Search Results (2,138)

Red algae (Rhodophyta) are an ancient lineage of photosynthetic eukaryotes that play important roles in marine ecosystems. However, genomic information for many species within the order Rhodymeniales remains limited, particularly for mitochondrial genomes. In this study, we sequenced, assembled, and analyzed the complete mitochondrial genome (mitogenome) of Rhodymenia intricata to investigate its genome organization, gene content, and phylogenetic position within Rhodymeniales. The mitogenome of R. intricata is a circular DNA molecule of 26,213 bp containing 49 genes, including 25 protein-coding genes (PCGs), 21 tRNA genes, and 3 rRNA genes. The genome shows a strong A + T bias (70.9%) and positive AT and GC skews, typical of red algal mitogenomes. Comparative analysis with other Rhodymeniales mitogenomes revealed generally conserved gene content and organization, with several lineage-specific features such as the presence of the rpl20 gene, an additional open reading frame (orf148), and three rRNA genes (rnl, rns, and rns5). Codon usage analysis indicated a preference for leucine and isoleucine codons and dominant start and stop codons (ATG and TAA). Phylogenetic analysis based on a concatenated dataset of 23 mitochondrial PCGs strongly supported the monophyly of Rhodymeniales and confirmed the close relationship between R. intricata and R. pseudopalmata. Overall, this study presents the first complete mitogenome of R. intricata and expands mitogenomic resources for Rhodymeniales, providing new insights into mitogenome evolution and phylogenetic relationships in red algae. Full article

Background: Wastewater surveillance has emerged as an effective tool for population-level pathogen monitoring. Its application to mutations associated with resistance to antivirals remains comparatively underdeveloped. We assessed the wastewater epidemiology framework using SARS-CoV-2 as a model pathogen to evaluate spatial, temporal, and therapeutic class-specific resistance dynamics. Methods: We analyzed about 10,000 SARS-CoV-2-positive wastewater samples from six Ontario public health regions collected between October 2021 and July 2024. Fifty-five mutations were screened, comprising therapeutic resistance-associated mutations and a biologically distinct group of immune-evasion mutations. Mutations detected in ≥10 samples at ≥1% frequency were retained for spatiotemporal analysis using LOESS smoothing and Kruskal–Wallis testing. Results: Twelve mutations met the inclusion thresholds. S:E340D, associated with reduced susceptibility to sotrovimab was geographically widespread but transient and low-frequency. Five remdesivir-associated polymerase mutations were sporadic with sharp localized peaks, including two mutations exceeding 99% frequency in isolated catchments. Three nirmatrelvir-associated protease mutations were detected, with ORF1a:Q3452K showing significant regional variation. FLiRT and FLuQE immune-evasion mutations were most persistent and abundant. LOESS smoothing showed distinct temporal patterns among mutations, while Kruskal–Wallis testing identified significant regional variation for ORF1a:Q3452K and the three immune-evasion mutations. Conclusions: These findings demonstrate that wastewater surveillance enables population-scale monitoring of antiviral resistance and immune escape-associated mutations and offers a scalable model for broader surveillance. Full article

Severe coronavirus disease 2019 (COVID-19) is characterized by acute immune dysregulation, with monocytes playing a central role in driving inflammation and disease severity. However, the transcriptional and post-transcriptional regulatory mechanisms underlying monocyte dysfunction in severe COVID-19 remain unexplored. In the study, an integrative analysis of paired bulk RNA-seq and miRNA-seq datasets was performed together with independent single-cell RNA-seq (scRNA-seq) data from convalescent individuals with a history of ICU or non-ICU COVID-19. Pooled cell proportions descriptively indicated a higher proportion of classical monocytes and lower proportions of non-classical monocytes, B cells and dendritic cells in individuals with a history of ICU disease; however, none of these differences was statistically significant in patient-level analyses after multiple-testing correction. Using the miRSCAPE framework, miRNA expression was inferred at single-cell resolution and identified distinct cluster-specific inferred miRNA expression patterns. Differential expression analysis of classical monocyte populations identified 284 nominally significant differentially expressed genes between convalescent ICU and non-ICU samples. Integration of miRNA-mRNA correlation analysis with experimentally validated interactions and independent assessment highlighted a focused regulatory network centered on ZMAT3, RHOB and HLA-DQA1. Host–pathogen interaction analysis identified database-supported SARS-CoV-2-host interactions involving ORF3a-RHOB and nucleoprotein-RHPN2, with additional host–host interactions connecting RHPN2, HLA-C and HLA-DQA1. Collectively, these findings provide a computational framework for investigating inferred miRNA associations of monocyte inflammatory pathways associated with prior COVID-19 severity and highlight regulatory interactions that warrant further experimental validation. Full article

Bovine coronavirus (BCoV) is a key pathogen causing calf diarrhea and bovine respiratory diseases, bringing sustained economic losses to the cattle industry. As an RNA virus, BCoV possesses high mutation and recombination capacities, leading to prominent genomic genetic diversity. The genome contains hypervariable and conserved regions, with the S (especially S1), HE and Open Reading Frame (ORF4) genes serving as major variation hotspots linked to viral antigenicity, tissue tropism shift and immune evasion. Host immune pressure drives strong positive selection on S protein antigenic variation. This review discusses existing research limitations and proposes future directions including genomic surveillance, reverse genetics verification and broad-spectrum vaccine development to support BCoV prevention and control. Full article

C-type lectins (CTLs) are important pattern recognition receptors (PRRs) involved in innate immune defense in crustaceans. In this study, a novel C-type lectin, designated LvCTL, was identified and functionally characterized from Litopenaeus vannamei. Sequence analysis revealed that the open reading frame (ORF) of LvCTL is 495 bp in length and encodes a 164-amino acid polypeptide containing a typical C-type lectin-like domain but lacking the classical carbohydrate-binding motifs. Notably, two novel EPF motifs were identified in the CTLD region, suggesting a potential non-canonical ligand recognition pattern. Phylogenetic analysis revealed that LvCTL clustered with other crustacean CTLs. Tissue expression analysis revealed that LvCTL exhibited the highest transcript level in the hepatopancreas. After immune stimulation, LvCTL expression in the hepatopancreas was significantly regulated by LPS, Vibrio parahaemolyticus, Staphylococcus aureus, WSSV and Poly(I: C), indicating its involvement in antimicrobial immune responses. RNA interference assays showed that knockdown of LvCTL significantly increased the bacterial burden and reduced the survival rate of shrimp after V. parahaemolyticus infection. Furthermore, recombinant LvCTL displayed broad bacterial binding activity to Gram-negative and Gram-positive bacteria and induced calcium-dependent agglutination of V. parahaemolyticus and S. aureus. Taken together, these results demonstrate that LvCTL functions as an important immune recognition molecule and contributes to antibacterial defense against V. parahaemolyticus in L. vannamei. These findings broaden our understanding of the functional diversity of shrimp CTLs and their contributions to crustacean innate immunity. Full article

(1) Background: The Hepatitis B virus (HBV) is characterized by extensive genetic diversity, including low-frequency variants that contribute to disease progression. We aimed to characterize low-frequency variants and evaluate their potential clinical impact. (2) Methods: We utilized 104 HBV near-full-length sequences generated using next-generation sequencing (NGS) from people living with HIV (PLHIV). We used an in-house bioinformatics suite (HBVgenomeR v5.9.7) to filter for low-frequency variants (5–50%), which were compared to escape and drug resistance mutations (DRMs) and hepatocellular carcinoma (HCC)-associated mutations reported at the consensus level. Unclassified variants were characterized by HBV open reading frames (ORFs) to determine mutation frequency per genomic region. (3) Results: A total of six escape mutations were detected in 8/104 (7.7%) sequences, with _surfaceN131T being the most prevalent (5/8). We also observed six DRMs in 30/104 (28.8%), with _rtV173L being the most prevalent (21/30). Truncation mutations were also observed with _rtA181T/_sW172* and _rtM204I/_sW196L being the most prevalent. A total of 8/104 (7.7%) sequences had four variants associated with HCC. The _xP46S was the highest observed HCC-associated mutation at 5/8. We report 1152 unique uncharacterized variants across all ORFs, and these were found in 94/104 (90.4%) sequences. The RNaseH domain had the highest burden (330/1152, 28.6%). (4) Conclusions: Deep sequencing results identified clinically significant mutations, including those below the 20% detection limit of traditional sequencing, that would go unreported. This highlights the possible underreporting of mutational burden in people living with HBV/HIV, indicating the importance of deep sequencing to aid in HBV/HIV understanding and management. Full article

TDP-43 dysfunction is a defining feature of amyotrophic lateral sclerosis (ALS), yet no biofluid biomarker directly measures its functional activity. We developed a serum-based homogeneous time-resolved FRET (hTR-FRET) assay that quantifies TDP-43 RNA binding activity using synthetic UU-rich RNA probes. We analyzed 1080 serum samples from controls, sporadic ALS, and genetic subgroups (C9orf72, SOD1) across multiple biorepositories. Cross-sectionally, TDP-43 functional activity was elevated in ALS (mean 390 a.u.) versus controls (302 a.u.), yielding AUC = 0.79. Genotype means were 392 a.u. (sporadic), 382 a.u. (C9orf72), and 323 a.u. (SOD1); a 366 a.u. threshold achieved 95% specificity against controls. Longitudinally, Target ALS showed a modest but significant inverse correlation between TDP-43 activity and ALSFRS-R, while other cohorts exhibited similar non-significant trends. Elevated signal in serum likely reflects increased extracellular release of probe-competent TDP-43 species during cell death and exosomal shedding, rather than restored intracellular nuclear splicing function. This assay provides a proof-of-concept platform for the direct functional measurement of probe-competent TDP-43 species in serum. While it demonstrates moderate group-level discrimination, individual diagnostic performance requires prospective validation. The assay may support exploratory applications in genotype stratification and progression monitoring in future clinical studies. Full article

Currently, there is no effective ELISA detection methods for orf virus (ORFV) based on recombinant proteins and monoclonal antibodies(mAbs). This research aims to express recombinant B2L protein (rB2L) of ORFV and prepare high specificity monoclonal antibodies targeting rB2L. After codon optimization for ORFV B2L gene, the optimized gene fragment was chemically synthesized and ligated into a prokaryotic pET-30a(+) vector for expression and purification of rB2L. Taking the purified rB2L as immunogen, three stable hybridoma cell lines (1F2, 2B3 and 3H6) secreting anti-ORFV mAbs were obtained through indirect ELISA combined with immunofluorescence. Subtype identification results indicated all three antibodies carried κ light chains with heavy chain subtypes IgG1, IgG2b and IgG2a, respectively. Indirect ELISA titer detection showed all three mAbs possessed titers higher than 1:6400. Among them, mAb 2B3 achieved the highest titer up to 1:25,600. Western blot verified that mAb 2B3 could specifically bind both rB2L and ORFV. Indirect immunofluorescence assay also confirmed mAb 2B3 could recognize viral antigens inside ORFV-infected primary goat testicular cells. These data suggest that rB2L and its specific monoclonal antibody 2B3 provide core experimental materials for developing an effective ORFV detection method. Full article

Goose astrovirus genotype 2 (GAstV-2) is an important pathogen associated with gosling gout, and rapid detection is useful for early diagnosis and field surveillance. In this study, a visual assay for GAstV-2 detection was developed by combining one-step reverse transcription multienzyme isothermal rapid amplification (MIRA) with the nucleic acid cleavage activity of Pyrococcus furiosus Argonaute (PfAgo). MIRA primers and specific guide DNAs were designed based on a conserved region of the GAstV-2 ORF1b gene, and the PfAgo reaction conditions were optimized. The optimal reaction contained 1.0 μM gDNA, 0.6 μM PfAgo, and 1.0 mM MnCl₂. Using recombinant pUC57-ORF1b plasmid DNA as the template, the lowest detectable plasmid concentration under the tested conditions was 1.0 × 10⁰ copies/μL. In the specificity assay, only GAstV-2 produced a positive signal, with no cross-reaction observed with GAstV-1, Tembusu virus, H9-subtype avian influenza virus, goose circovirus, fowl adenovirus serotype 4, or goose parvovirus. The assay was further tested with 23 clinical samples suspected of GAstV-2 infection. In a preliminary evaluation of 23 clinical samples, the RT-MIRA-PfAgo results were concordant with those obtained by conventional RT-PCR and RT-qPCR. Overall, the RT-MIRA-PfAgo assay provided sensitive and specific GAstV detection within a short time, without requiring programmed thermal cycling or an expensive real-time PCR instrument for routine endpoint detection. This method may be useful for GAstV-2 detection in basic laboratories and field settings. Full article

The SWEET (Sugars Will Eventually be Exported Transporter) family, a class of sugar transporters identified in recent years, plays important roles in plant growth and development. Increasing evidence suggests that SWEET genes are also involved in plant responses to abiotic stresses, including salt stress. However, the genome-wide composition and salt-responsive functions of SWEET genes in Elymus nutans Griseb. have not been systematically characterized to date. This study presents the first genome-wide identification of the SWEET gene family in E. nutans and functional characterization of EnSWEET15 in salt stress response, providing candidate genes and a theoretical basis for improving salt tolerance in this species. A total of 12 EnSWEET genes were identified from the E. nutans genome and were unevenly distributed across eight chromosomes. Phylogenetic analysis classified them into four subfamilies, and promoter analysis revealed abundant cis-elements related to hormone signaling and stress responses, suggesting roles in abiotic stress adaptation. Expression analysis showed that EnSWEET15 was significantly upregulated in both roots and leaves under salt stress. The full-length EnSWEET15 coding sequence (930 bp ORF) was cloned and functionally characterized. Heterologous overexpression in Arabidopsis thaliana indicated that EnSWEET15 enhances salt tolerance. Transgenic lines showed reduced reactive oxygen species (ROS) accumulation and increased activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), compared with wild-type plants under salt stress. Overall, this study identifies EnSWEET15 as a positive regulator of salt tolerance via modulating ROS homeostasis, providing novel insights into SWEET-mediated salt stress response in perennial forages and valuable genetic resources for salt-tolerant breeding of E. nutans. Full article

Age estimation using DNA methylation analysis is one of the tools currently used in forensic science to facilitate human identification. A total of 117 buccal swab samples from individuals aged 18 to 67 years were analyzed using a custom panel encompassing 610 CpG sites across 12 age-related genes. The analysis was performed using the amplicon-based technology Ion AmpliSeq™, which incorporates Ion Torrent S5 sequencing (Thermo Fisher Scientific, Waltham, MA, USA). The resulting methylation data were used to develop and compare age-prediction models based on Watson-strand, Crick-strand, and combined Watson + Crick methylation matrices. After candidate CpG selection and model reduction, the final Watson-strand model used 5 CpG sites from the age-related genes C1orf132, ELOVL2, KLF14, PDE4C and TRIM59 and was fitted using Elastic Net regression with alpha grid search, lambda selection and internal cross-validation. Model validation yielded prediction results with a mean absolute error of 4.42 years for the Watson matrix, 4.75 years for the Crick matrix, and 5.08 years for the WC matrix. These results allow for the evaluation of the suitability of using Ion AmpliSeq™ technology in forensic age estimation studies based on DNA methylation analysis. Full article

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a major infectious disease that poses a severe threat to the global swine industry. To investigate the role of miRNAs in the infection and susceptibility of PRRSV, four miRNA libraries were constructed and sequenced from PRRSV-infected and mock-infected of Tibetan pigs and Large White pigs at 7 days post-infection. A novel miRNA, miR-novel-80, was differentially expressed between PRRSV-infected and mock-infected porcine alveolar macrophages from 2 pig breeds. Importantly, the over-expression of miR-novel-80 inhibited the replication of a PRRSV-1 strain and multiple lineages (L1, L5, and L8) of PRRSV-2 strains in a dose-dependent manner. Bioinformatic predictions and experimental validation demonstrated that miR-novel-80 restricts viral replication through a dual antiviral mechanism. Directly, it targets the PRRSV nsp1-coding region within the ORF1a to suppress viral proliferation. Indirectly, miR-novel-80 specifically down-regulates the expression of host factor CXXC finger protein 4 (CXXC4). This reduction relieves the suppression of the Wnt/β-catenin signaling pathway, which in turn activates NF-κB-dependent innate immune responses to further inhibit PRRSV infection. Collectively, this study investigates the biological characteristics of miR-novel-80 and unveils its underlying molecular mechanisms in restricting PRRSV infection in vitro. However, its biological function and anti-PRRSV therapeutic effect in vivo need further investigation. Full article

The RNA exosome plays a critical role in surveilling nuclear mRNA biogenesis and regulating co-translational mRNA decay in the cytoplasm. Unlike canonical translation, repeat-associated non-AUG (RAN) translation of a GGGGCC hexanucleotide repeat expansion (HRE) within an intron of the C9orf72 locus leads to the synthesis of neurotoxic dipeptide-repeat (DPR) proteins, contributing to the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis (C9-ALS/FTD). However, it remains unclear whether aberrant RAN translation is monitored and regulated co-translationally or how C9orf72 HRE (C9-HRE) mRNA is degraded during this process. Here, we demonstrate that RAN translation triggers the rapid decay of C9-HRE mRNA. During this process, the RNA exosome engages the translating ribosome-C9-HRE mRNA complex to mediate RAN translation-coupled mRNA decay. Moreover, overexpression of EXOSC3, a key subunit of the RNA exosome cap, promotes RAN translation-coupled decay of C9-HRE mRNA and suppresses DPR production. In iPSC-derived neurons, a reduction in EXOSC3 levels blocks C9-HRE mRNA decay in a translation-dependent manner, further confirming its role in RAN translation surveillance. These findings highlight the essential function of the RNA exosome, particularly EXOSC3, in mitigating RAN translation-associated toxicity and preventing pathological DPR production. This work provides insights into potential therapeutic strategies for C9-ALS/FTD and may have broader implications for other disorders involving RAN translation. Full article

Background/Objectives: Salmonella enterica (S. enterica) is a major poultry-associated foodborne pathogen and a persistent public health concern. The global rise in antimicrobial resistance has accelerated the search for alternative control strategies, including the use of bacteriophages. However, their successful application requires a comprehensive evaluation of their biological performance, genomic safety, and functional proteins. This study aimed to characterize Salmonella phage WP110 and assess its potential as a biocontrol agent in broiler-associated production systems. Methods: Phage WP110 was evaluated against 251 S. enterica isolates from broiler-related sources. Adsorption kinetics, one-step growth, environmental stability (temperature and pH), and effective multiplicity of infection (MOI) were determined using Salmonella Kentucky S1H28. Whole-genome sequencing (WGS) and bioinformatic analyses were performed for genome annotation, taxonomic classification, and safety evaluation. In addition, protein structural prediction of a putative endolysin (WP110-gp057) was conducted using AlphaFold2, followed by structural comparison and molecular docking with peptidoglycan. Biocontrol efficacy was evaluated in contaminated rice husk, chicken meat, and on non-food materials. Results: Phage WP110 demonstrated a broad lytic spectrum, lysing 248/251 S. enterica isolates (98.8%). It adsorbed rapidly (within 3–15 min) to host cells and exhibited a latent period of ~20 min with a burst size of 134 particles per infected cell. Phage WP110 remained stable at 4–45 °C and pH 5–11 but was inactivated at ≥75 °C and pH 2. Complete bacterial inactivation in broth assay was achieved at an MOI of 10⁴. Genomic analysis revealed a 110,216 bp linear dsDNA genome (39.74% GC) comprising 204 ORFs, 25 tRNAs, and long direct terminal repeats, with no detectable antibiotic resistance genes. Phylogenetic and intergenomic analyses classified phage WP110 as a novel species within the genus Epseptimavirus. Structural modeling of WP110-gp057 revealed conserved catalytic residues and high structural similarity to T5 endolysin, while docking analysis supported a structurally plausible interaction with peptidoglycan at the predicted active-site groove, consistent with its proposed role in host cell wall degradation. In application models, phage WP110 significantly reduced Salmonella contamination in rice husk (up to 4.3 log CFU/g), chicken meat (up to 1.7 log CFU/g), and on non-food material surfaces (0.7–1.5 log CFU reduction). Conclusions: Phage WP110 is a broad-host-range lytic phage with favorable infection kinetics, environmental robustness, and genomic safety. Its functionally supported endolysin and strong antibacterial efficacy across broiler-associated matrices highlight its potential as a biocontrol agent for Salmonella mitigation in poultry value chain. Full article

The production of multiple proteins using a single open reading frame (sORF)/polyprotein system is a powerful strategy for coordinated multi-protein expression in eukaryotes. The most widely used approach relies on 2A peptides, but conventional 2A systems suffer from several limitations, and their viral origin makes them less than ideal for commercial crop biotechnology applications. Self-excising protein (SEP) modules are a promising alternative that enables coordinated production of multiple proteins from a single ORF encoding a polyprotein precursor. SEPs provide distinct advantages over conventional systems and effectively address many of the limitations inherent to the 2A approach. An SEP module is a fusion protein composed of an N-terminal excising domain (NED) and a C-terminal excising domain (CED) joined by a peptide linker. Using this architecture, a panel of SEP modules has been developed by pairing an engineered intein (serving as the NED) with various CEDs, including a second engineered intein, a 2A-like peptide, and ubiquitin. These modules release multiple proteins from the polyprotein precursor with nearly stoichiometric expression and clean cleavage. Coordinated coexpression using SEPs has been successfully demonstrated in several eukaryotic systems, including yeast, mammalian cells, and plants. This review offers a comprehensive analysis of the SEP technology while underscoring its major applications. Full article