Search Results (396)

Streptococcus parauberis is a major pathogen responsible for streptococcosis in both marine and freshwater fish species, causing substantial economic losses in aquaculture. The increasing prevalence of multidrug resistance has highlighted the urgent need for alternative disease control strategies. Interference with bacterial quorum sensing (QS) systems represents a promising approach. This study aimed to identify and biochemically characterize an Rgg-family transcriptional regulator and evaluate its potential as a target for quorum sensing-related regulatory interference in vitro. We hypothesized that this Rgg regulator may function as a quorum sensing-associated transcription factor capable of promoter binding and modulation by small molecules. Bioinformatic analyses were used to identify the rgg gene encoding an Rgg-family transcriptional regulator and predict its structural features. The gene was cloned, heterologously expressed, and purified. Promoter binding activity was examined using electrophoretic mobility shift assay (EMSA), and key amino acid residues were identified through site-directed mutagenesis. The inhibitory effect of the cyclic peptide cyclosporin A (CsA) on Rgg-promoter binding was further assessed. The rgg gene (864 bp) encoding a 287-amino-acid protein (34.1 kDa) was successfully identified and expressed. Purified Rgg specifically bound to its own promoter region in a concentration-dependent manner. Mutations at conserved arginine residues R12 and R15 within the helix-turn-helix DNA-binding domain abolished promoter binding activity. Furthermore, CsA disturbed Rgg-promoter binding in a dose-dependent manner. This study provides the first in vitro characterization of an Rgg-family transcriptional regulator in fish-derived S. parauberis. The findings expand current understanding of Rgg-family regulators potentially associated with quorum sensing in aquatic streptococci and provide a preliminary basis for further investigation of quorum sensing-related regulatory interference strategies for controlling streptococcal diseases in aquaculture. Full article

Soybean (Glycine max) is a critically important crop for oil, protein, feed, and food security in China. Expanding soybean cultivation into high-latitude regions represents one of the most direct and effective strategies to increase total production. In the present study, we employed KASP (Kompetitive Allele-Specific PCR) marker technology to systematically analyze 18 variant loci across 14 flowering-time genes in 443 soybean germplasm accessions adapted to high-latitude conditions in Arctic Village (Beiji Cun), Mohe City (>53° N), northeastern China. Our results revealed clear functional-tier-dependent selection gradients: key mutation sites (frequency > 96%) in upstream photoreceptors and core circadian clock genes, such as E2 and GmPRR3a, were nearly fixed in the population, whereas downstream flowering genes such as GmFT5b and GmFT2b remained under dynamic selection. Combinatorial analysis of early-maturity allelic variants identified 178 distinct genotype combinations, including six dominant types (n ≥ 10). Field phenotypic analysis demonstrated that the cumulative number of early-maturity alleles was significantly negatively correlated with flowering time, with specific allele combinations such as FT5a^A + FKF1b-hap3^T exhibiting particularly strong flower-promoting effects. A set of 80 highly enriched super-early-maturity accessions, including extreme materials such as MHL22002, were identified, providing valuable genetic resources and a theoretical framework for elucidating the flowering regulatory mechanisms of high-latitude soybean and for breeding super-early-maturing varieties. Full article

Chronic lymphocytic leukemia (CLL) is uncommon in Asia, and longitudinal genomic data from Asian cohorts are limited. We conducted serial whole-exome sequencing (WES) in a multicenter Korean cohort of newly diagnosed, elderly CLL treated with chlorambucil–obinutuzumab to evaluate mutational heterogeneity and clonal hematopoiesis of indeterminate potential (CHIP) during treatment and follow-up. Tumor-only variants were filtered, restricted to nonsynonymous or loss-of-function coding/splice-site mutations, and summarized as a binary patient-by-gene matrix for principal component analysis (PCA), trajectory analysis, and k-means clustering. CHIP was defined as ≥1 qualifying mutation in a prespecified CHIP gene set. Baseline PCA was more compact in patients with complete response at end of treatment, whereas partial response or progressive disease cases were more dispersed. PCA trajectories were compact and directionally consistent in complete responders, more dispersed in partial responders, and highly heterogeneous without a dominant direction in progressive disease. Clustering identified dispersed and compact clusters, and CHIP-associated mutations were enriched in the dispersed cluster (55.6% vs. 8.3%, Fisher’s exact p = 0.0086). In paired samples collected 3–5 months after end of treatment, CHIP status changed in some patients. Serial WES may provide complementary information to treatment response, although these observations require confirmation in larger cohorts. Full article

The development of a universal SARS-CoV-2 vaccine holds great promise for achieving broad and durable protection against existing and future coronavirus variants. The identification, selection, and rational redesign of conserved viral epitopes constitute the direct immunological foundation of universal SARS-CoV-2 vaccine development. The breadth and durability of protection are therefore primarily determined at the level of antigen and epitope design, whereas adjuvants, delivery platforms, and routes of administration serve as enabling and amplifying components rather than primary drivers of universality. Accordingly, this review discusses key determinants of universal vaccine design, including antigen selection, adjuvant utilization, and route of administration. The spike protein, particularly its receptor-binding domain, is a major antigenic target, but its high mutation rate challenges long-term vaccine efficacy. Strategies focusing on conserved epitopes in antigen designs show potential to elicit cross-neutralizing immune responses. Nanoparticle-based vaccines capable of presenting multiple homologous or heterologous antigens have demonstrated enhanced immunogenicity, broad protection in preclinical models and safety in clinical trials. The addition of next-generation adjuvants further amplifies humoral and cellular immunity beyond the capabilities of traditional aluminum-based adjuvants. Moreover, mucosal vaccine delivery may provide superior local protection at viral entry sites and limit transmission. Importantly, integrating these technological advances with epitope-centered antigen design and immunological data from vaccinated individuals will accelerate the identification of conserved epitopes and inform future vaccine design. A multidisciplinary approach combining optimized antigen engineering, novel adjuvant systems, and innovative delivery strategies is essential for the realization of a broadly protective universal SARS-CoV-2 vaccine. Full article

Male genitourinary (mGU) malignancies, including prostate, bladder, kidney, testicular, and penile cancers, represent a clinically and epidemiologically significant subset of global cancer burden. Although well-established etiological factors such as genetic mutations, androgen signaling, and environmental exposures contribute to tumorigenesis, the underlying mechanisms remain ill-defined. Recent advances in next-generation sequencing and metagenomics technologies have facilitated a deeper understanding of the human microbiome, revealing its potential role in carcinogenesis. While the gut microbiome has been extensively studied, emerging evidence indicates that site-specific microbial communities within the genitourinary (GU) tract may significantly influence cancer susceptibility, progression, and therapeutic outcomes. Accordingly, this review aims to comprehensively summarize the current evidence examining the relationship between the GU microbiome and the development, progression, and treatment of mGU cancers. To provide the specific context, relevant publications were collected from Google Scholar, PubMed, Science Direct, Dimension AI, and EBSCO Host using specific keywords such as “bladder cancer”, “dysbiosis”, “genitourinary”, “genitourinary cancer”, “microbiome”, “pathogens”, “penile cancer”, “prostate cancer”, “renal cancer”, “testicular cancer”, “urogenital microbiome”. We did not add any limits to the publication date during the inclusion of papers. However, it is noteworthy that the initial reports, including the aforementioned keywords, have been published since 2015. Emerging evidence highlights a significant association between the dysbiosis of the GU microbiome and the development of mGU cancers. Notably, an increase in bacterial richness and species diversity has been correlated with a rapid progression of these cancers, suggesting that such features may be explored as potential candidate biomarkers. Advanced sequencing and meta-omics technologies have enabled the identification of distinct microbial signatures with emerging diagnostic, prognostic, and therapeutic potential. Despite these advancements, the understanding of the functional and mechanistic roles of microbiota, particularly within the penile and seminal environments, remains limited. Full article

Background/Objectives: Coxsackievirus B4 (CVB4), a member of the Enterovirus genus and the Picornaviridae family, is a significant pathogen causing several human diseases such as pancreatitis, myocarditis, cardiomyopathy and type 1 diabetes. Despite its clinical impact, no vaccines or specific antiviral therapies are currently available. This study investigates the attenuation of CVB4 virulence through targeted mutations in the domain V of the IRES (Internal Ribosome Entry Segment) sequence present in the 5′ UTR (Untranslated Region) of the viral genome. Materials and Methods: We engineered six CVB4E2 mutants by introducing single nucleotide mutations in domain V of the IRES sequence using PCR-based site-directed mutagenesis assays. Mutants were rigorously evaluated in vitro for their replicative capacities on HeLa cell culture and for their in vitro translation efficiencies in standard rabbit reticulocyte lysates supplemented with HeLa cell S10 extracts. Using different strategies of immunization and lethal challenges in a Balb/c mice model, we evaluated the immune responses elicited by the most attenuated C488A mutant strain. Results: The obtained results demonstrated that the live-attenuated C488A mutant with the single mutation C to A at nucleotide position 488 of the viral IRES sequence exhibited a significant reduction in vitro of both viral productivity and translation efficiency. The oral immunization with the live-attenuated C488A mutant induced a potent immune response and protected Balb/c mice against lethal infection challenge with a pathogenic strain. Conclusions: These findings underscored the critical role of IRES in CVB4 virulence and highlighted the use of the live-attenuated C488A mutant strain as a promising candidate for developing a live-attenuated vaccine against CVB4 infections. Full article

Background: The emergence of polymyxin-resistant, carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a critical challenge to clinical management. This study aimed to delineate the molecular mechanisms driving the acquisition of polymyxin resistance in CRKP. Methods: We analyzed polymyxin-susceptible and polymyxin-resistant CRKP isolates obtained from a single patient. Antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentrations. Whole genome sequencing was employed to identify variations in two-component systems and to screen for mcr genes, which were involved in polymyxin resistance. Differential gene expression was assessed using RNA sequencing and validated by quantitative real-time PCR. Furthermore, site-directed mutagenesis was utilized to confirm the causal role of specific mutations in conferring the resistant phenotype. Results: An L96P mutation in the PhoQ protein was found in the polymyxin-resistant CRKP isolate. Compared with the PhoQ wild-type, this mutation significantly upregulated expression of phoP/Q, pmrD, and arnBCADTEF operon-related genes. A corresponding L96P mutant was subsequently constructed in the polymyxin-susceptible ATCC 13883 strain via site-directed mutagenesis. Antimicrobial susceptibility testing confirmed that the PhoQ L96P mutation elevates the minimal inhibitory concentrations of colistin and polymyxin B to 64 mg/L and >32 mg/L, respectively, from a baseline of 0.5 mg/L. Conclusions: The PhoQ L96P mutation is a pivotal driver of polymyxin resistance in CRKP, primarily mediated through the upregulation of the arnBCADTEF operon. Full article

As one of the five major indigenous yellow cattle breeds in China, Qinchuan cattle are characterized by stable genetic performance and desirable meat quality. However, compared with imported commercial breeds, Qinchuan cattle have a relatively slow growth rate. Therefore, improving the growth rate of Qinchuan cattle has become a top priority in Qinchuan cattle breeding. The CDC10 (Septin 7) gene, an important member of the Septin family, participates in various cellular physiological processes including intracellular substance transport, cell division, cell cycle regulation, and apoptosis. Studies have repeatedly mapped the CDC10 gene to quantitative trait loci influencing growth-related traits, such as body weight and carcass weight in many beef cattle breeds. Previous study has also demonstrated the high expression of CDC10 in JB cattle with high performance for carcass weight, however, the association between CDC10 and growth-related traits in Qinchuan cattle remain unclear. Therefore, in this study, we selected five individuals each from Chinese Simmental, Mongolian cattle, Luxi cattle, and Qinchuan cattle for direct sequencing, aiming to identify mutations within the CDC10 gene of native Chinese yellow cattle. Subsequently, we performed genotyping of 367 Qinchuan cattle using the MassARRAY technology, followed by genetic diversity analysis of the identified mutations and association analysis between these sites and growth-related traits of Qinchuan cattle. This study demonstrated high expression of the CDC10 gene in Qinchuan cattle with high performance for carcass weight. Furthermore, we identified the g.61303052G>C and c.225A>G SNPs in the promoter and exon regions, respectively, as being significantly associated with multiple growth-related traits in Qinchuan cattle. The c.225A>G SNP was also found to alter the secondary structure of the CDC10 protein. These findings provide reliable molecular markers for enhancing the growth rate of Qinchuan cattle and establish a solid theoretical foundation for the development of the beef cattle industry. Full article

Monoacylglycerols (MAGs) are significant intermediate byproducts in the hydrolysis of oils and fats. The accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes. Therefore, the development of efficient biocatalysts for the specific MAG conversion is of great industrial importance. The lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application. In this study, a combinatorial protein engineering strategy was employed to overcome these limitations. We integrated fusion protein technology with rational design to enhance both the functional expression and catalytic efficiency of AOL. Firstly, the superfolder green fluorescent protein (sfGFP) was fused to the N-terminus of AOL. The results indicated that the sfGFP fusion tag significantly improved the solubility and stability of the enzyme, preventing the formation of inclusion bodies. The fusion protein sfGFP-AOL exhibited a MAG conversion rate of approximately 65%, confirming the positive impact of the fusion tag on enzyme developability. To further boost catalytic performance, site-directed mutagenesis was performed based on structural analysis. Among the variants, the mutant sfGFP-Y92Q emerged as the most potent candidate. In the MAG conversion, sfGFP-Y92Q achieved a conversion rate of 98%, which was not only significantly higher than that of sfGFP-AOL but also outperformed the widely used commercial immobilized lipase, Novozym 435 (~54%). Structural modeling and docking analysis revealed that the Y92Q mutation optimized the geometry of the active site. The substitution of Tyrosine with Glutamine at position 92 likely enlarged the substrate-binding pocket and altered the local electrostatic environment, thereby relieving steric hindrance and facilitating the access of the bulky MAG substrate to the catalytic center. In conclusion, this work demonstrates that the synergistic application of sfGFP fusion and rational point mutation (Y92Q) can dramatically transform the catalytic properties of AOL. The engineered sfGFP-Y92Q variant serves as a robust and highly efficient biocatalyst for MAG degradation. Its superior performance compared to commercial standards suggests immense potential for cost-effective applications in the bio-manufacturing of high-purity fatty acids and biodiesel, offering a greener alternative to traditional chemical processes. Full article

Based on PCR with complementary primer pairs and Pfu DNA polymerase, QuickChange site-directed mutagenesis has been widely employed, but its efficiency varies from mutation to mutation. An alternative strategy relies on partially overlapping primer pairs with 3′-overhangs, and this strategy has led to the recent development of P3a and P3b site-directed mutagenesis, in which the use of SuperFi II and Q5 polymerases raises the mutagenesis efficiency to ~100%. It is unclear whether these two DNA polymerases also improve the QuickChange method. Herein, we have evaluated this possibility by engineering 46 mutations on seven expression plasmids, two of which possess extremely GC-rich sequences. As Pfu DNA polymerase is a slow enzyme, its replacement with SuperFi II and Q5 polymerases reduced PCR length. Moreover, the average efficiency for each of the seven plasmids ranged from 48% to 69%, thereby outperforming the original QuickChange method. However, this efficiency is still lower than that from the P3a and P3b methods, supporting the superiority of primer pairs with 3′-overhangs. Analysis of the incorrect plasmids from the improved QuickChange method revealed frequent insertions at primer sites. The insertions were derived from primers and varied from mutation to mutation, with certain sites much more prone to such insertions. In comparison, these insertions occurred at a much lower frequency with the P3a and P3b methods, suggesting that primer pairs with 3′-overhangs enhance mutagenesis efficiency by reducing the likelihood to introduce insertions at primer sites. Thus, this study improves the QuickChange mutagenesis method, supports the superiority of the P3a and P3b methods, and uncovers a novel molecular mechanism by which the efficiency of PCR-based mutagenesis with completely overlapping primer pairs is negatively affected. Full article

Liquid biopsy is moving beyond mutation-centric assays to multimodal frameworks that integrate cell-free DNA (cfDNA) signals with additional analytes such as circulating tumor cells (CTCs) and extracellular vesicles (EVs). In this review, we summarize emerging technologies across analytes for early cancer detection, emphasizing sequencing and error-suppression strategies and the growing evidence for multi-cancer early detection (MCED), tissue-of-origin (TOO) inference, diagnostic triage, and longitudinal surveillance. At low tumor fractions, fragmentomic and methylation features preserve tissue and chromatin context; when combined with radiomics using deep learning, they support blood-first, high-specificity risk stratification, increase positive predictive value (PPV), reduce unnecessary procedures, and enhance early prediction of treatment response and relapse. Building on these findings, we propose a pathway-aware workflow: initial blood-based risk scoring, followed by organ-directed imaging, and targeted secondary testing when indicated. We further recommend that model reports include not only discrimination metrics but also calibration, decision-curve analysis, PPV/negative predictive value (NPV) at fixed specificity, and TOO accuracy, alongside multi-site external validation and blinded dataset splits to improve generalizability. Overall, liquid biopsy is transitioning from signal discovery to deployable multimodal decision systems; standardized pre-analytical and analytical workflows, robust error suppression, and prospective real-world evaluations will be pivotal for clinical implementation. Full article

Nanoparticles interact dynamically with proteins, often leading to adsorption-induced conformational changes that alter protein function and contribute to corona formation. Here we investigated the adsorption and unfolding of a model protein GB1 on latex nanoparticle surfaces using a combination of mutational analysis, equilibrium binding assays, stopped-flow kinetics and Φ-value interpretation. Seven site-directed variants of GB1 were studied to dissect residue-specific contributions to adsorption energetics. Fluorescence binding isotherms revealed that D46A and T53A mutations weakened surface affinity, while kinetic analysis demonstrated that D46A reduced adsorption rate by ~6-fold and produced a dramatic unfolding/refolding shift, identifying Asp46 as a key docking site. Φ-value analysis further highlighted Asp46 and Thr53 as central residues in the adsorption transition state, whereas mutations in the hydrophobic core or distal loops had negligible effects. These results support a dock–loosen–unfold mechanism in which electrostatic recognition initiates binding, followed by hydrophobic exposure and hairpin stabilization. This residue-level sampling of key sites advances mechanistic understanding of protein–nanoparticle interactions and suggests strategies for tuning surface charge to control corona formation. Our approach provides a generalizable method to map adsorption transition states, with implications for designing safer nanomaterials, predicting protein corona composition, and harnessing protein unfolding in biosensing applications. Full article

In all arrestins, the gate loop is the central part of the lariat loop, which has an unusual shape and participates in maintaining the basal conformation. The gate loop supplies two out of five charges that constitute a stabilizing intramolecular interaction, aspartates in the polar core between the two domains. To elucidate the functional role of individual gate loop residues, we performed comprehensive site-directed mutagenesis and tested the effects of mutations on arrestin-1 binding to its preferred target, phosphorylated light-activated rhodopsin, and unphosphorylated activated form. Out of 34 mutations tested, 24 and 25 affected the binding to phosphorylated and unphosphorylated rhodopsin, respectively. Manipulation of residues following polar core aspartates reduced preference for phosphorylated over unphosphorylated light-activated rhodopsin as dramatically as replacing these negatively charged aspartates with positively charged arginine. The data show that numerous lariat loop residues play distinct roles in arrestin-1 binding and its exquisite preference for phosphorylated light-activated rhodopsin. Full article

Human growth hormone (GH) exerts its pleiotropic effects by binding to its receptor (GHR), leading to receptor dimerization and activation. We combined structural, evolutionary, and genetic analyses to elucidate the critical determinants of GH-GHR interaction and the impact of disease-causing mutations. Protein contact analysis revealed the specific amino acid residues involved in two distinct binding interfaces between GH and two chains of GHR. ConSurf analysis demonstrated significant sequence conservation in the receptor-binding regions of GH across species, highlighting their functional importance. A comprehensive list of known disease-causing mutations in GH was compiled and mapped to these binding interfaces and conserved regions. Computational site-directed mutagenesis (SDM) analysis predicted the impact of several mutations on protein stability, revealing both stabilizing and destabilizing effects. Sequence comparisons with orthologs from various species further supported the evolutionary conservation of key functional residues. Integrated analysis of contact residues between GH and GHR showed a strong correlation between receptor-binding residues, evolutionary conservation, and the occurrence of disease-associated mutations. These findings underscore the critical role of specific GH residues in mediating high-affinity interactions with its receptor and how mutations in these conserved contact points can disrupt binding affinity and/or protein stability, ultimately leading to growth disorders. This multi-faceted approach provides valuable insights into the molecular mechanisms underlying growth hormone deficiency and related syndromes. Full article

Aspergillus flavus is both an agricultural and clinical pathogen, notable for its ability to contaminate crops with aflatoxins and cause invasive aspergillosis. The increasing emergence of azole resistance in A. flavus poses a serious challenge to food safety and human health. Although mutations in ergosterol biosynthesis genes have been reported in resistant isolates, their functional contributions remain largely unvalidated. In this study, we investigated the role of the CYP51A Y119F mutation in azole resistance. Site-directed mutants were generated using PCR-based gene editing, and their susceptibility to antifungal agents was assessed through Clinical and Laboratory Standards Institute broth microdilution and agar diffusion assays. The Y119F mutation reduced susceptibility specifically to voriconazole and isavuconazole, while susceptibility to itraconazole and posaconazole remained unchanged. To explore the structural basis of this phenotype, molecular dynamics simulations were performed. The mutant protein exhibited greater fluctuations and reduced conformational stability compared to the wild-type enzyme. Tunnel analysis further indicated that the Y119F substitution caused narrowing and shortening of the main access tunnels to the heme-binding pocket, likely impairing azole access and binding. The combined biochemical and structural analyses suggest that Y119F represents a primary resistance-conferring mutation that modifies the structural dynamics of CYP51A. Full article