Search Results (244)

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host-encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Here, we report the activity results of 147 REases on dZ-modified PCR DNA. Among the enzymes tested, 53% are resistant or partially resistant, and 47% are sensitive when their restriction sites contain one to six modified bases. Sites with four to six dZ substitutions are most likely to resist Type II restriction. Our results support the notion that dZ-modified phage genomes evolved to combat host-encoded restriction systems. dZ-modified DNA can also reduce phage T5 exonuclease degradation, but has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into an E. coli plasmid, the level of dZ incorporation reached 19–20% of adenosine positions. dZ levels further increased to 29–44% with co-expression of a DNA polymerase gene from the same phage. High levels of dZ incorporation in recombinant plasmid are possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work expands our understanding of the dZ modification of DNA and opens new avenues for engineering restriction systems and therapeutic applications. Full article

Flag leaf angle (FLA) in rice (Oryza sativa L.) is one of the important traits affecting F₁ seed production by mechanization. Here, we report the map-based cloning and functional characterization of the FLA1 (FLAG-LEAF-ANGLE 1) gene, which resides at a major-effect quantitative trait locus (QTL). Through cell morphological observations and exogenous hormone treatment assays, we demonstrate that gibberellin (GA) modulates rice FLA by altering both the number of cell layers and cell length. Combining genetic and molecular biological analyses with genetic complementation and gene overexpression assays, we elucidated and validated the biological function of FLA1. In addition, we found that FLA1 is constitutively expressed and encodes a protein localized to both the cell membrane and nucleus. Via RT-qPCR assays, we further demonstrated that the FLA1^fla-R allele from the rice accession fla-R enhances GA biosynthesis by upregulating the expression of CLA1 and GA20ox2. Furthermore, yeast two-hybrid assays revealed that auxin-repressed protein 1 (ARP1) interacts with FLA1, suggesting a potential role of this interaction in the modulation of rice FLA. Collectively, our results demonstrate that optimizing rice FLA via molecular manipulation of FLA1 can resolve the problem of flag leaf shearing during F₁ hybrid rice seed production without compromising F₁ hybrid seed yield, thereby facilitating mechanized F₁ hybrid rice seed production. Full article

Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host–virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5′-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5′-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development. Full article

Cervical cancer (CC) can be prevented through continuous screening and the timely detection of cervical intraepithelial neoplasia (CIN) using immunohistochemistry techniques to identify biomarker expressions. In a previous study, we proposed nuclear REST loss as a biomarker in precancerous lesions and CC; however, no validated antibodies are available for detecting REST in cytology or cervical tissues. Although we have developed an IgM-type anti-REST monoclonal antibody capable of detecting REST in liquid-based cytology cells, it was not useful for the detection of REST in cervical tissues by immunohistochemistry. The main objective of this study is to generate single-chain variable fragments (scFvs) for the clinical evaluation of REST in cervical tissues from women with CIN and CC. Using RNA from an IgM-producing hybridoma anti-REST, we conducted RT-PCR and PCR to obtain the coding sequences for the variable regions of the heavy and light chains. These sequences were joined with a linker to create a single-chain antibody. The scFv was then cloned into the pSyn1 vector, expressed in E. coli TG1, and purified through chromatography. Subsequently, it was characterized using immunological methods to assess its biological activity and employed to evaluate REST expression in cytological samples and cervical tissues. The anti-REST scFv represents an innovative detection tool that retains the antigen recognition of the parental IgM while overcoming its size limitation, enabling tissue penetration and detection of REST in cervical samples. Its application facilitates the identification of REST in cervical samples, reinforcing REST’s potential as a diagnostic biomarker for CC and CIN. Full article

Background: Dihydroflavonol 4-reductase (DFR) is pivotal for anthocyanin biosynthesis and plays a crucial role in plant development and stress adaptation. However, a systematic characterization of the DFR gene family is lacking in Lonicera japonica Thunb. Methods: In the present study, based on genome and transcriptome data of L. japonica, the research identified six LjDFR gene family members throughout the entire genome. Results: The LjDFR genes were located on Chr.04 and Chr.09 and the full-length coding sequences of LjDFR1-LjDFR6 were cloned. Subcellular localization analysis showed that LjDFRs are primarily found at the cell membrane and in the nucleus. Phylogenetic analysis showed closer clustering of LjDFR genes with Capsicum annuum and Camellia sinensis. Promoter analysis linked LjDFR genes to light response, hormone signaling, and stress-responses. qRT-PCR analysis demonstrated tissue-specific and stage-specific expression patterns among LjDFR members. Notably, LjDFR2 expression was significantly higher in the intensely pigmented tissues of Lonicera japonica Thunb. var. chinensis (Wats.) Bak. compared to L. japonica. Coupled with its phylogenetic proximity to the anthocyanin-related CsDFRa and CaDFR5 genes, this suggests that LjDFR2 may be positively correlated with anthocyanin accumulation. Additionally, the expression of LjDFR2 and LjDFR4 was markedly induced by both drought and salt stress, indicating their roles in abiotic stress responses. Conclusions: This research provides a foundation for further functional studies of LjDFR genes in anthocyanin biosynthesis and stress resistance and offers candidate genes for molecular breeding of L. japonica. Full article

Small Cajal body-associated RNAs (scaRNAs) are essential for biochemical modification of spliceosomal RNAs and spliceosome function. Changes in scaRNA expression level have been associated with developmental issues, including cancer and congenital heart defects (CHDs), although the mechanism remains unclear. Small Cajal body-associated RNA 1 (scaRNA1) guides pseudouridylation at uridine 89 (Ψ89) of the spliceosomal RNA U2, a highly conserved modification that may be critical for spliceosome function. To investigate the role of scaRNA1 in splicing regulation, CRISPR-Cas9 genome editing was used to introduce targeted deletions in the scaRNA1 locus in HEK293T cells. Edited clones were identified by T7 endonuclease I assay and confirmed by Sanger sequencing. Pseudouridylation at Ψ89 was quantified using CMC-based reverse transcription followed by quantitative PCR, and global mRNA splicing alterations were assessed by RNA sequencing. Clones harboring scaRNA1 disruptions exhibited a significant reduction in Ψ89 pseudouridylation, consistent with impaired scaRNA1 function. Transcriptome analysis (of mRNA from two clones) revealed >300 protein coding genes with significant changes in transcript isoform level, including >100 genes related to RNA-binding activity. These results indicate that scaRNA1 disruption alters spliceosomal function and leads to substantial changes in mRNA splicing. The dysregulated splicing of RNA-binding proteins may impair RNA processing and gene expression programs required for normal development, providing new insight into how noncoding RNA dysfunction may contribute to developmental pathogenesis. Full article

The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications. Full article

Bean common mosaic virus (BCMV; Potyvirus phaseovulgaris) is one of the primary viruses that severely impacts the yield and quality of common beans (Phaseolus vulgaris L.) and has a worldwide distribution. Utilizing small RNA sequencing and RT-PCR validation, this study identified widespread co-infection by multiple viruses in field-collected common bean samples, with BCMV being the dominant viral species. A novel isolate, designated DY9, was obtained from these field samples. Pathotype characterization confirmed DY9 as pathotype PG-III, while previous studies reported all other PG-III members as Bean common mosaic necrosis virus (BCMNV). Whole-genome sequencing and phylogenetic analysis revealed that DY9 was genetically closer to BCMV and diverged significantly from known PG-III isolates. Based on these findings, we constructed an infectious clone of DY9. To address the genetic instability of Potyvirus in the Escherichia coli (E. coli) expression system, we discovered that inserting Intron 2 (derived from the NiR gene of P. vulgaris, GenBank: U10419.1) at position 2431 of the HC-Pro gene and targeting Intron 1 (derived from the ST LS1 gene of Solanum tuberosum, GenBank: X04753.1) at position 4240 of the CI gene significantly improved the stability of the cloning vector. The clone was verified to systemically infect common bean plants and induce typical mosaic symptoms. Infectivity was validated through RT-PCR, RT-qPCR, Western blotting, and transmission electron microscopy. This study represents the first successful construction of an infectious clone for pathotype PG-III BCMV, providing a critical reverse genetics tool for dissecting viral pathogenesis and identifying resistance genes. These findings not only expand the genetic diversity of BCMV but also offer a methodological reference for constructing infectious clones of Potyvirus species. Full article

Staphylococcus aureus is a globally crucial foodborne pathogen that can cause diarrhea, vomiting, and bloodstream infection in immunocompromised individuals. S. aureus has three predominant sequence types (STs) (ST7, ST188 and ST398) that are prevalent clones in both food and clinical cases. This study aimed to screen ST-specific targets for S. aureus ST7, ST188 and ST398, and then developed a novel rapid and accurate assay for the detection of these three predominant S. aureus STs in food. A total of 505 Staphylococcus strain genome sequences including 371 sequences of 58 different STs and 134 other non-target S. aureus ST genome sequences were subjected to pan-genome analysis; we successfully screened five novel ST-specific targets (group_10498 and group_10499 target for S. aureus ST7, group_9415 and group_9419 target for S. aureus ST188, group_9911 target for S. aureus ST398). The excellent specificity and sensitivity of all the targets were confirmed by PCR assays. Based on these molecular targets, mPCR and qPCR methods were developed for specifically identifying S. aureus’ three predominant STs without non-target bacterial interference. The limits of detection (LODs) for the mPCR assay in artificially contaminated milk were determined to be 10⁴ CFU/mL for ST7, 10⁵ CFU/mL for ST188, and 10⁴ CFU/mL for ST398, while the LODs achieved by the qPCR method were 8.6 × 10² CFU/mL, 1.2 × 10² CFU/mL, and 6.4 × 10³ CFU/mL, respectively. The testing results for actual food samples suggested that the developed mPCR or qPCR assays could be used as an alternative to standard MLST analysis, for the rapid and reliable identification of S. aureus STs. The novel molecular detection technology established in this study provides an efficient and reliable detection method for the prevention and control of predominant S. aureus ST contamination in food and has important application potential and promotion prospects. Full article

Apricot (Prunus armeniaca L.) exhibits a gametophytic self-incompatibility (GSI) system. To identify the S-genotypes of the main apricot cultivars, including 133 native Chinese cultivars and 35 foreign accessions, PCR was performed using a combination of five primers based on the conserved regions of Prunus S-RNase genes. After cloning and sequencing the PCR products, the S-genotypes of all 168 apricot cultivars were determined. A total of 46 different S-RNase alleles, with 15 new alleles, were identified. For all 168 accessions, the top five most frequent S-alleles were S₈, S₁₁, S₉, S₁₆, and S₅₃. S₁₁, S₈, and S₁₆ were the most frequent in Chinese cultivars, and S₉, S₈, and S₂ were mostly found in European accessions. For Chinese apricot cultivars, the distribution of S-alleles among five geographic regions was also investigated. In Northwest China, S₁₆ was the most frequent S-allele. In the Xinjiang region, S₆₆, S₄₉, and S₁₄ were the top three most frequent S-alleles. In North China, S₈, S₁₁, and S₅₃ were the top three most frequent S-alleles. In addition, the self-compatible type, S_C, was not detected in these 133 Chinese accessions. Finally, the phylogenetic tree of apricot S-alleles indicated that there are four groups of S-RNase genes (S₉₇/S₁₀₆, S₁₄/S_14a/S₆₆, S₉/S₁₇/S₄₄, and S₂₃/S₅₃) presenting a very close relation. These results provide more data on the S-genotypes of apricot accessions, which can support future breeding programs by aiding in the selection of the appropriate parents and contributing to efficient orchard design by combining cultivars with suitable pollinizers. Full article

The construction of infectious clones (ICs) is essential for studying viral replication, pathogenesis, and host interactions. Milk vetch dwarf virus (MDV), a nanovirus with a multipartite, single-stranded DNA genome, presents unique challenges for IC development due to its segmented genome organization. To enable functional analysis of its genome, we constructed full-length tandem-dimer-based ICs for all eight MDV genomic segments. Each segment was cloned into a binary vector and co-delivered into Nicotiana benthamiana, Nicotiana tabacum, Vicia faba, and Vigna unguiculata plants via Agrobacterium-mediated inoculation. Systemic infection was successfully reconstituted in all host plants, with PCR-based detection confirming the presence of all viral segments in the infected leaves of nearly all tested plants. Segmental accumulation in infected plants was quantified using qPCR, revealing non-equimolar distribution across hosts. This study establishes the first complete IC system for MDV, enabling reproducible infection, replication analysis, and quantitative segment profiling. It provides a foundational tool for future molecular investigations into MDV replication, host interactions, and viral movement, advancing our understanding of nanovirus biology and transmission dynamics. Full article

Antimicrobial resistance (AMR), particularly due to extended-spectrum β-lactamases (ESBLs) and carbapenemases (CPs), poses a critical threat to global health. This study aimed to characterize the molecular epidemiology, resistance profiles, and genomic features of ESBL- and CP-producing Escherichia coli and Klebsiella pneumonaie (ESBL/CP-Ec/Kp) isolates from a Spanish hospital (2020–2024) and explore links to environmental reservoirs like white storks foraging at a nearby landfill. A total of 121 clinical Ec/Kp isolates (55 ESBL-Ec, 1 CP-Ec, 35 ESBL-Kp, 17 CP-Kp, 13 ESBL+CP-Kp) underwent phenotypic testing, PCR, and whole-genome sequencing (WGS). Analyses included phylogenomics (cgMLST), detection of AMR genes, plasmid typing, and comparative genomics. Among ESBL-Ec, bla_CTX-M-15 was the most prevalent (60.0%), and one CP-Ec carrying bla_NDM-5 was identified. WGS of 44 selected ESBL/CP-Ec isolates revealed a variety of AMR genes, and 56.8% of isolates carried class one integrons (56.8%). IncF-type plasmids predominated, and 84.1% of isolates were assigned as ExPEC/UPEC. The lineage ST131 dominated (75%), with IncF-bla_CTX-M-15-carrying plasmids. Among the 18 ESBL/CP-Kp isolates sequenced, the lineage ST307 was the most frequent (44.4%), followed by ST15 and ST11, carrying a diversity of AMR determinants and plasmids (IncFIB(K), IncL, ColpVC). Virulence included ybt loci in ICEKp; hypervirulence genes were absent. Genomic analysis of 62 clinical isolates (44 Ec, 18 Kp) showed close phylogenetic links to stork-derived strains, with ST131-Ec and ST307-Kp from humans and birds differing just by ≤22 and ≤10 ADs, respectively, with a conserved plasmid content (i.e., IncL-bla_OXA-48, IncFIB(K)-bla_CTX-M-15). High-risk ESBL/CP-Ec/Kp clones persist across clinical and environmental contexts. WGS-based surveillance is key for understanding AMR spread and guiding interventions. Results support a One Health approach to combat AMR through cross-sector collaboration. Full article

Enhancing the specificity and applicability of PCR-based genome-walking methods is highly desirable. A new and universal genome-walking tool, called center degenerated walking-primer PCR (CDWP-PCR), is presented in this study. CDWP-PCR involves adopting a center degenerated walking primer (cdWP) in the secondary/tertiary round of amplification. This cdWP is generated by degenerating the seven central nucleotides of the normal walking primer (nWP) used in primary PCR to NNNNNNN (where N includes the bases A, T, C, and G). Clearly, a partially complementary structure is formed between the two primers. Accordingly, the primary CDWP-PCR non-target products defined by the nWP are diluted in secondary/tertiary CDWP-PCR, as these non-targets have difficulty in annealing with the cdWP; conversely, the primary target product can still be efficiently amplified. The working performance of the proposed CDWP-PCR is verified through cloning of the unknown flanks of three known genes. All the clear DNA bands in the tertiary CDWP-PCRs are confirmed to be correct, and the largest DNA band is 8.0 kb. Overall, CDWP-PCR can be considered as a reliable supplement to existing genome-walking methods. Full article

Prolonged expression of gene-editing components in CRISPR-modified plants can interfere with phenotypic analysis of target traits, increase the risk of off-target mutations, and lead to unnecessary metabolic burden. To mitigate these issues in peanut (Arachis hypogaea L.), callus-specific promoters were screened to restrict Cas9 expression to the callus stage, minimizing its activity in regenerated plants. In this study, six callus-specific genes in peanut were identified by mining RNA sequencing datasets and validating their expression profiles using quantitative reverse transcriptase PCR. The promoters of Arahy.H0FE8D, Arahy.WT3AEF, Arahy.I20Q6X, Arahy.ELJ55T, and Arahy.N9CMH4 were cloned and assessed for their expression activity. Beta-glucuronidase (GUS) histochemical staining confirmed that all five promoters were functional in peanut callus. Further investigation revealed their ability to drive cytosine base editing via a deaminase-nCas9 fusion protein, with all promoters successfully inducing precise base substitutions in peanut. Notably, P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-ELJ55T, and P_Ah-N9CMH4 exhibited comparable or higher editing efficiencies than the commonly used cauliflower mosaic virus 35S promoter. These findings provide valuable tools for improving the biosafety of CRISPR-based genome editing in peanut breeding programs. Full article

The immune landscape of tumor-draining lymph nodes (TDLNs) plays a critical role in shaping antitumor responses and influencing prognosis in oral squamous cell carcinoma (OSCC). Among patients with lymph node (LN) metastasis, clinical outcomes vary widely, yet reliable biomarkers for prognostic stratification remain limited. This study aimed to identify immune features in tumors and LNs that differentiate between favorable and poor outcomes in OSCC patients with nodal metastasis. We analyzed T cell receptor (TCR) CDR3 repertoires and the expression of immune-related genes in primary tumors and paired sentinel LNs from OSCC patients who underwent tumor resection and lymphadenectomy. Patients were divided into three groups: Group A (no nodal metastasis), Group B1 (metastasis without recurrence), and Group B2 (metastasis with recurrence). TCR diversity was assessed using the Shannon index. The expression of immune-related genes (e.g., CD3E, CD4, CD8B, FOXP3, CTLA4, IL2, IL4) was measured by quantitative PCR and normalized to GAPDH. TCR diversity was lower in tumors than in non-metastatic LNs, reflecting clonal expansion. Metastatic LNs exhibited tumor-like diversity, suggesting infiltration by tumor-reactive clones. Tumor gene expression did not differ across groups, but LNs from metastatic cases showed the reduced expression of several immune genes. Notably, CD3E, CD8B, CTLA4, IL2, and IL4 distinguished B1 from B2. The immune profiling of LNs offers superior prognostic value over tumor analysis in OSCC patients with LN metastasis. LN-based evaluation may aid in postoperative risk stratification and personalized postoperative management and could inform decisions regarding adjuvant therapy and follow-up strategies. Full article