Search Results (256)

This study applied a guided culturomics workflow to isolate and characterize gut-associated yeasts as probiotic candidates. Culture conditions were optimized using Dixon agar and Modified Schädler Agar with a moderate antibiotic cocktail (colistin, chloramphenicol, vancomycin) to suppress bacteria without impairing yeast growth, combined with incubation at 37 °C under anaerobic conditions to mimic the intestinal environment. From three healthy donors, 305 isolates were recovered (MSA: 193; Dixon: 112). After excluding pseudohyphal morphotypes and PCR positive Candida isolates, 127 non-Candida strains remained. Safety screening (hemolysis, DNase, coagulase) reduced the pool to 26 safe isolates. Simulated gastrointestinal stress tests showed that 20 out of 26 exhibited at least 50 percent survival under pH 2.0 or 0.5% bile salts. Functional assays revealed strain-specific antimicrobial activity against Staphylococcus aureus, Escherichia coli including O157:H7 and Salmonella species, with several isolates (Y6, Y22, Y42, Y48, Y55, Y56, Y73, Y105, Y127) showing broad and strong inhibition. Two isolates Y44 and Y55 further demonstrated robust bile and acid tolerance (>50% survivability) in both conditions. All isolates displayed intracellular but not extracellular bile salt hydrolase activity, indicating a viability dependent cholesterol lowering potential. Overall, the workflow minimized bacterial interference while enriching for safe and functional yeasts. Full article

Human infections caused by pathogenic bacteria remain a major global health concern. Among them, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi are particularly prevalent and associated with significant morbidity and mortality. While antibiotics have long been the cornerstone of bacterial infection treatment, the widespread and often inappropriate use of these drugs has led to the emergence of multidrug-resistant (MDR) strains. This escalating resistance crisis underscores the urgent need for alternative therapeutic strategies. Amid the escalating global antimicrobial-resistance crisis, a genome-wide screen of 1800 Saccharomyces cerevisiae knockouts identified a TAD1-deficient mutant whose cell-free supernatant (CFS) rapidly eradicates multidrug-resistant E. coli, S. aureus, K. pneumoniae, and S. typhi in vitro. CFS disrupts pathogenic biofilms, downregulates biofilm-associated genes, and exerts bactericidal activity by triggering intracellular reactive oxygen species (ROS) accumulation and compromising envelope integrity. Probiotic profiling revealed robust tolerance to an acidic pH and physiological bile, high auto-aggregation, and efficient co-aggregation with target pathogens. In both Galleria mellonella and murine infectious models, administration of CFS or live yeast significantly increased survival, attenuated intestinal histopathology, and reduced inflammatory infiltration. These data establish the TAD1-knockout strain and its secreted metabolites as dual-function antimicrobial-probiotic entities, offering a sustainable therapeutic alternative to conventional antibiotics against multidrug-resistant bacterial infections. Full article

Biosurfactants (BSs) are natural, biodegradable compounds crucial for replacing synthetic emulsifiers in the food industry, provided their production costs can be reduced through the use of sustainable and low-cost substrates. This study evaluated the viability of licuri oil as a carbon source for BS production by Candida utilis and assessed the product’s functional stability in food formulations. Production kinetics confirmed the yeast’s efficiency, reducing the water surface tension to a minimum of 31.55 mN·m⁻¹ at 120 h. Factorial screening identified a high carbon-to-nitrogen ratio as the key factor influencing ST reduction. The isolated BS demonstrated high surface activity, with a Critical Micelle Concentration of 0.9 g·L⁻¹. Furthermore, the cell-free broth maintained excellent emulsifying activity (E₂₄ > 70%) against canola and motor oils across extreme pH, temperature, and salinity conditions. Twelve mayonnaise-type dressings were formulated, utilizing licuri oil, and tested for long-term physical stability. Six formulations, featuring the BS in combination with lecithin and/or egg yolk, remained stable without phase segregation after 240 days of refrigeration, maintaining a stable pH and suitable microbiological conditions for human consumption. The findings confirm that the valorization of licuri oil provides a route to produce a highly efficient and robust BS, positioning it as a promising co-stabilizer for enhancing the shelf-life and natural appeal of complex food emulsions. Full article

Soybean cyst nematode (SCN, Heterodera glycines) is one of the major pathogens of soybean worldwide. We utilized the CHIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) data from the transgenic GmMYB29 strain (Glycine Max roots). We then performed enrichment analysis using KEGG and GO to identify potential candidate genes within the promoter-binding region. A targeted regulatory relationship between the GmMYB29 and GmPP2C-37like genes was further identified using the dual-luciferase Assay (Luciferase, LUC) and yeast one-hybrid Assay (Y1H). Hairy roots with target gene overexpression and gene-edited hairy roots were generated, and their resistance to soybean cyst nematode (SCN) was evaluated. Meanwhile, the presence of reciprocal genes with GmPP2C-37like was determined by the yeast two-hybrid library screening method. The targeting relationship between GmMYB29 and GmPP2C-37like genes was further validated through the Y1H assay and LUC assay. Based on phenotypic assessments of SCN, transgenic soybean roots overexpressing GmPP2C-37like exhibited significantly enhanced resistance to SCN 3 compared to wild-type. Further analysis revealed that GmPP2C-37like collaborates with other regulatory factors to modulate soybean resistance against SCN. Yeast two-hybrid library (Y2H) screening identified 18 interacting proteins. These findings not only illuminate the functional role of GmPP2C-37like but also provide a foundation for dissecting its molecular network. Moreover, the results offer promising candidate genes for enhancing SCN resistance and optimizing soybean resilience through targeted genetic strategies. Full article

The vitality of pollen significantly influences the efficiency of pollination and microspore embryogenesis. Mining genes associated with pollen vitality will help accelerate pepper (Capsicum annuum L.) breeding progress via genetic engineering. PEX (pollen extensin-like), a member of the LRX (leucine-rich repeat extensin) family, is predominantly expressed in pollen and participates in regulating pollen vitality. However, its function and regulatory factors in pepper remain elusive. In this study, GUS histochemical staining results revealed that pepper CaPEX3 could be expressed in petals, sepals, anthers, and pollens of transgenic tomato (Solanum lycopersicum L.) lines expressing CaPEX3 promoter::GUS. Moreover, inhibition of the CaPEX3 by virus-induced gene silencing (VIGS) in pepper resulted in reduced pollen germination rate and viability, while overexpression of CaPEX3 in tomato significantly enhanced germination rate and pollen viability. In addition, TRANSPARENT TESTA GLABRA 1 (CaTTG1) and Nuclear transcription factor Y subunit C9 (CaNFYC9) were screened out and identified as the upstream regulatory transcription factors of CaPEX3 through yeast one-hybrid (Y1H) screening and dual-luciferase reporter (Dual-LUC) assays. Taken together, the identification of transcription factors may reveal a more comprehensive mechanism underlying CaPEX3-mediated enhancement of pepper pollen viability. This study not only provides genetic resources for pollen viability research but also establishes a theoretical foundation for pepper breeding. Full article

Isoflavone synthase (IFS) is the key enzyme in isoflavonoid biosynthesis and has been functionally characterized in numerous plant species. Glycyrrhiza species, valued for their medicinal properties, accumulate flavonoids with significant physiological activities. Among these, isoflavones play crucial roles in plant growth, development and stress responses. However, the IFS gene family in Glycyrrhiza remains poorly understood. In this study, we identified 10, 9 and 9 IFS genes in G. uralensis, G. inflata and G. glabra, respectively. Phylogenetic analysis classified these genes into four distinct clades (Clade A–D). Further characterization included chromosomal localization, gene structure, conserved motifs, cis-acting elements and synteny analysis. Using yeast one-hybrid (Y1H) screening, dual-luciferase assays and an electrophoretic mobility shift assay (EMSA), these results revealed that auxin response factor 4 (GgARF4) directly binds to the isoflavone synthase 9 (GgIFS9) promoter and activates its expression. Following indole-3-acetic acid (IAA) treatment, RNA-seq revealed that in the differentially expressed genes (DEGs), the genes involved in isoflavonoid and flavonoid biosynthesis pathways were significantly enriched. The result of quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that GgIFS9 was strongly induced by IAA. β-Glucuronidase (GUS) assays confirmed that IAA activates the expression of the GgIFS9 promoter in Nicotiana tabacum. Our findings reveal that, through GgARF4 and its downstream-activated gene GgIFS9, IAA may promote flavonoid synthesis in G. glabra. This study provides novel insights into the auxin-mediated regulation of secondary metabolism in Glycyrrhiza species. Full article

Background/Objectives: Mutations in NACHT, LRR and PYD domain-containing protein 2 (NLRP2) and NLRP7 genes, members of the NOD-like receptor (NLR) family of innate immune sensors, result in recurrent miscarriages and reproductive wastage in women. These genes have been identified to be maternal effect genes in humans and mice regulating early embryo development. Previous research in vitro suggests that NLRP2 and NLRP7 regulate DNA methylation and/or immune signaling through inflammasome formation. However, the exact mechanisms underlying NLRP2 and NLRP7 function are not well defined. Methods: To determine the interacting proteins required for NLRP2/NLRP7-mediated regulation of DNA methylation, yeast 2-hybrid screens, coimmunoprecipitation, and FRET studies were performed and verified the ability of novel protein interactions to affect global DNA methylation by 5-methylcytosine-specific ELISA. Results: Various methodologies employed in this research demonstrate a novel protein interaction between human ErbB3-binding protein 1 (EBP1, also known as proliferation-associated protein 2G4 (PA2G4) and NLRP2 or NLRP7. In addition, NLRP2 and NLRP7 regulate EBP1 gene expression. Functionally, global DNA methylation levels appeared to decrease further when NLRP2 and NLRP7 were co-expressed with EBP1, although additional studies may need to confirm the significance of this effect. Conclusions: Since EBP1 is implicated in apoptosis, cell proliferation, DNA methylation, and differentiation, our discovery significantly advances our understanding of how mutations in NLRP2 or NLRP7 may contribute to reproductive wastage in women through EBP1. Full article

The fw2.2 (fruit weight 2.2) gene negatively regulates cell division and significantly influences fruit size, but its regulatory mechanisms in pears remain unclear. Here, we investigated how pear FWL (fw2.2-like) genes control cell division using Duli pear, Korla fragrant pear, and Yali pear. During the cell division phase, fluorescence in situ hybridization (FISH) revealed stronger expression of FWL1 and FWL5 in smaller fruits compared to larger ones, with both genes localized in the core and flesh tissues. Gene silencing experiments demonstrated that silencing of FWL5 leads to a significant increase in the number of cells, with a concomitant enlargement of the fruit. Yeast two-hybrid screening identified 147 proteins interacting with FWL5, showing substantial overlap with FWL1 interactors. Key candidates included metallothionein-like protein (MT) and Ole-e-1, with the latter displaying a positive correlation with fruit size during cell division. Bimolecular fluorescence complementation (BiFC) confirmed direct interactions between Ole-e-1 and both FWL1/FWL5. Functional analysis indicated the Ole-e-1 gene family has diverse roles in pear development. We propose that Ole-e-1 interacts with FWL genes to modulate cell division, thereby determining final fruit size. This study uncovers a novel regulatory axis linking cell cycle control and fruit size in pears. Full article

Verticillium wilt, caused by Verticillium dahliae, severely threatens various crops and trees worldwide. This study aimed to characterize the function of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum (ER) degradation)-domain-containing protein, VdCUE, in V. dahliae, which exhibits sequence divergence between the defoliating strain XJ592 and the non-defoliating strain XJ511. We generated ∆VdCUE-knockout mutants and evaluated their phenotypes in growth and virulence. Functional analyses included verifying the signal peptide activity of VdCUE, testing its ability to induce cell death or inhibit BAX-induced cell death in Nicotiana benthamiana leaves, and identifying host targets via yeast two-hybrid screening. The ∆VdCUE mutants showed reduced formation of melanized microsclerotia but no other obvious growth defects. Cotton plants infected with the ∆VdCUE mutants exhibited a significantly lower disease index and defoliation rate. VdCUE was confirmed to be secreted via a functional signal peptide, but it neither triggered cell death nor inhibited BAX-induced cell death. Three putative host targets were identified and supported by AI-based three-dimensional structural modeling, including tRNA-specific 2-thiouridylase, peptidyl-prolyl cis-trans isomerase, and 40S ribosomal protein, which may mediate VdCUE-dependent virulence regulation. These findings reveal VdCUE as a key virulence factor in V. dahliae, contributing to our understanding of its pathogenic mechanism. Full article

The emergence of human coronaviruses has led to three epidemics or pandemics in the last two decades, collectively causing millions of deaths and thus highlighting a long-term need to identify new antiviral drug targets and develop antiviral therapeutics. In this study, a compound library was screened to uncover novel potential inhibitors of coronavirus replication. Three lead compounds, designated #16-14, #16-23, and #16-24, which were Ixazomib and its analogs, were identified based on their potent antiviral activity and minimal cytotoxicity. These compounds were found to inhibit the immunoproteasome subunit LMP7, a target whose subcellular localization and expression are altered in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-infected Huh7 cells. Yeast two-hybrid assays and co-immunoprecipitation further revealed that LMP7 interacts with the viral proteins Nsp13 and Nsp16. In addition, Nsp13 and Nsp16 disrupted the expression of LMP7 in response to pathogen attacks. Functional studies showed that LMP7 knockout in BEAS-2B-ACE2 cells resulted in enhanced replication of attenuated SARS-CoV-2, highlighting the role of this subunit in restricting viral replication. Taken together, these findings position LMP7 as a novel therapeutic target and highlight Ixazomib and its analogs as potential antiviral agents against current and future coronavirus threats. Full article

The early flowering and less lateral branches in watermelon hold significant agricultural value. The synergistic effects of these traits provide an ideal template for watermelon plant architecture improvement. However, the molecular regulatory networks underlying the development of lateral organs (including branches and flowers) in watermelon remain unclear. In this study, we found ClTFL1 knockout lines significantly promote flowering time and inhibit lateral branching and tendril formation, while also leading to a mild apical flower phenotype. These findings indicate that the function of ClTFL1 in watermelon is more extensive than that of its homologous genes in Arabidopsis, rice, and tomato. Through yeast two-hybrid screening, we identified the interacting proteins of ClTFL1, including members of the 14-3-3 family ClGRF8, ClGRF9, and ClGRF12. Bimolecular fluorescence complementation (BiFC) assays further demonstrated ClTFL1 could directly interact with the ClGRF8, ClGRF9, and ClGRF12 protein. The knockout of ClGRF8 and ClGRF12 leads to reduced lateral branches and early flowering. These phenotypes are highly consistent with those of ClTFL1 knockout mutants. Our findings demonstrate the important role of the ClTFL1-ClGRFs module in regulating lateral organ development and flowering time in watermelon, offering important targets for watermelon plant architectural modification and molecular breeding. Full article

Dendrobium huoshanense, an endangered orchid species, is renowned for its polysaccharides with vast pharmacological significance in stems. Phosphomannomutase (PMM) critically regulates polysaccharide accumulation. Transcriptional regulation of DhPMM remains poorly characterized. This study employed a yeast one-hybrid (Y1H) system to identify upstream regulators of DhPMM. The 2.15 kb DhPMM promoter was cloned, revealing multiple stress- and hormone-responsive cis-elements (e.g., ABRE, MYC, ERF). A high-complexity Y1H library (3.60 × 10⁹ CFU) was constructed with insert sizes averaging 1–2 kb. Screening using aureobasidin A (AbA)-resistant Y1HGold [pAbAi-DhPMM] identified 11 candidate clones, including four transcription factor families (DOF, NAC, ERF, BES1). Interactions were rigorously confirmed by pairwise Y1H showing AbA-resistant growth and dual-luciferase assays demonstrating DhPMM activation. This represents the first functional cDNA library resource for D. huoshanense and identification of TFs interacting with DhPMM. The discovery of TFs belonging to DOF, NAC, ERF, and BES1 families as DhPMM regulators elucidated the transcriptional network underlying polysaccharide biosynthesis. This establishes a transcriptional framework for engineering polysaccharide biosynthesis in D. huoshanense. Full article

Fusarium head blight (FHB), caused by Fusarium graminearum (F. graminearum), has become one of the most devastating wheat diseases, severely impacting both grain yield and quality. The resistance gene TaHis (encoding a histidine-rich calcium-binding protein), located at the major FHB resistance locus Fhb1, has been demonstrated to confer FHB resistance in wheat, although its underlying mechanism remains unclear. In this study, we screened a wheat yeast two-hybrid (Y2H) library and identified TaU11/U12-35K, a core component of the U12-type spliceosome (U11/U12 small nuclear ribonucleoprotein), as a novel interacting partner of TaHis. Their physical interaction was further confirmed by both Y2H and bimolecular fluorescence complementation assays. Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS)-mediated knockdown of TaU11/U12-35K significantly enhanced FHB resistance in both resistant (Bainong 4299) and susceptible (Bainong 5819) cultivars compared to controls. Expression profiling revealed that TaU11/U12-35K was significantly downregulated upon F. graminearum infection in both cultivars, with consistently lower basal expression levels in Bainong 4299, suggesting a negative correlation between TaU11/U12-35K expression and FHB resistance. Collectively, our results demonstrate that TaU11/U12-35K physically interacts with TaHis and functions as a negative regulator of FHB resistance. This study provides new insights into the molecular mechanism of TaHis-mediated FHB resistance in wheat. Full article

This study aimed to identify and evaluate yeasts and lactic acid bacteria (LAB) isolated from Mexican cocoa mucilage (Theobroma cacao) and coffee pulp (Coffea arabica) for their potential use as sourdough starter co-cultures to improve bread quality. Functional screens included assessments of amylolytic, proteolytic, and phytase activities, CO₂ production, acidification capacity, and exopolysaccharide (EPS) synthesis. Saccharomyces cerevisiae YCTA13 exhibited the highest fermentative performance, surpassing commercial baker’s yeast by 52.24%. Leuconostoc mesenteroides LABCTA3 showed a high acidification capacity and EPS production, while Lactiplantibacillus plantarum 20B3HB had the highest phytase activity. Six yeast–LAB combinations were formulated as mixed starter co-cultures and evaluated in sourdough breadmaking. The B3Y14 co-culture (LABCTA3 + YCTA14) significantly improved the bread volume and height by 35.61% and 17.18%, respectively, compared to the commercial sourdough starter, and reduced crumb firmness by 59.66%. Image analysis of the bread crumb revealed that B3Y14 enhanced the crumb structure, resulting in greater alveolar uniformity and a balanced gas cell geometry. Specifically, B3Y14 showed low alveolar regularity (1.16 ± 0.03) and circularity (0.40 ± 0.01), indicating a fine and homogeneous crumb structure. These findings highlight the synergistic potential of selected allochthonous yeast and LAB strains in optimizing sourdough performance, positively impacting bread texture, structure, and quality. Full article

Coffee pulp, the primary residue generated during the wet processing of Coffea arabica L., is frequently applied directly to fields as a crude soil amendment. However, this practice often lacks proper microbial stabilization, limiting its agronomic potential and posing risks due to the presence of phytotoxic compounds. In Colombia, disease-resistant varieties such as Coffea arabica L. var. Castillo and var. Cenicafé 1, developed by the National Coffee Research Center (Cenicafé), are the amongst the most widely cultivated varieties in the country; however, despite their widespread adoption, the microbial ecology of postharvest residues from these varieties remains poorly characterized. This study aimed to isolate and functionally characterize native microbial communities from the pulp of Coffea arabica var. Castillo and var. Cenicafé 1, and to evaluate their role in postharvest processing and organic waste management. Fresh pulp samples were collected from a wet-processing facility located in tropical mid-elevation zones. A total of 53 microbial isolates were recovered using culture-dependent techniques on selective media targeting yeasts, lactic acid bacteria (LAB), and filamentous fungi. Amplicon sequencing of the 16S rRNA gene (V3–V4 region) and ITS1 region was conducted to profile bacterial and fungal communities, revealing diverse microbial consortia dominated by Aspergillus, Lactobacillus, Leuconostoc, Pichia, and Saccharomyces species. Enzymatic screening indicated high pectinolytic and cellulolytic activity. Composting trials using inoculated pulp showed a ~40% reduction in composting time and improved nutrient content. These findings support the use of native microbiota to enhance composting efficiency and postharvest valorization, contributing to more sustainable and circular coffee systems. Full article