both enzymes was constructed, and the optimal strain, Escherichia coli 02, was selected. Key reaction conditions for whole-cell biocatalysis were systematically optimized, and a scale-up experiment was performed in a 1 L fermenter using a fed-batch strategy. The product was quantitatively analyzed and structurally verified using gas chromatography (GC) and nuclear magnetic resonance (NMR) spectroscopy.Results The selected AtQOR exhibited a specific activity of 10.35 U/mg in catalyzing the conversion of p-hydroxybenzylidene acetone to RK. Under optimal reaction conditions, E. coli 02 produced 37.88 g/L of raspberry ketone from 40 g/L p-hydroxybenzylidene acetone in 12 h, with a conversion rate of 93.54%. Using a fed-batch strategy in a 1 L fermenter, the RK titer reached 54.32 g/L in only 10 h, with a conversion rate of 89.43%, and the product was confirmed to be a high-purity target compound.Conclusion The dual-enzyme cascade catalytic system constructed in this study substantially overcomes the existing titer bottleneck in RK biosynthesis, providing an efficient, stable, and scalable technological solution for its industrial green manufacturing.

Abstract:Aeromonas veronii is a Gram-negative pathogenic bacterium that causes various diseases in aquatic animals and humans, posing a serious threat to aquaculture and public health. The type Ⅵ secretion system (T6SS) is a key virulence factor determining the pathogenicity of A. veronii. It is known that the bacterial enhancer-binding protein (bEBP) VasH is responsible for regulating the T6SS function, while whether this regulatory relationship exists in A. veronii remains unknown. Objective To elucidate the impacts of VasH on T6SS expression and function in A. veronii, thereby providing a theoretical basis for deciphering the mechanism of T6SS-mediated pathogenicity of A. veronii and for the subsequent prevention and control of A. veronii infections. Methods With A. veronii C4 as the wild-type strain, the VasH-deficient mutant strain Δ vasH and the complemented strain Δ vasH/p- vasH were constructed via a homologous recombination strategy. RT-qPCR was employed to measure the relative expression levels of T6SS genes in each strain to clarify the effect of VasH on T6SS gene expression. Growth curve establishment, transmission electron microscopy (TEM) for observing bacterial morphology, and the crystal violet assay for biofilm quantification were performed to determine the influences of VasH on pathogenic characteristics. An in vitro bacterial competition assay and a zebrafish model for determining the median lethal dose (LD ₅₀) were employed to assess the comprehensive effects of VasH on bacterial competitiveness and pathogenicity. Results The VasH-deficient mutant strain Δ vasH and the complemented strain Δ vasH/p- vasH were successfully constructed. The deletion of VasH resulted in a decrease ( P<0.05) in the expression of genes encoding both T6SS structural and effector proteins, indicating that VasH was responsible for regulating T6SS gene expression. The deletion of vasH did not significantly affect the growth or surface morphology/structure of A. veronii. However, it led to a significant increase in the biofilm formation and a significant decrease in in vitro bacterial competitiveness. The zebrafish infection assay showed that the LD ₅₀ of Δ vasH was 1.58×10 ¹¹, which was 2.15 times that (7.34×10 ¹⁰) of the wild type. Moreover, at an infection concentration of 5×10 ⁸ CFU/mL, the death rate of zebrafish decreased from 33.3% in the wild-type infection group to 0 following infection with Δ vasH, indicating that deletion of vasH attenuated the virulence of A. veronii. Conclusion In A. veronii, VasH positively regulates T6SS gene expression. It may not be involved in regulating the growth state or external morphology of the pathogen but significantly influences the biofilm formation and competitiveness of A. veronii, thereby modulating the overall virulence during infection of the zebrafish host. This study provides essential research tools and lays a preliminary foundation for further exploration of the molecular mechanisms by which VasH mediates pathogenicity through regulating T6SS activity and function in A. veronii.

Abstract:Objective To investigate the effects of luxS deletion on the biofilm formation and stress tolerance of Listeria monocytogenes and to elucidate the role of luxS in regulating environmental adaptability of this pathogen.Methods With Listeria monocytogenes ATCC 19112 as the parental strain, a luxS-deleted mutant, a complemented strain, and an empty-vector control strain were constructed. Growth curve analysis, environmental stress tolerance assays and biofilm formation assays were performed to comparatively analyze phenotypic differences among the wild-type, mutant, and complemented strains.Results Compared with the wild-type strain, the luxS-deleted mutant exhibited reduced adaptability under adverse conditions, including low temperature, high osmotic pressure, oxidative stress, and acid stress. Its biofilm-forming ability decreased after 24 h and 48 h of incubation (P<0.01), accompanied by a reduction in the production of extracellular polymeric substances in the biofilm matrix. Further transcriptional analysis revealed that the expression levels of agrA, agrB, lmo2504, and sigB were downregulated (P<0.05), whereas that of ladR was upregulated (P<0.05) following luxS deletion. Under disinfectant stress, MTT assay showed that the metabolic activity of the luxS-deleted strain was lower than that of the wild-type and complemented strains after 48 h of biofilm cultivation (P<0.05). After 72 h, no significant differences were observed among the strains.Conclusion These findings demonstrate that luxS plays a critical role in regulating the environmental adaptability of L. monocytogenes and provide a novel theoretical basis for the development of future control and prevention strategies targeting this pathogen.

Abstract:Objective To characterize the successional dynamics of bacterial and fungal communities during leaf litter decomposition and elucidate the associations between keystone taxa and substrate component losses, thereby advancing our understanding of microbial regulation during litter decomposition in forest ecosystems.Methods We established an indoor microcosm with the leaf litter from Alnus cremastogyne to track microbial community succession during the litter decomposition. High-throughput sequencing, chemical composition analysis, and bacterial-fungal cross-domain co-occurrence network analysis were integrated to characterize microbial community dynamics, identify keystone taxa, and test their associations with loss rates of major chemical components across four decomposition stages (initial, 45%, 75%, and 90% mass loss).Results At the phylum level, bacterial and fungal community composition showed only minor changes in relative abundance across stages, with Pseudomonadota (relative abundance of 50%- 80%) dominating bacterial communities and Ascomycota (>95%) dominating fungal communities. In contrast, pronounced stage-dependent succession was observed at the genus level. Decomposition stage accounted for substantial variations in bacterial (R²=0.573, P<0.001) and fungal (R²=0.377, P<0.001) community structures. With the progression of decomposition, cross-domain networks exhibited increased nodes and connectivity and shifted from loose to modular structures, with positive correlations consistently exceeding negative correlations. The number of keystone taxa increased over time and became progressively dominated by fungi. During the first three decomposition stages, the number of bacterial nodes accounted for approximately 75% of total nodes, whereas at the 90% decomposition stage, the number of fungal nodes increased markedly (from 103 to 320), resulting in comparable proportions of bacterial and fungal nodes. At the 45% decomposition stage, keystone bacterial taxa were primarily associated with losses of non-structural components and hemicellulose, whereas at the 75% and 90% decomposition stages, keystone fungal taxa were more closely associated with cellulose and lignin losses. Functional predictions further supported this stage-specific division of labor, indicating stronger potential of structural carbon degradation at the late stages of decomposition.Conclusion Bacteria and fungi jointly participate in A. cremastogyne leaf litter decomposition with temporally differentiated contributions. Bacteria play a more prominent role during early and middle stages, whereas fungi become increasingly important at late stages. These findings indicate coordinated shifts in microbial community structure and resource utilization rather than simple taxonomic replacement, contributing new insights into microbial regulation during litter decomposition.

Abstract:Objective The gut microbiota plays crucial roles in host energy metabolism, immune regulation, and environmental adaptation. However, seasonal variations in the structure, function, and assembly mechanisms of the gut microbiota in the plateau zokor (Eospalax baileyi) remain poorly understood.Methods This study employed Illumina high-throughput sequencing technology to perform 16S rRNA (V3-V4 region) sequencing on 17 gastrointestinal content samples of plateau zokors, thus investigating the effects of season (spring vs. winter) on the diversity, function, and assembly processes of the gut microbiota.Results The alpha diversity and richness of the gut microbiota in plateau zokors were higher in spring than in winter (P<0.05), and the gut microbiota structure in winter exhibited extremely significant differences (P<0.001). At the phylum and genus levels, the relative abundance of certain dominant microbial taxa differed between seasons (P<0.05). PICRUSt functional prediction revealed that at Level 1, metabolism was the dominant pathway, with the genetic information processing, environmental information processing, human diseases, and organismal systems pathways being enriched in the spring group compared with the winter group (P<0.05). Further analysis of metabolic pathways at Level 2 indicated that pathways related to carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, metabolism of other amino acids, and metabolism of terpenoids and polyketides were higher in spring than in winter (P<0.05). The neutral community model (NCM) showed more extensive dispersal of the gut microbiota among individuals in winter (Nm=228.75) than in spring (Nm=216.68). The normalized stochasticity ratio (NST) was less than 0.5 in the spring group, indicating dominance by deterministic processes, while it was greater than 0.5 in the winter group, suggesting dominance by stochastic processes, with significant differences between groups (P<0.001). Further iCAMP analysis revealed that drift and dispersal limitation were the primary ecological processes governing microbial community assembly in both seasons, and the relative contributions of these ecological processes differed between spring and winter (P<0.001).Conclusion Season significantly affects the structure, composition, function, and assembly mechanisms of the gut microbiota in plateau zokors. During winter, plateau zokors may save energy by reducing the metabolic activity and information-processing functions of the gut microbiota. In addition, plateau zokors may adjust taxa abundance of the gut microbiota to cope with environmental changes and maintain intestinal homeostasis. These findings provide important insights into the mechanisms by which wild plateau rodents adapt to high-altitude and cold environments from the gut microbiota.

Abstract:Microbial antioxidant functions hold significant application potential in the development of holistic health resources, yet their spatial distribution patterns across continuous ecological gradients remain poorly understood.Objective To analyze the diversity and spatial distribution characteristics of antioxidant function-related genes in prokaryotes within the Lasha Mountain watershed.Methods A spatially exhaustive nine-grid sampling strategy was employed, with 117 sampling points established across the watershed and its 12 sub-basins. PICRUSt2 was used for functional prediction based on high-throughput sequencing data of the 16S rRNA gene.Results A total of 38 genes associated with antioxidant compound synthesis were identified and categorized into 12 functional groups. The diversity and total abundance of antioxidant function-related genes were unevenly distributed within the Lasha Mountain watershed, with the downstream region exhibiting higher gene diversity and abundance. The genes associated with non-enzymatic antioxidant compounds dominated both in functional category richness (7 categories) and abundance (0.005 270-0.007 657), with glutathione and thioredoxin genes exhibiting the highest abundance. The overall abundance variation of individual genes (CV=125.85) primarily originated within sub-basins (CV=77.23). After the antioxidant function-related genes were classified by synthesized compounds, the pattern of variation reversed.Conclusion The antioxidant function-related genes of prokaryotes exhibit spatial distribution differences in the watershed, and the downstream area—characterized by multiple coupled stress conditions—is a hotspot for the diversity and total abundance of such genes. This study provides an ecological basis for the targeted screening of high-quality antioxidant strains.

Abstract:Objective To explore genes related to the regulation of yeast spore germination, we previously screened for mutants defective in maintaining the quiescent state of spores.Methods Using the temperature-sensitive yeast mutant J33 with defective spore quiescence maintenance as the research material, heterozygous diploid strains were constructed through tetrad dissection and haploid fusion, and genetic segregation analysis was performed to clarify the genetic regulatory pattern of mutant phenotypes. The yeast genomic library complementation screening was applied to identify candidate mutant genes, and gene cloning and sequence alignment were conducted to confirm the mutation site. Furthermore, recombinant plasmids carrying wild-type and mutant TIP20 were constructed for functional complementation assays, and site-directed mutagenesis was used to verify the correlation between TIP20 mutation and strain phenotypes.Results Functional complementation experiments demonstrated that introduction of the wild-type TIP20 restored the growth at 37 ℃ and the normal spore germination phenotype of J33, while the mutant TIP20 and empty vector had no such effect. Site-directed mutagenesis analysis further confirmed that this single-base mutation was T1219G, a direct cause of the TS phenotype and defective spore germination of J33.Conclusion TIP20 is involved in the regulation of yeast spore germination. TIP20 encodes a subunit of the tethering complex involved in endoplasmic reticulum-Golgi retrograde transport. However, TIP20 is likely a multifunctional protein, and its mammalian homologue RINT1 has been reported to regulate the cell cycle checkpoint. Thus, TIP20 may also be involved in regulating the quiescent state of yeast cells.

Abstract:The efficient conversion of lignocellulosic biomass is at the core of ensuring the economic feasibility of biorefineries, where the comprehensive utilization of xylose serves as a determinant of overall conversion efficiency. Pichia kudriavzevii has demonstrated significant potential in industrial bioprocessing owing to its tolerance to low pH, high temperatures, and environmental stressors. However, its innate deficiency in xylose assimilation severely restricts its application in biomass valorization.Objective To elucidate the molecular mechanisms underlying the silencing of xylose metabolism in P. kudriavzevii E1, thereby providing a theoretical basis for the bioconversion of lignocellulosic feedstocks.Methods The xylose assimilation capacity of P. kudriavzevii E1 was evaluated, and its genome was analyzed to identify the genes and metabolic bottlenecks associated with xylose assimilation. Comparative transcriptomics was employed to characterize the differential expression of metabolic genes before and after the introduction of a xylose transporter. Furthermore, the heterologous expression of genes involved in efficient xylose metabolism was performed to verify specific rate-limiting steps within the pathway.Results Bioinformatics analysis, coupled with the functional restoration of xylose uptake via heterologous transporter expression, confirmed that the lack of high-affinity xylose transporters was the primary limiting factor for xylose assimilation in P. kudriavzevii E1. Although three genes—PkXYL1, PkXYL2, and PkXKS1—encoding core enzymes of the xylose redox pathway were natively present in the P. kudriavzevii E1 genome, in vitro enzymatic assays revealed that the low relative activity of PkXR was a critical cause of substrate accumulation and slow xylose metabolism. Comparative transcriptomics of the engineered strain P. kudriavzevii E1-Xpg4562 indicated that yeast cells underwent profound metabolic reprogramming in xylose-containing media, preferentially activating ribosome biogenesis and oxidative phosphorylation. However, the significant downregulation of TAL1 and the insufficient transcriptional response of genes in the pentose phosphate pathway (PPP) resulted in inefficient PPP flux. This prevented the effective redirection of carbon flux into glycolysis, thereby obstructing downstream xylose metabolism. Finally, quantification of the expression of key xylose metabolism genes identified by transcriptomics further demonstrated that the uncoordinated transcriptional regulation of essential downstream genes hindered overall metabolic efficiency.Conclusion The silencing of xylose metabolism in P. kudriavzevii E1 results from the combined effects of deficient substrate transport, low endogenous catalytic activity, and uncoordinated transcriptional regulation. This study provides a crucial theoretical foundation for the precision engineering of xylose metabolic pathways in non-conventional industrial yeasts.

Abstract:Microbial fertilizers are an important component of green agricultural inputs, playing a key role in reducing chemical fertilizer use, improving arable land quality, and mitigating agricultural emissions. Using the registration certificate data of microbial fertilizers in China from 2000 to 2025, this paper systematically analyzes the structural characteristics and regional patterns of China’s microbial fertilizer industry and takes Guangdong Province as a case to examine the development bottlenecks of this industry in Guangdong. The study finds that China’s microbial fertilizer industry has gone through three stages: slow start, steady growth, and explosive expansion, forming a clustered pattern centered on Shandong, Hebei, Heilongjiang, and Henan provinces. However, three major structural contradictions are becoming increasingly prominent. Specifically, the products are highly concentrated in microbial inoculants, bioorganic fertilizers, and compound microbial fertilizers; microbial strain resources are highly homogenized; and enterprises are scattered, small, and weak. The number of registration certificates in Guangdong Province (334) accounts for only 2.83% of the national total (11 813) and is merely 13.82% of that of Shandong Province (2 416), which ranks first in China. Nearly 40% of enterprises are in a state of one certificate for one product, indicating a severe mismatch between the industrial scale and Guangdong’s status as a major agricultural province. In response to the above problems, this paper proposes recommendations from six aspects: elevating the policy position of microbial fertilizers, mining and protecting microbial strain resources, reforming registration management, optimizing the enterprise ecosystem, bridging the gap in technology transfer, and exploring the paths tailored for Guangdong, aiming to provide a reference for the high-quality development of the microbial fertilizer industry in China and especially Guangdong Province.

Abstract:Objective To efficiently express and purify wild-type and three active site-deleted variants (ENO1-M1, ENO1-M2, and ENO1-M3) of ENO1 through the insect cell/baculovirus expression system, thus providing a theoretical basis for subsequent functional research, antibody development, and inhibitor screening.Methods Wild-type and active site-deleted genes of ENO1 with a C-terminal 6×His tag were cloned into the pFastBac-HTB vector via molecular cloning and then transformed into DH10Bac competent cells to obtain recombinant Bacmid through screening. Recombinant baculovirus particles were transfected into ExpiSf9 insect cells to generate the P0 virus stock, which was then amplified to produce high-titer P1 virus for target protein expression. Expression products were purified by affinity chromatography (e.g., nickel column) and identified by SDS-PAGE, Coomassie Brilliant Blue staining, and Western blotting. The anti-ENO1 monoclonal antibody, single-chain antibody, and chimeric antibody were used for specific detection by Western blotting. On the basis of the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) under the catalysis by ENO1, the specific activity of ENO1 was measured by UV spectrophotometry at 240 nm.Results All recombinant plasmids and bacmids were successfully constructed, and high-level expression of recombinant proteins was achieved in insect cells. High-purity recombinant proteins were obtained, and Western blotting analysis confirmed their reactivity. The deletion of active sites of the enzyme did not affect the immunoreactivity. The specific activity of wild-type ENO1 was 691.28 U/mg, while those of the three deletion variants were significantly reduced. ENO1-M2 (with deletion of GSHAGNK at residues 156-162) and ENO1-M3 (with deletion of SPDPSRYI at residues 262-270) exhibited particularly pronounced losses of enzyme activity.Conclusion We successfully expressed and purified both wild-type and active site-deleted ENO1 proteins with retained reactivity by using the baculovirus expression vector system. The results confirm that GSHAGNK and SPDPSRYI are two key active sites of this enzyme, laying both theoretical and material foundations for further exploring the functions of ENO1 and developing its specific inhibitors.

Abstract:Litopenaeus vannamei is one of the crustacean species with the highest production and economic value in the global aquaculture industry. Its health status is closely linked to the community succession and balance of the microbiota in the aquaculture system, and the stable supply of seeds is one of the fundamental and core components of the high-quality development of the shrimp aquaculture industry. Characterizing the pattern of bacterial community succession during the early developmental stages of shrimp is an essential prerequisite for achieving microbiome-based regulation in larviculture.Objective To establish a standardized bacterial community dataset covering the entire cycle of L. vannamei larviculture, thereby providing data support for systematic investigations on the microbial communities during shrimp larvae development.Methods Focusing on the complete developmental stages of L. vannamei larvae, larval shrimps and rearing water samples were collected from the larviculture system of the Zhejiang Mariculture Research Institute following standardized sampling and laboratory processing protocols. Bacterial community datasets were constructed by high-throughput 16S rRNA gene amplicon sequencing combined with the standard Dix-seq amplicon analysis pipeline.Results This dataset encompassed the raw paired-end sequencing reads from 102 samples (48 larval samples and 54 rearing water samples) across the entire cycle of shrimp larviculture, comprising a total of 204 fastq.gz files and 4 709 988 raw paired-end reads. After quality control, 4 255 994 effective sequences and 202 505 zero-radius operational taxonomic units (ZOTUs) were obtained. Data generation strictly followed standardized protocols, with multi-point composite sampling ensuring sample representativeness of microbial samples, and unified sample collection and nucleic acid extraction procedures were employed. All analytical parameters were publicly disclosed through the parameter card mechanism of the Dix-seq pipeline, guaranteeing the reproducibility and reliability of the data analyses.Conclusion The samples in this dataset cover the key developmental nodes of L. vannamei larvae, achieving synchronous monitoring of the bacterial communities throughout the entire larviculture period. It provides fundamental data for exploring bacterial community succession and host-environment interaction mechanisms during the early developmental stages of shrimp, thereby facilitating the development of green larviculture technologies based on microbiome regulation. Additionally, it serves as a benchmark for the standardized management and analysis of similar high-throughput sequencing data, which is of great significance for enhancing the standardization and operability of microbiome research in agricultural ecosystems.