Search Results (7,467)

Background: Cytomegalovirus (CMV) viral load monitoring forms the basis of preemptive treatment strategies in patients undergoing solid organ and hematopoietic stem cell transplantation. This study aimed to evaluate the analytical performance and inter-method agreement of a laboratory-developed CMV real-time PCR (qPCR) test compared to a commercial reference method using plasma samples. Methods: A total of 100 EDTA plasma samples were analyzed in parallel using a laboratory-developed CMV qPCR test and the reference method (Roche Cobas^® CMV). Analytical sensitivity was determined us-ing synthetic DNA cloned into the pUC57 plasmid backbone containing the US17 region of the CMV genome, and the limit of detection (LoD₉₅) was calculated using probit regression analysis. The relationship between the quantitative results obtained from clinical samples was evaluated using the Spearman rank correlation coefficient, while inter-method clinical agreement was assessed using the Bland–Altman method. Results: The limit of detection (LoD₉₅) of the laboratory-developed CMV qPCR test, as determined by probit regression analysis, was 63.8 copies/µL. A weak and statistically non-significant correlation was ob-served between the laboratory-developed CMV qPCR test and the reference method in Spearman rank correlation analysis of samples for which numerical quantitative results were available from both methods (ρ = 0.32; p = 0.22; n = 16). Bland–Altman analysis showed a mean difference of −0.48 log₁₀ units, with the vast majority of measurements falling within the 95% limits of agreement. Conclusions: The assay demonstrated measurable analytical performance and inter-method agreement; however, its use for quantitative viral load monitoring, particularly at low CMV DNA levels, should be interpreted with caution. Full article

Triterpenoid saponins are important bioactive compounds synthesized through the isoprenoid pathway, in which 2,3-oxidosqualene serves as a precursor of triterpenoid saponins. In this study, we identified and characterized eight oxidosqualene cyclase (PlOSC) genes in Paeonia lactiflora using molecular cloning and bioinformatic analyses. Full-length cDNAs of PlOSCs (PlOSC1–PlOSC8) were cloned, and the protein sequences exhibited significant similarities to known cyclases, including β-amyrin synthase and cycloartenol synthase. Phylogenetic analysis revealed distinct groups of PlOSCs corresponding to lupeol, β-amyrin, and cycloartenol synthases. Sequence alignment confirmed the presence of highly conserved motifs, including the “SDCTAE” and “QW” motifs, which are crucial for cyclization and stability in PlOSCs. To determine the functional roles of PlOSCs, we conducted functional expression studies in Saccharomyces cerevisiae. The results showed that PlOSC3 and PlOSC6 are monofunctional β-amyrin synthases that produce β-amyrin in yeast culture, as confirmed through GC-MS analysis. Further investigation of PlOSC gene expression in various tissues indicated that PlOSC3 was predominantly expressed in roots, whereas PlOSC6 was highly expressed in leaves. Corresponding metabolite analyses revealed that triterpenoid accumulation was significantly higher in roots than in leaves, suggesting tissue-specific biosynthesis and accumulation patterns in triterpenoid biosynthesis. These findings contribute to our understanding of the regulation of triterpenoid biosynthesis in P. lactiflora and provide insights into the functional roles of OSCs in triterpenoid and nortriterpenoid formation. Full article

Minimal residual disease (MRD) is the most robust independent prognostic biomarker for pediatric acute lymphoblastic leukemia (ALL). Conventional MRD detection assays suffer from insufficient sensitivity and inherent technical limitations, failing to identify ultra-low-level leukemic blasts and thereby contributing to disease relapse. Next-generation sequencing (NGS)-based MRD detection (NGS-MRD) overcomes these drawbacks by targeting immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements and enabling the precise quantification of residual leukemic clones. In recent years, NGS-MRD has undergone extensive technological optimization in target panel design, result interpretation and sample type expansion, and has been validated for its clinical utility in therapeutic threshold definition, prognostic stratification, post-therapy monitoring and treatment adjustments in pediatric ALL. This review synthesizes the latest technological refinements and clinical applications of NGS-MRD in pediatric ALL, critically discusses the current challenges that limit its routine clinical use, and proposes future research directions to address these issues. Full article

Maize (Zea mays L.), a typical thermophilic crop originating from tropical regions, exhibits an inherent sensitivity to low-temperature stress. Cold stress severely restricts maize seed germination, seedling growth, the physiological metabolism, and the final grain yield, which greatly limits its geographical cultivation range and sustainable industrial development. Elucidating the molecular regulatory mechanisms underlying maize cold tolerance and excavating cold-resistant functional genes are essential for the molecular breeding of cold-tolerant maize varieties and expanding maize planting areas in high-latitude and low-temperature-prone regions. In this study, using the strongly cold-tolerant maize inbred line B144 as the experimental material, we cloned the ZmMAPKKKA gene (NCBI accession: LOC103651289) and systematically screened and verified its cold-stress-specific interacting proteins via multiple molecular biological assays. The full-length coding sequence (CDS) of ZmMAPKKKA is 1134 bp, encoding a 377-amino-acid protein with a predicted molecular weight of 40.37 kDa. The quantitative real-time PCR (qRT-PCR) results demonstrated that the ZmMAPKKKA expression was significantly upregulated by 16.56-fold in maize roots after 12 h of low-temperature treatment, indicating a tissue-specific and robust cold response in root tissues. A total of 25 interacting proteins were identified through yeast two-hybrid screening, among which three stress-responsive proteins, including a protein kinase (LOC100286253), a protein phosphatase 2C (PP2C) (LOC542176), and a NAC transcription factor (LOC118474710), were selected for subsequent verification. The Pull-Down, Co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays consistently confirmed that ZmMAPKKKA specifically interacts with these three proteins both in vitro and in vivo under cold stress conditions. This study is the first to construct a ZmMAPKKKA-centered protein interaction module in the maize mitogen-activated protein kinase (MAPK) cascade under cold stress, establishing a novel kinase–phosphatase–transcription factor regulatory cascade that improves the current understanding of cold signal transduction mechanisms in maize. Homologous genes of ZmMAPKKKA in gramineous crops including rice (Oryza sativa) and sorghum (Sorghum bicolor) have been proven to participate in diverse abiotic stress responses, suggesting the conserved functional roles of MAPKKK family genes across gramineous species. Collectively, our findings provide comprehensive insights into the molecular mechanism of the maize MAPK signaling pathway mediating cold stress adaptation and supply valuable functional gene resources for cold-tolerant maize germplasm innovation and molecular breeding. Full article

This study investigated rhamnolipid synthesis in Pseudomonas aeruginosa ATCC 27853. Two constitutive promoters, P_rpsJ and P_oprL, were isolated and cloned upstream of the rhlABRI and rmlBDAC gene clusters to evaluate their impact on rhamnolipid titers. The overexpression of rhlB, driven by the P_rpsJ promoter, significantly enhanced rhamnolipid production. Subsequent glycine-scanning mutagenesis of RhlB identified an optimal variant (RhlBM328G), which increased the titer 1.82-fold (to 24.6 g·L⁻¹) compared to the wild type, achieving a product yield of 0.39 g·g⁻¹. Characterization of the extracted rhamnolipids revealed a critical micelle concentration of 1 mg/L, a corresponding surface tension of 53.9 mN/m, and a hydrophilic–lipophilic balance (HLB) value of 14. This HLB value indicated that the synthesized rhamnolipids possess superior hydrophilicity, robust oil-in-water emulsifying capabilities, and excellent solubilization and dispersion properties. Furthermore, molecular docking and molecular dynamics simulations demonstrated that in the RhlBM328G mutant, the nucleophilic attack distances between the substrates and the catalytic moiety are optimized for catalysis, thereby boosting rhamnolipid production. Full article

Litter decomposition regulates the quantity and quality of plant-derived carbon (C) and nitrogen (N) inputs to soil and is closely associated with microbial community structure. However, how elevated ozone (O₃) and nitrogen (N) addition interactively affect residual litter inputs and their associations with soil microbial communities remains poorly understood, especially in agroforestry systems. Here, we conducted a 12-month in situ litter decomposition experiment using two poplar clones (107 and 546) under ambient or elevated O₃ with or without N addition (60 kg N ha⁻¹ yr⁻¹) at an O₃-FACE platform in northern China. Litter mass and chemical traits were measured during decomposition, and endpoint soil microbial community structure was characterized using phospholipid fatty acid (PLFA) profiling. Treatment effects and litter–microbe associations were evaluated using linear mixed-effects models, correlation analysis, and redundancy analysis (RDA). Endpoint litter mass remaining was significantly affected by O₃, clone identity, and their interactions with N addition, while endpoint litter chemical traits showed trait-specific responses. PLFA-derived microbial community indices also showed treatment- and clone-dependent responses, particularly in bacterial groups, AM fungi, and the fungal-to-bacterial ratio. Endpoint litter mass remaining showed the strongest statistical association with PLFA-derived microbial community structure, whereas individual nutrient concentrations showed weaker independent effects. These findings suggest that O₃- and N-induced changes in residual litter quantity and quality are associated with shifts in PLFA-derived microbial community structure. Because PLFA characterizes microbial community structure rather than process rates, these findings should be interpreted as evidence of structural microbial reorganization associated with altered residual litter inputs, rather than direct evidence of changes in C or N cycling rates. Full article

Henosepilachna vigintioctopunctata is a major pest of solanaceous crops. Matrix metalloproteinase 2 (MMP2) is a zinc ion-dependent endopeptidase that plays a crucial role in the remodeling process of the extracellular matrix (ECM) within cells. However, the function of HvMMP2 in H. vigintioctopunctata remains unknown. In this study, we cloned and characterized the HvMMP2 gene in H. vigintioctopunctata and investigated its function using RNA interference (RNAi). HvMMP2 exists as two transcript variants that differ at the 5′ end. HvMMP2 is highly expressed in the prepupal stage, the pupal stage and the intestine. Silencing HvMMP2 expression in fourth-instar larvae led to approximately 54% mortality at the prepupal stage, with the remaining larvae dying after pupation. RNAi with HvMMP2 in third-instar larvae did not affect their development to the fourth instar, but caused mortality in the majority of larvae during the prepupal and pupal stages, and most of these pupae exhibited wing deformities. Examination of these stunted larvae by dissection showed that their fat bodies were abnormally shaped and that yellow uric acid crystals had accumulated in the Malpighian tubules. Collectively, our findings indicate that HvMMP2 plays a critical role in pupation and eclosion in H. vigintioctopunctata and support HvMMP2 as a potential molecular target for further RNAi-based control studies. Full article

Endophytic fungi are promising sources of novel antiviral compounds, and the crude extract from Alternaria alternata PO4PR2 has previously shown anti-HIV-1 activity. This study evaluated its efficacy against integrase strand-transfer inhibitor (INSTI)-resistant HIV-1 and its mechanism of action. Key resistance mutations (Y143H, G118R, N155H, and R263K) were introduced into the HIV-1 pNL4.3 clone via site-directed mutagenesis and confirmed through Sanger sequencing. Viral infectivity was assessed in TZM-bl cells, while cytotoxicity was measured using an MTT assay. Antiviral activity was determined through a luciferase-based assay, and integration inhibition was evaluated using integrase activity assays and Alu-gag nested PCR. The extract demonstrated potent inhibition of resistant mutants, with low IC₅₀ values (0.02971–0.1652 μg/mL), and showed minimal cytotoxicity (CC₅₀ = 300 μg/mL), maintaining over 80% cell viability. It inhibited integrase activity by 67%, specifically targeting the strand-transfer step, and significantly reduced integrated viral DNA. Molecular docking of 14 compounds identified coumarin derivatives as key bioactive metabolites, exhibiting mutation-tolerant binding within the integrase catalytic pocket. Overall, these findings highlight PO4PR2 as a promising source of compounds for developing new therapies targeting drug-resistant HIV-1 integrase. Full article

Macrophages play a crucial role in health and disease. Currently, reporter mice for tracking alternatively activated macrophages in vivo are lacking. We designed a transgenic mouse model in which luminescence and fluorescence proteins, click beetle red luciferase (CBRED2) and mKate2, report on the expression of the Mrc1/Cd206 promoter, active in the monocyte/macrophage population. The mouse line was named B6Mrc1-mKate2-CBRED2. Using this novel mouse model, we were able to develop in vitro assays to validate transgenic macrophage polarization and test them with compounds of repolarization potency. Furthermore, in the in vivo assays, we exploited the migratory and infiltrative potency of macrophages for detecting tumor locations via optical imaging. In fact, macrophages can act as universal cancer markers, as they infiltrate primary and secondary tumors, stimulating or suppressing tumor growth. We first characterized transgenic mice for reporter expression ex vivo, followed by the generation of luminescence-based assays to reflect the polarity of differentiated macrophages, and lastly, we visualized reporter macrophages accumulating and infiltrating the tumor microenvironment (TME) of murine pancreatic ductal adenocarcinoma (PDAC) at multiple time points. We found that the extent of macrophage recruitment and retention was dependent on the infiltrative T-cell and dendritic cell populations present in the TME, reflecting the immunologically hot or cold nature of the PDAC clones, respectively. In conclusion, the ability to optically detect light-emitting macrophages can be applied not only for cancer studies but also in the context of inflammatory diseases. Full article

The flower color of Rhododendron is primarily determined by anthocyanin biosynthesis, with cytochrome P450 CYP75 family members, particularly flavonoid 3′,5′-hydroxylase (F3′5′H), playing a central role. However, the composition and functional characterization of CYP75 genes in Rhododendron remain insufficiently explored. This study performed genome-wide identification of the CYP75 gene family using the Rhododendron simsii reference genome and functionally characterized the corresponding F3′5′H homolog cloned from Rhododendron × hybridum petals (red cultivar and pink cultivar). Seven RsCYP75 genes were identified, categorized into two subfamilies: RsCYP75A (A1–A5) and RsCYP75B (B1–B2), with a prominent cluster on chromosome 13. All encoded proteins contained a conserved cytochrome P450 domain and typical heme-binding motifs. Among these, RhCYP75A2 showed the highest expression level in red petals at full blooming period and was designated as RhF3′5′H. RhF3′5′H encodes a basic membrane protein with the characteristic F3′5′H motif, with its transcript most abundant in flowers. Transient overexpression of RhF3′5′H in red R. × hybridum petals resulted in a 9.74-fold increase in its transcript levels and a 1.25-fold increase in anthocyanin content compared to that in the control accompanied by the up-regulation of CHS, F3H, DFR and ANS. Conversely, RhF3′5′H silencing reduced anthocyanin accumulation but increased CHS and F3H transcript levels, suggesting a compensatory transcriptional response in the upstream anthocyanin pathway. Moreover, RhF3′5′H was heterologously expressed in E. coli Rosetta as an MBP fusion protein, purified, and identified by LC-MS/MS and ELISA. The protein showed the ability to promote anthocyanin accumulation. Molecular docking analysis demonstrated that RhF3′5′H can bind to naringenin and dihydrokaempferol. These results confirm that RhF3′5′H is a functional F3′5′H-type CYP75A enzyme and a positive regulator of anthocyanin accumulation in Rhododendron petals. This work enriches the CYP75 gene catalog in Rhododendron and provides candidate genes for future studies on flower color regulation and molecular breeding. Full article

Background/Objectives: Cadherin-13 (CDH13), part of the cadherin family, is attached to the plasma membrane through glycosylphosphatidylinositol. CDH13 plays essential roles in the development of the neurological and vascular systems and is a risk factor for neural and cardiovascular diseases. CDH13 is expressed on the plasma membrane in both mature and uncleaved precursor forms with the prodomain. Although several anti-CDH13 monoclonal antibodies (mAbs) are available for basic research, there have been no reports of anti-CDH13 mAbs that can detect both the mature form and the uncleaved precursor in flow cytometry. Methods: We developed novel anti-human CDH13 mAbs (named Ca₁₃Mabs) using the mature form of CDH13-expressed cells as an antigen. Results: Among Ca₁₃Mabs, a clone, Ca₁₃Mab-17 (IgG_2b, κ) specifically recognized the mature and uncleaved precursor CDH13-overexpressed Chinese hamster ovary-K1 (CHO/CDH13) cells with no detectable cross-reactivity toward 21 other cadherins by flow cytometry. Ca₁₃Mab-17 also detected endogenous CDH13 in human glioblastoma (LN229 and U87MG) and lung mesothelioma (NCI-H2052) cell lines. The dissociation constant (K_D) value of Ca₁₃Mab-17 for LN229 was estimated at 4.1 × 10⁻⁸ M. Furthermore, Ca₁₃Mab-17 detected both the mature and uncleaved precursor CDH13 in Western blotting. It also identified new blood vessels and glioblastoma cells by immunohistochemistry. Conclusions: Ca₁₃Mab-17 is a versatile tool for detecting both mature and uncleaved precursor forms of CDH13 and has potential for tumor diagnosis and therapy. Full article

Recently, the existence of a new class of plant phosphotransfer proteins (HPts) with transmembrane (TM) domains was predicted by a large-scale bioinformatics method. These non-canonical proteins belong to the multistep phosphorelay (MSP) signal transduction system. The gene for one of these predicted TM-HPt was first cloned from tea (Camellia sinensis L.) plant cells. The membrane localization of the encoded protein (TM-CsHPt1) was confirmed using confocal microscopy and immunoblotting. These proteins were detected in the endoplasmic reticulum-enriched but not plasma membrane-enriched fractions. Using the BiFC method, the ability of TM-CsHPt1 to homodimerize was shown, similar to classical soluble HPt. However, heterodimerization between canonical and non-canonical CsHPts was not detected. Furthermore, TM-CsHPt1 was capable of specific interaction with the Arabidopsis cytokinin (CK) receptor AHK3, but not its paralogs AHK2 and AHK4. The obtained data are compatible with the involvement of TM-CsHPt1 in CK signaling (which utilizes the MSP system), possibly through a suggested non-canonical membrane branch. In addition, the key components of the CK signaling system in C. sinensis were uncovered and characterized by bioinformatics and phylogenetic analysis. The putative functions of the predicted MSP membrane branch in the tea plant are discussed. Full article

Soil salinization is one of the major abiotic stresses limiting agricultural production. As an economically important fruit tree worldwide, grapevine generally exhibits weak salt tolerance. Therefore, identifying key stress-tolerance genes is of great significance for improving stress resistance in grapevines. In this study, the transcription factor gene VvWRKY57, which is induced by salt stress, was cloned from the grape cultivar Vitis vinifera ‘Shine Muscat’. Its function under salt stress was systematically evaluated via heterologous overexpression in Arabidopsis thaliana. The full-length CDS of the VvWRKY57 gene is 915 bp, encoding a protein of 305 amino acids. The protein contains a typical WRKY conserved domain, belongs to group II of the WRKY family, and is localized in the nucleus and cytoplasm. Expression pattern analysis showed that VvWRKY57 was expressed in roots, stems, and leaves of grapevine. Based on this expression profile, transgenic Arabidopsis thaliana plants overexpressing VvWRKY57 were generated to further investigate its role in salt tolerance. Subsequent salt tolerance assays revealed that, compared with wild-type plants, the overexpression lines exhibited stronger resistance phenotypes under salt stress. This study demonstrates for the first time that grape-derived VvWRKY57 functions in enhancing salt tolerance in model plants, providing a novel genetic resource and theoretical basis for crop salt-tolerance molecular breeding using this gene. Full article

dTDP-L-rhamnose (Deoxythymidine diphospho-L-rhamnose) is a crucial active sugar nucleotide that serves as the key glycosyl donor for the synthesis of rhamnose-containing polysaccharides in bacteria, holding broad application potential in pathogen-associated molecular mimicry and vaccine development. In this study, the rhamnose synthase gene cluster (Pa-RmlABCD) was successfully cloned for the first time from the marine bacterium Pseudoalteromonas agarivorans Hao 2018. Four key enzymes—Glc-1-P thymidylyltransferase (Pa-RmlA), dTDP-glucose-4,6-dehydratase (Pa-RmlB), dTDP-4-keto-6-deoxyglucose 3,5-epimerase (Pa-RmlC), and dTDP-4-keto-rhamnose reductase (Pa-RmlD)—were heterologously expressed in Escherichia coli. A one-pot four-enzyme synthesis system was constructed, and the successful synthesis of dTDP-L-rhamnose was verified by Q Exactive Focus. After correction for recovery (92% ± 2%), the actual yield reached 3.47 mg/L with a conversion rate of 53.4% ± 1.1%. Combined with bioinformatics analysis, tertiary structure modeling, and molecular docking simulations, the sequence characteristics, substrate binding modes, and catalytic mechanisms of Pa-RmlABCD were systematically elucidated. By characterizing the marine-derived Pa-RmlABCD system and achieving efficient one-pot synthesis, this work opens up a new avenue for the sustainable production of dTDP-L-rhamnose, with the potential to alleviate the current industrial supply constraints. Full article

Yeast surface display is a powerful strategy for enzyme immobilization and whole-cell biocatalysis; however, the intracellular processing of heterologous enzymes during secretion and anchoring remains poorly understood. In this study, a GH5 endoglucanase gene (eglS, 1.4 kb) from Bacillus subtilis, originally isolated from a paper mill effluent, was cloned into the pYD1 vector and expressed in Saccharomyces cerevisiae EBY100 using the Aga1–Aga2 surface display system. The recombinant strain produced clear degradation halos on carboxymethyl cellulose (CMC) plates, confirming cellulolytic activity at the whole-cell level. Zymographic analysis revealed multiple active enzyme forms depending on the cellular fraction analyzed. Intracellular extracts displayed active bands ranging from 70 to 57 kDa, consistent with immature or partially processed Aga2 fusion proteins, whereas cell wall-associated fractions showed active bands between 55 and 35 kDa, suggesting proteolytic processing during secretion and surface anchoring. The apparent specific activity of the cytoplasmic fraction was 5.33 ± 0.31 U mg⁻¹, while the cell wall-associated fraction exhibited a higher apparent specific activity (58.4 ± 10.1 U mg⁻¹). Although these values were obtained from non-purified fractions and therefore do not represent intrinsic enzymatic constants, they indicate a relative enrichment of catalytically active enzyme in the cell wall-associated fraction, consistent with functional surface display. The presence of multiple active enzyme forms and the enhanced catalytic efficiency observed in the cell wall-associated fraction suggest that the engineered yeast strain may serve as a promising whole-cell biocatalyst, with potential applications in consolidated bioprocessing (CBP) strategies for lignocellulosic biomass conversion. Full article