Search Results (177)

Background/Objectives: Because human prostate cancer (PCa) typically exhibits slow tumor growth, establishing reliable PCa tumor models is often time-consuming and unpredictable, thereby limiting the efficiency of preclinical theranostic research. To overcome this limitation, this study employed a non-viral PiggyBac transposon system to introduce triple-reporter genes into PSMA-expressing C4-2 cells, generating orthotopic and subcutaneous xenograft models that allow noninvasive, real-time monitoring of PCa progression and treatment response. Methods: Reporter-engineered C4-2 3R cells were generated by co-transfecting constructs encoding the reporter cassette and PB transposase, followed by enrichment through fluorescence microscopy and fluorescence-activated cell sorting (FACS) and implantation orthotopically or subcutaneously into mice. Tumor growth and treatment response to a single 2 Gy X-ray dose followed by 14.8 MBq of ¹⁷⁷Lu-PSMA-617, or to each monotherapy, were monitored weekly using an IVIS imaging system. Imaging findings were validated by tumor dissection and hematoxylin and eosin (H&E) staining, while PSMA expression was assessed by Western blotting and ¹⁸F-PSMA-1007 PET/CT. Results: C4-2 3R cells stably expressed the triple reporter genes (mRFP, luc2, and HSV1-tk), generating detectable orthotopic bioluminescence within one week and persisting for at least five weeks. In contrast, subcutaneous implantation generated only transient luc2 signals with no tumor formation. X-ray exposure did not increase total PSMA levels but induced the redistribution of PSMA to the cell membrane. Combined external beam radiotherapy (EBRT) and ¹⁷⁷Lu-PSMA-617 treatment produced higher ¹⁸F-PSMA-1007 uptake and stronger tumor suppression, with minimal residual tumor mass, as compared to single-treatment or control groups. Conclusions: This preliminary investigation suggests that the C4-2 3R model provides a practical and trackable tool for investigating slow-growing PCa tumors and evaluating PSMA-targeted therapies, either alone or in combination with EBRT. Full article

Pseudorabies virus (PRV) is an important α-herpesvirus. However, the chromatin state and transcriptional regulatory mechanisms of its genome upon entry into host cell nuclei remain poorly understood. In this study, we employed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to analyze chromatin accessibility and the transcriptome of the PRV genome in PK15 cells at multiple time points post-infection. ATAC-seq analysis revealed that the proportion of viral reads increased progressively over time, from 0.01% at 4 h to approximately 1.09% and 13% at 8 h and 12 h, respectively. A total of 112 ATAC peaks were identified, distributed across the PRV genome without apparent low-accessibility regions. Fragment length analysis demonstrated that the PRV genome does not adopt the regularly phased nucleosome organization typical of the host genome. RNA-seq analysis detected 66 expressed PRV genes, which were classified into four kinetic expression clusters, corresponding to immediate-early/early (IE/E), early (E), early-to-late transitional (E-L), and late (L) gene groups. Virus–host correlation analysis revealed significant associations between PRV gene expression and multiple host inflammation-related genes. Integrated multi-omics analysis further confirmed the concordance between chromatin accessibility dynamics and transcriptional activity changes. Collectively, these findings indicate that the PRV genome maintains a predominantly open and accessible chromatin state throughout lytic infection, providing new insights into the epigenetic regulatory mechanisms of α-herpesviruses. Full article

Antibiotic resistance genes (ARGs) have been widely recognized as emerging environmental contaminants due to their potential threats to public health and ecosystems. Owing to the extensive use of antibiotics in clinical, agricultural, and aquaculture settings, large quantities of ARGs are continuously introduced into the natural environment. As a result, aquatic environments—particularly surface waters—act as important reservoirs and transmission pathways for ARGs. However, despite the Yellow River being a major surface river in northern China, the occurrence and sources of ARGs in this river remain poorly understood. In this study, shotgun metagenomic sequencing was applied to investigate the composition of ARGs, mobile genetic elements (MGEs), and virulence factors (VFs) in three water sources in the Baotou section of the Yellow River. A total of 35 types of ARGs, 3 types of MGEs, and 14 categories of VFs were identified across all samples. Among them, multidrug resistance genes, peptide resistance genes and glycopeptide resistance genes exhibited relatively high abundances, indicating their widespread distribution in the study area. In terms of MGEs, recombinases and transposases were dominant, suggesting their important roles in gene mobilization. Meanwhile, VFs were mainly associated with metabolic functions, immune modulation, and adherence, reflecting the potential pathogenic risks in the aquatic environment. Co-occurrence networks revealed extensive associations between ARGs and MGEs, as well as ARGs and VFs. Many positive correlations were observed, suggesting possible ecological associations between ARGs and MGEs and potential co-selection between ARGs and VFs. Overall, this study provides valuable insights into the distribution patterns and co-occurrence patterns of ARGs, MGEs, and VFs in the Yellow River and offers a scientific basis for the management and control of antibiotic resistance pollution in large river systems. Full article

Hypertension threatens the health of aging captive giant pandas, yet its molecular signatures remain poorly characterized. Here, multi-omics sequencing was applied to explore the molecular regulatory process of hypertension and the pharmacodynamic effect of levamlodipine on this endangered species. Six aged giant pandas were divided into hypertensive and normotensive groups (three hypertensive and three normotensive pandas) based on clinical phenotypes and blood pressure measurements. A multi-omics approach was employed, including blood RNA-seq, Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), single-cell RNA-seq (scRNA-seq) of peripheral blood mononuclear cells, and time-series RNA-seq following levamlodipine administration. Our transcriptomic analysis revealed a statistically significant decline in ACE2 transcript abundance (Padj < 0.05), which suggests a possible shift in renin–angiotensin system signaling linked to hypertensive status in giant pandas. Single-cell analysis of 88,693 cells revealed that hypertension-associated genes were predominantly enriched in monocytes and T cells, implicating immune cell activation. Time-dependent transcriptional changes after levamlodipine administration. Temporal gene dynamics showed early activation of metabolic pathways followed by delayed inhibition of ion channels and calcium signaling. This study provides a transcriptional molecular perspective into the pathogenesis of hypertension in giant pandas, which is conducive to developing more effective antihypertensive treatment strategies, thereby protecting the health of this endangered species. Full article

The widespread use of antimicrobial biocides and metals has led to the continuous accumulation of biocide and metal resistance genes (BMRGs) in the environment. The issue is of growing concern, as it reduces the efficacy of these agents and poses a potential threat to coastal ecological security. However, the extent of coastal BMRG pollution, its transmission mechanisms, and the influence of seasonal variations on its assembly remain poorly understood. In this study, metagenomic sequencing was employed to investigate BMRGs, microbiomes, and mobile genetic elements (MGEs) within the subtropical nearshore ecosystem of the Beibu Gulf during the autumn and winter seasons. A total of 33 BMRG types and 457 subtypes were detected, with higher subtype diversity in winter than in autumn (440 vs. 326 subtypes). Notably, genes resistant to multi-biocides exhibited the highest diversity, whereas those resistant to both biocides and metals were the most abundant. Co-occurrence network analysis showed that 22 of the 23 detected BMRGs in the winter network were associated with MGEs, especially transposase-related elements such as tnpA. Path modeling indicated that BMRG abundance was more strongly associated with bacterial community composition in autumn, whereas MGE-related variables showed stronger associations in winter. These findings suggest a pronounced seasonal shift in the underlying mechanisms shaping BMRG dynamics, with bacterial communities playing a dominant role in autumn and MGEs playing a more critical role in winter. This seasonal shift highlights the need for season-specific monitoring of BMRGs, coastal pollution control, and resistance-risk management in subtropical coastal ecosystems. Full article

Clostridium perfringens is a major foodborne pathogen in which the genomic localization of the enterotoxin gene, cpe, plays an important epidemiological role. In this study, four isolates associated with independent foodborne outbreaks in South Korea were analyzed using complete genome sequencing. All isolates were cpe-positive, including three strains carrying chromosomal cpe (c-cpe) and one strain carrying plasmid-borne cpe (p-cpe). To provide a broader genomic context, complete genomes retrieved from the National Center for Biotechnology Information database were also analyzed. Most cpe-positive strains carried p-cpe, whereas c-cpe strains were relatively uncommon. Whole-genome analysis revealed a distinct separation between c-cpe and p-cpe strains based on conserved core-genome features and virulence gene profiles. In c-cpe strains, the cpe gene was consistently located between the nadA–C operon and a downstream nucleobase transporter gene and was flanked by IS1470 family transposases, suggesting a conserved chromosomal structure and a possible vertical inheritance. Conversely, p-cpe strains carried cpe on conserved pCW3-like plasmids, indicating that horizontal gene transfer mediated by a specific plasmid lineage contributes to cpe dissemination across diverse genetic backgrounds. Overall, these findings show that cpe localization is associated with distinct genomic patterns in C. perfringens. Full article

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology. Full article

Indole, a volatile metabolite produced by bacterial tryptophanase (encoded by tnaA) during tryptophan metabolism, contributes to oral malodor and may influence the progression of periodontitis. However, the genetic features underlying strain-specific indole production and its association with bacterial virulence remain unclear. Analysis of a previously published periodontitis cohort revealed that periodontitis severity was associated with salivary indole-related metabolic signatures, which were positively correlated with the abundance of Porphyromonas gingivalis (P. gingivalis). Further analysis showed that W83, a reference strain previously reported to exhibit relatively high virulence-associated characteristics, produced significantly higher levels of indole than ATCC 33277 under the experimental conditions. Comparative genomic analysis of 36 complete P. gingivalis genomes showed that the amino acid sequences of TnaA were highly conserved. However, the transposase region adjacent to tnaA differed among strains: previously reported high-virulence strains, including W83, W50, and A7436, harbored the IS5-family transposase ISPg1, whereas several low-virulence reference strains carried the IS982-family transposase IS195. In saliva samples from periodontitis patients, ISPg1 expression was positively correlated with tnaA expression, and both were associated with periodontal clinical parameters. Together, these findings indicate that transposase-associated genomic variation near tnaA is associated with strain-specific tryptophan-indole metabolism, virulence-associated gene expression, and periodontal clinical parameters, while direct causality remains to be established in future functional studies. Full article

The FAR-RED IMPAIRED RESPONSE 1 (FAR1) and FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) transcription factors, together with other members of the FAR1-RELATED SEQUENCE (FRS) and FRS-RELATED FACTOR (FRF) families, represent a striking example of transposable element domestication in plants. Derived from ancient Mutator-like element (MULE) transposases, these proteins have been repurposed as transcriptional regulators throughout the plant kingdom. FHY3 and FAR1 were first identified in Arabidopsis thaliana as positive regulators of phytochrome A (phyA) signaling. They participate in the coordination of light signaling with the circadian clock, chlorophyll biosynthesis, hormone pathways, stress responses, flowering time, shoot branching, leaf senescence, seed dormancy, and phosphate homeostasis. At the molecular level, FHY3 and FAR1 regulate gene expression mainly by binding to the conserved FHY3/FAR1-binding site, FBS, with the sequence CACGCGC, in the promoters of target genes. They also act through protein interactions with key signaling regulators, including HY5, PIFs, EIN3, TOC1, and SPL transcription factors. In this review, we summarize the molecular basis of FHY3/FAR1 gene family function, discuss the roles and mutant phenotypes of characterized family members, and highlight recent advances from other plant species beyond Arabidopsis. Collectively, this gene family illustrates how domesticated transposase-derived proteins have evolved into key regulators of plant development and environmental adaptation. Full article

Atypical Salmonella enterica strains that evade conventional detection pose significant challenges to food safety surveillance. A hydrogen sulfide (H₂S)-negative and serologically untypable S. enterica strain (SF1060) was detected by qPCR from cooked farmed mussels in Galicia, Spain, and characterized using phenotypic and genomic approaches. Despite typical biochemical profiles, SF1060 failed to produce black colonies on Xylose Lysine Deoxycholate (XLD) agar and lacked detectable somatic antigens by conventional serotyping. Hybrid genome assembly using nanopore and illumina sequencing yielded a closed chromosome and five plasmids. In silico analyses identified the strain as S. Senftenberg ST14. Comparative genomics revealed a chromosomal inversion at the rfb operon (encoding enzymes needed to synthesize deoxysugars and O antigens) mediated by IS5-family transposase ISEc68, which truncated the rfbD gene and separated the remaining rfb genes at rfbD, disrupting O-antigen biosynthesis, explaining the inconclusive phenotypic serotyping results. The phs operon responsible for H₂S production lacked premature stop codons, suggesting the H₂S-negative phenotype may result from an alternative mechanism. This study demonstrates how whole-genome sequencing resolves identification of atypical strains that fail culture-based detection and emphasizes the critical need for molecular surveillance methods in seafood safety programs, particularly in regions where atypical S. enterica variants may be endemic. Full article

Pathogenicity islands (PAIs) are regions of bacterial genomes that harbor genes encoding virulence factors. Identifying molecules that enhance pathogenicity is crucial for understanding the mechanisms pathogens employ to cause disease and their evolution. Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a pathogenic microorganism that causes caseous lymphadenitis (CLA) in sheep and goats. Despite its prevalence in Mexico, its genetic material has not been analyzed for virulence factors acquired through horizontal gene transfer. Therefore, the aim of this study was to characterize the complete genomes of Mexican C. pseudotuberculosis strains and identify virulence-related genes harbored with PAIs. Seventeen strains of C.pseudotuberculosis biovar ovis isolated from Mexico were whole-genome sequenced using illumina technology, assembled de novo with SPAdes, and annotated using Prokka. PAIs were predicted with GIPSy based on genomic signatures associated with horizontal gene transfer, including G + C deviation, codon usage, virulence factors, transposases, and tRNA-flanking regions. Positive selection was assessed using POTION v1.2 by identifying orthologous groups enriched in non-synonymous substitutions. This represents the first comprehensive PAI analysis of Mexican C. pseudotuberculosis strains, identifying 14 putative pathogenicity islands harboring 51 virulence-associated genes. Additionally, positive selection analysis identified five coding sequences, including radA and rpiB, that are undergoing adaptive evolutionary changes. These findings elucidate the pathogenic mechanisms and genomic plasticity of Mexican C. pseudotuberculosis strains. They also highlight novel genetic targets for vaccine and therapeutic development against CLA. Full article

Water availability and light conditions are among the most important environmental factors affecting tree growth and development. The FAR1/FHY3 (FRS) gene family consists of transposase-derived transcription factors that are widely involved in light signaling and responses to environmental stresses. Although FRS genes have been characterized in several plant species, a comprehensive analysis in P. bournei is still lacking. In this study, we performed the first comprehensive genome-wide analysis of the FRS gene family in P. bournei, including physicochemical characterization, chromosomal localization, phylogenetic analysis, gene structure and conserved motif analysis, protein structure prediction, promoter cis-element analysis, organ/tissue expression profiling, and RT-qPCR analysis under PEG-induced osmotic stress, full-light, and shade treatments. A total of 21 PbFRS genes were identified and found to be unevenly distributed across 11 chromosomes. Phylogenetic analysis, together with Arabidopsis thaliana and Zea mays FRS proteins, clustered the family members into five clades, including one P. bournei-specific clade, suggesting lineage-specific expansion and possible functional diversification. Structural analyses revealed both conserved and divergent features among PbFRS members. Promoter analysis identified diverse cis-acting elements related to light, temperature, hormones, and stress responses, suggesting that PbFRS genes may have diverse regulatory potentials in response to environmental signals. Organ/tissue expression profiling further revealed clear differences in expression patterns among family members. In addition, RT-qPCR analysis showed that several genes, including PbFRS9, PbFRS10, PbFRS12, PbFRS13, PbFRS16, and PbFRS18, exhibited transcriptional responses to PEG-induced osmotic stress, full-light, and shade treatments. These results indicate that these genes may serve as candidates for future functional studies, although their direct roles in stress tolerance require further validation. Overall, these results provide the first systematic overview of the PbFRS gene family and identify transcriptionally responsive candidate genes for future functional studies in P. bournei. Full article

Cell lineage relationship studies in developmental and regenerative biology have been greatly advanced using techniques such as fluorescent labeling driven by cell-type-specific promoters. Nevertheless, unbiased non-invasive tools for distinguishing cell lineages are inevitably desired. Mitochondrial DNA (mtDNA) exhibits wide-range single-nucleotide polymorphisms (SNPs) among individual cells. Here, we aim to distinguish cell types in organs/tissues of the same individual and in the regenerated liver based on the use of mtDNA SNPs. For this, two approaches—“Mitochondrial Alteration Enrichment and Sequencing” (MAESTER) and “mitochondrial single-cell assay for transposase-accessible chromatin with sequencing” (mtscATAC-seq)—were adopted to facilitate the detection of mtDNA SNPs in single cells. With MAESTER, we show that specific cell types in the liver and spleen of the same individual can be successfully defined using collective individual-specific markers composed of panels of unique mtDNA SNP combinations. For its application, we performed partial hepatectomy (PH) on a Krt19:Dre^ERT2/+;R26:Rox-ZsGreen-Stop-Rox-tdTomato/+ mouse harboring tdTomato-labeled cholangiocytes following tamoxifen injection and demonstrated that utilizing panels of unique mtDNA SNP combinations detected by mtscATAC-seq in the pre-PH cholangiocytes as markers can faithfully trace the cell fate in the post-PH liver samples. Hence, this approach may serve as an unbiased tool for investigating cell lineage relationships in relevant research areas such as liver regeneration. Full article

Hospital wastewater treatment efficacy is conventionally assessed by total antibiotic resistance gene (ARG) abundance; however, whether this metric accurately reflects biosafety risk remains poorly defined. Using a one-year longitudinal metagenomic survey (bimonthly sampling; n = 18 per group), we simultaneously profiled the resistome, virulome, and mobilome of hospital wastewater influent and effluent; stratified functional gene abundances by genomic origin; quantified ARG–mobile genetic element (MGE) colocalization; and characterized multicategory gene cocarriage across the 15 most abundant pathogenic species. Although the abundance of total strict ARGs decreased significantly in the effluent (p = 0.038), the abundances of metal resistance genes and virulence factors increased concurrently (both p < 0.01), and 8 of the 20 ARG subtypes were enriched rather than removed. This decline was driven exclusively by a reduction in the number of plasmid-encoded ARGs (p < 0.001), whereas genes encoding chromosomal virulence factors, metal resistance genes, biocide resistance genes, and MGEs were significantly enriched in the effluent (all p < 0.05). The normalized ARG–MGE colocalization rate was significantly greater in the effluent (p = 0.028), with a concurrent shift toward transposase-mediated chromosomal mobilization. Pathogen-associated metagenomic assemblies of clinically relevant species exhibited synchronous multicategory resistance coenrichment in the effluent, which is consistent with coselection under antibiotic, biocide, and metal pressures. Total ARG abundance is fundamentally decoupled from biosafety risk in treated hospital wastewater, warranting integrated surveillance beyond ARG-centric metrics. Full article

In this study, we systematically analyzed the dynamic changes in chromatin accessibility and the transcriptional responses in the spleen of largemouth bass (Micropterus salmoides) following infection with iridovirus (LMBV) using the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq). Based on post-infection survival status, largemouth bass were classified into a resistant group (SR) and a susceptible group (SS). A total of 11,317 differentially accessible regions were identified between the two groups, among which the chromatin accessibility of core promoter regions was entirely increased in the SR group, suggesting that chromatin remodeling in these regions may directly participate in the transcriptional regulation of immune-related genes. Functional enrichment analysis revealed that genes associated with differentially accessible regions were significantly enriched in immune-related pathways such as autophagy, apoptosis, Toll-like receptor signaling, and NOD-like receptor signaling. Motif analysis further identified that transcription factors significantly enriched in the SR group included CTCF and heterodimers composed of multiple members of the ETS and FOX transcription factor families. Through integrative analysis, seven transcription factors (CTCF, Spi1, ETV2::FOXI1, FOXJ2::ELF1, FOXO1::ELK1, SPIC, and FOXO1::ELF1) were found to be significantly enriched in core promoter regions. To further screen for differentially expressed genes directly regulated by chromatin accessibility changes, an overlapping analysis was performed between 629 predicted target genes and 2656 differentially expressed genes (DEGs), resulting in the identification of 71 candidate genes. Among these, three immune-related genes (irf4a, btk, and nfil3-2) belonging to the ETS and FOX families were identified. This study reveals the dynamic chromatin accessibility landscape of largemouth bass in response to LMBV infection and demonstrates that increased chromatin accessibility in core promoter regions is closely associated with the resistant phenotype. Heterodimers of ETS and FOX family transcription factors may participate in antiviral immune responses by regulating the expression of key immune genes such as irf4a, btk, and nfil3-2, providing potential epigenetic molecular markers for disease resistance breeding in fish. Full article