Search Results (866)

Background/Objectives: The emergence of antimicrobial-resistant (AMR) pathogens in aquaculture ecosystems poses a significant risk to both food security and human health. Shewanella species are recognized as significant AMR reservoirs, yet their prevalence and resistance mechanisms within a shrimp-related ecosystem remain poorly characterized. This study aimed to perform a genotypic and phenotypic characterization of S. algae VK101, isolated from wild-caught black tiger shrimp (Penaeus monodon) broodstock. Methods: A complete 5.21 Mb genome was generated using a hybrid Illumina and Oxford Nanopore sequencing approach. Antimicrobial susceptibility was evaluated for 21 antibiotics via Minimum Inhibitory Concentration (MIC) testing. Comparative pangenomics and genome-wide association studies (GWAS) across 125 S. algae genomes were conducted to identify novel resistance determinants. Results: MIC analysis revealed that VK101 was resistant to ampicillin (>16 µg/mL) and colistin (8 µg/mL), while showing intermediate susceptibility to imipenem and ciprofloxacin. In silico analysis identified 205 antimicrobial resistance genes (ARGs), including a perfect hit for the fluoroquinolone resistance gene qnrA3. Notably, no mcr genes were detected. Although VK101 exhibited moderate resistance (8 µg/mL), GWAS across the broader S. algae population linked a specific lptA mutation (K140N) to high-level resistance (64 µg/mL). Other GWAS-identified genes (e.g., czcA, ampC, and oprM) likely represent indirect associations driven by genetic linkage or clade-specific markers rather than direct causal factors. Conclusions: These findings highlighted the presence of multidrug-resistant S. algae in wild-caught P. monodon broodstock, reflecting the occurrence of antimicrobial resistance in aquatic environments. Colistin resistance in these isolates was primarily mediated by chromosomal variants rather than mobile mcr elements, indicating the need for integrated genomic surveillance within the aquaculture value chain. Full article

Saffron (Crocus sativus L.) is a high-value crop vulnerable to potyvirus infections threatening its yield and quality. In this study, we combined Oxford Nanopore long-read sequencing with exploratory transcriptomic profiling to characterize the saffron virome and to describe expression profiles associated with two distinct infection histories: (i) saffron plants experimentally inoculated with cucumber mosaic virus (CMV; Cucumovirus CMV) and turnip mosaic virus (TuMV; Potyvirus rapae) under controlled greenhouse conditions, and (ii) saffron plants naturally infected by diverse viruses. We identified six plant-infecting viral families in both conditions, including Potyviridae, Geminiviridae, Caulimoviridae, Tymoviridae, Aspiviridae, and Partitiviridae. Transcriptomic profiling revealed distinct expression profiles associated with each infection background. Given the limitations of the experimental design, gene expression differences are interpreted descriptively. We describe pathway enrichments associated with antiviral responses. Naturally infected plants exhibited a broad-spectrum, tolerance-based response characterized by the upregulation of photosynthesis-related genes, calcium-mediated signaling components, and stress-responsive transcription factors. In contrast, virus-inoculated plants activated a targeted antiviral program involving RNA silencing, autophagy, ubiquitin-mediated proteolysis, and hormonal regulation. Both GO and KEGG enrichment analyses supported these findings, highlighting photosynthesis and metabolic flexibility in naturally infected plants versus hypersensitive response, RNA surveillance, and lignin biosynthesis in virus-inoculated plants. This work provides a comprehensive view of the saffron virome and offers a hypothesis-generating overview of transcriptional responses associated with natural versus experimental virus infections. These findings advance the understanding of the saffron virome and provide a valuable resource for breeding virus-resistant cultivars. Full article

To date, the problem of mastitis in cattle remains relevant for both the industrial sector and scientific research. Despite numerous active investigations, the causes of this disease have not been fully established. It is postulated that several factors may be involved, such as bacterial pathogens, animal husbandry practices, and weather and climatic conditions. In this study, we selected cows from farms in Yakutia to investigate microbial isolates present in the milk of cows affected by mastitis and treated with antibiotics. Five identified Staphylococcus aureus isolates were investigated using whole-genome sequencing (Illumina sequencing and nanopore sequencing), followed by analysis of virulence factors in the genomes and cultural properties of the isolates. The profile of S. aureus virulence genes (exotoxins, cytotoxins, superantigen-like proteins, adhesins) was identified via WGS. Hemolysin gene (hla) was detected in all isolates. An investigation of the cultural properties of the isolates, specifically through hemolysis of rabbit erythrocytes and Western blot analysis of the culture liquid of S. aureus, revealed different expression levels of alpha-hemolysin among the strains. One isolate (17-21) exhibited the highest secretion level of about 320 ± 37 ng, both in the hemolysis test and immunoblotting assay. An investigation of the isolates’ antibiotic resistance showed that all isolates exhibited multidrug resistance, as confirmed by the presence of antibiotic resistance genes in these isolates. One isolate (7-7) exhibited the broadest range of phenotypic resistance and was resistant to all tested antibiotics (except clindamycin). Phylogenetic analysis suggested that the evolution of these isolates occurred independently in their respective ecological niches, although their transfer from cattle to humans, and vice versa, is possible. Isolates 7-7, 18-22, 33-40, and 35-42 are most typical to Yakutian cattle, while isolate 17-21 might have been introduced from a different region. To the best of our knowledge, this is the first in-depth study into a range of S. aureus isolates associated with mastitis infection in Yakutian cattle. Full article

Providencia stuartii (Ps) is a clinically significant opportunistic pathogen often associated with “difficult-to-treat resistance” (DTR) infections due to pan-resistance to first-line antimicrobials. We report the clinical diagnosis and rapid genomic characterization of strain Ps-CMC-4104, recovered from a human splenic abscess in a patient with infected necrotizing pancreatitis. To resolve the complex genetic architecture of this strain, we utilized hybrid sequencing combining Oxford Nanopore (long-read) and Illumina (short-read) technologies. Analysis revealed a 4,504,925 bp circular chromosome featuring a unique genomic resistance island (GRI) closely related to Salmonella SGI1. Notably, the PsGRI contains multiple copies of NDM-1 and PER-1 carbapenem-resistance and -inhibitor genes, a repetitive structure typically unresolvable by standard short-read methods. Additionally, a large 278,489 bp low-copy circular plasmid harbored single copies of these carbapenemase and extended-spectrum β-lactamase genes alongside other antimicrobial resistance determinants and ISCR1 insertion sequences. Nanopore technology allowed us to precisely identify the duplications, providing critical insights into the strain’s pan-resistant phenotype. This study serves as proof-of-concept for the importance of integrating long-read sequencing into clinical workflows to identify complex resistance mechanisms in DTR pathogens, facilitating targeted antimicrobial stewardship and infection control. Full article

Background/Objectives: Castanopsis tibetana Hance (C. tibetana) is a valuable timber species in southern China. Its chloroplast and nuclear genomes have been characterized, but its mitochondrial genome (mitogenome) remains unknown. This study assembles and characterizes the first complete mitogenome of C. tibetana, elucidating its structural and evolutionary features. Methods: A hybrid approach combining Oxford Nanopore long reads and Illumina short reads was used. The mitogenome was assembled via iterative seed-based mapping and annotated via GeSeq and tRNAscan-SE. Repeats were identified via MISA, TRF, and REPuter. The RNA editing sites were predicted with the PREP suite. Phylogenetic analysis was performed on 14 conserved protein-coding genes from 13 species via maximum likelihood and Bayesian inference. Results: The mitogenome is a 554,078 bp circular molecule (GC 45.27%) encoding 51 genes (32 PCGs, 16 tRNAs, 3 rRNAs). It contains 202 simple sequence repeats (37.1% tetrameric). We predicted 53 C-to-U RNA editing sites, most frequently in nad7 and nad5. Codon usage showed bias, with 28 codons having RSCU > 1. Twenty fragments (6001 bp, 1.08% of the mitogenome) were transferred from the chloroplast. Phylogenomic analysis placed C. tibetana within Fagaceae, close to other Castanopsis species. Conclusions: This study provides the first comprehensive characterization of the C. tibetana mitogenome, revealing its structural architecture, repetitive landscape, RNA editing profile, and phylogenetic placement. These findings offer valuable genomic resources for understanding mitogenome evolution in Fagaceae and support future research on the conservation genetics and molecular breeding of this important tree species. Full article

Tick-borne encephalitis virus (TBEV) is primarily transmitted by Ixodes spp. and poses significant health risks, leading to morbidity and mortality in humans. Two of the five subtypes, Siberian and Far Eastern are known to circulate in Mongolia. In 2021, Ixodes persulcatus ticks were collected from Bulgan aimag (province) using flagging and dragging methods and subsequently screened for TBEV using PCR. Positive samples underwent sequencing using an Oxford Nanopore Technologies-based hybrid capture approach, resulting in two coding-complete TBEV genomes from separate tick pools. Phylogenetic analysis classified both genomes within the Siberian subtype, grouping them with other Mongolian sequences from I. persulcatus collected in 2014, 2020, 2021, and 2023. The study sequences, PX654173 and PX654174, showed high genetic similarity (99.9% and 99.8%, respectively) to the sequence PQ479142, obtained from I. persulcatus ticks in Selenge, Mongolia, in 2021. The estimated time to most recent common ancestor (TMRCA) of the Siberian genotype was approximately 981 CE (95% HPD: 646–1347) with the emergence of a distinct Mongolian clade of TBEV around 1888 CE (95% HPD: 1834–1934). These findings highlight the value of expanded whole-genome sequencing to improve our understanding of TBEV’s genetic diversity and evolutionary history in Central Asia. Full article

Diagnostic testing of foot-and-mouth disease virus (FMDV) currently utilizes reverse transcription quantitative PCR (RT-qPCR) to detect the presence of viral RNA and double antibody sandwich ELISAs (DAS-ELISAs) to determine viral serotype. Serotype identification is critical to support informed vaccine selection to combat outbreaks. While DAS-ELISAs are capable of serotype identification, the test suffers from low sensitivity and requires a viral isolate for successful detection. In this study, we developed FMDV-ONTAPS: an Oxford Nanopore Technologies Amplicon P1 Sequencing protocol involving reverse transcription-PCR to amplify P1 of the FMDV genome, and Nanopore sequencing of the amplicons to provide genetic data for serotype and subtype/topotype identification. FMDV isolates representing all seven serotypes were successfully sequenced with this method. Additionally, the protocol successfully provided serotype identification from a variety of specimen matrices obtained from experimentally infected animals that included milk, serum, oral and nasal swabs, tissue suspensions, vesicular fluid, and oral fluid. The limit of detection for FMDV cell culture isolates was comparable for both sequencing and RT-qPCR detection. RT-qPCR Cq values for clinical samples evaluated ranged from 8 to 28.21. Sequencing was successful for all samples except for a single tissue suspension sample (Cq of 28.21). Identification of FMDV serotype in clinical samples is critical for effective outbreak response, and Nanopore sequencing offers a timelier and more sensitive alternative to DAS-ELISAs. Full article

Severe Fever with Thrombocytopenia Syndrome (SFTS) is caused by the SFTS virus (SFTSV) and is associated with a high mortality rate. Although previous studies have reported RNA modifications such as m6A on SFTSV RNA, an integrated analysis of native viral transcript architecture and multiple RNA modification types within infected cells remains lacking. Here, we used Oxford Nanopore direct RNA sequencing (DRS) to analyze native SFTSV RNA in infected cells, combining strand-specific alignment, isoform reconstruction through read endpoint clustering, isoform-level quantification, and signal-level modification identification using unmodified in vitro transcripts as a baseline. This approach allowed us to construct detailed maps of the L, M, and bidirectionally encoded S segments at single-molecule, isoform-level resolution. The results reveal a “length-layering” pattern in SFTSV transcription, anchored by recurrent 3′ termination hotspots: only a few full-length transcripts dominate expression, whereas multiple reproducible truncated isoforms were associated with discrete termination windows, a pattern less consistent with random degradation alone and suggestive of regulated transcript termination. At the single-nucleotide level, the modification landscape is predominantly Ψ (pseudouridine), followed by m⁵C (5-methylcytosine), with sparse m⁶A (N6-methyladenosine). Modification hotspots are co-located across isoforms at the same genomic coordinates, exhibiting segmental/strand asymmetry, with sharper peaks on (−) RNA. These patterns provide a testable framework and raise the possibility that transcript-boundary organization and site-constrained Ψ/m5C signals may be associated with variation in viral RNA output. More broadly, isoform proportions around termination hotspots and Ψ/m5C-enriched regions at conserved sites may serve as quantitative features for characterizing viral RNA organization and prioritizing targets for future functional investigation. Our single-molecule integrated map establishes a reproducible methodological framework for studying SFTSV RNA regulation and provides a resource for future work aimed at assessing how transcript boundaries and RNA modification patterns may relate to polymerase activity and virus–host interaction. Full article

Underutilised, nutrient-dense legumes in their sprouted form provide promising substrates for developing functional fermented foods capable of influencing gut microbial activity and metabolite production. This study evaluated the effects of probiotic lactic acid bacteria-fermented beverages derived from sprouted Bambara groundnut (Vigna subterranea) and cowpea (Vigna unguiculata) on gut microbiota composition and short-chain fatty acid (SCFA) production using an in vitro colonic fermentation model. The beverages were fermented with either Bifidobacterium animalis BB-12 (BCBF24) or Lactiplantibacillus plantarum 75 (BCL7524). During colonic fermentation, at 0, 12, 24, and 38 h, faecal slurries were collected for SCFA analysis using gas chromatography–mass spectrometry (GC-MS) and deoxyribonucleic acid (DNA) sequencing (Oxford Nanopore Technologies). Microbial diversity decreased, indicating selective enrichment of taxa. BCL7524 induced a major shift, significantly (p < 0.05) enriching Bacillota and driving Megasphaera to ~42% dominance within 24 h. This reflected cross-feeding from L. plantarum to lactate-utilising Megasphaera spp. Spearman correlation linked Megasphaera to a broad SCFA profile, including isobutyric, isovaleric, valeric, and hexanoic acids, with a significant (p < 0.05) positive correlation observed for hexanoic acid. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated proteolysis and mapped hexanoic acid to fatty acid biosynthesis pathways, suggesting chain-elongation activity contributing to hexanoate formation. In line with this, BCL7524 produced significantly (p < 0.05) higher levels of hexanoate (3–14 mM) and valerate (10–15 mM), supporting chain-elongation activity within the community. In contrast, BCBF24 enriched Actinomycetota and Bifidobacterium, correlating with acetate production (18–23 mM). This study demonstrates that specific synbiotic beverages can modulate gut microbial ecology and metabolic output under in vitro conditions. Full article

Liverwort organellar genomes are generally highly conserved, but the subclass Pellidae (simple thalloids) shows unusual variation. This ancient yet unexplored lineage of simple thalloid liverworts provides an excellent model for investigating organellar genome evolution. In this study, we assembled four new plastid and four new mitochondrial Pellidae genomes using Oxford Nanopore sequencing, supplementing 86 plastomes and 82 mitogenomes from databases. We assessed nucleotide diversity and codon usage, and inferred phylogenies using IQ-TREE with fossil-calibrated dating. Plastomes ranged 120.6–126.5 kb, and mitogenomes 109–180 kb, with Apopellia endiviifolia featuring an exceptionally reduced mitogenome (~109 kb). Native RNA sequencing enabled a revised annotation of the mitochondrial atp1 gene in Apopellia, revealing two introns (previously thought absent) and reducing the intergenic region share to 36.26%, the lowest known among liverworts. Comparative analyses revealed contrasting evolutionary dynamics between organelles: Plastomes displayed higher nucleotide diversity and phylogenetically inconsistent codon usage patterns, likely influenced by compositional bias, whereas mitogenomes were more conserved and largely consistent with established phylogenetic relationships among the orders. Phylogenomic analyses yielded discordant topologies: Chloroplast data recovered Pellidae as a monophyletic clade, whereas mitochondrial data placed Pelliales (Pellia/Apopellia) as basal Jungermanniopsida, rendering Pellidae paraphyletic. Within Pellidae-relevant clades, several major divergences were dated to the Carboniferous–Permian, but with systematic chloroplast–mitochondrial offsets. These results highlight recurrent organellar incongruence and the dynamic evolutionary history of Pellidae organellar genomes. Full article

Kombucha fermentation is driven by a Symbiotic Culture of Bacteria and Yeast (SCOBY), a cellulose-rich biofilm that hosts a complex microbial consortium. While most kombucha studies focus on the liquid beverage, the SCOBY pellicle itself remains underexplored, particularly with respect to species-level microbial resolution and its intrinsic biological activities. In this study, a commercial kombucha SCOBY was characterized using full-length 16S rRNA gene and ITS amplicon sequencing based on Oxford Nanopore Technology, enabling species-level taxonomic resolution. In parallel, hydroalcoholic and aqueous extracts of dried SCOBY biomass were evaluated for in vitro antioxidant activity (DPPH and ABTS assays), antidiabetic-related enzyme inhibition (α-glucosidase and dipeptidyl peptidase-4, DPP4), and anti-aging-related enzyme inhibition (tyrosinase and elastase). The SCOBY bacterial community was strongly dominated by acetic acid bacteria, with Komagataeibacter saccharivorans and Acetobacter tropicalis accounting for more than 60% of total reads, reflecting a biofilm structure optimized for cellulose production and oxidative metabolism. The yeast community showed marked unevenness, with Brettanomyces bruxellensis representing over 80% of reads, consistent with its known role in ethanol production and stress tolerance within kombucha systems. In vitro assays revealed that hydroalcoholic SCOBY extracts consistently exhibited higher biological activity than aqueous extracts across all tested assays. However, both extracts showed substantially lower potency than purified reference compounds, indicating moderate but measurable bioactivity typical of complex fermented matrices. These findings support the potential valorization of SCOBY as a fermentation-derived biomaterial and functional ingredient while underscoring the need for further chemical characterization, mechanistic studies, and biological validation beyond enzyme-based assays. Full article

Dynamic changes in gene and transcript expression represent a key factor in regulating the cyclical development of hair follicles. In this study, based on Nanopore sequencing (ONT-seq) data of skin tissue from three developmental stages (anagen (An), catagen (Cn), and telogen (Tn)) of Jiangnan cashmere goat hair follicles, this study presents a profile of candidate DETs implicated in cycle regulation by delineating their stage-specific expression patterns and dynamic expression trends from anagen to telogen. A large proportion of the candidate DETs were significantly enriched in functions related to fat synthesis, storage, or metabolism, with significance levels of p < 0.05 or p < 0.01. These significantly enriched DETs, which were generally upregulated from An to Cn or downregulated from Cn to Tn, support a model where accelerated intradermal fat deposition promotes the progression from An to Cn, while its subsequent decrease facilitates the transition from Cn to Tn. Concurrently, our results also suggest a potential role for dynamic changes in AS patterns in regulating the hair follicle cycle. This regulatory role of AS patterns is potentially mediated through affecting genes related to lipid synthesis/metabolism or cell structure/interaction. Notably, a broader range of fat synthesis, storage, or metabolism-related transcripts showed significant differential expression (p < 0.05) in the An vs. Cn group. Ultimately, by establishing this candidate DET profile, we aim to provide fresh perspectives for deciphering the complex molecular regulation of the hair follicle cycle and to identify new targets for genetically enhancing or molecularly breeding cashmere traits in cashmere goats. Full article

Canine distemper virus (CDV) affects both domestic and wild carnivores and is associated with a high mortality rate. The virus can cross species barriers, infecting a wide range of mammals, which raises concerns for both wildlife conservation and domestic animal health. During our study, we processed a total of n = 552 oral and rectal swab samples from n = 260 red foxes (Vulpes vulpes) and n = 16 golden jackals (Canis aureus). The samples were collected by the National Food Chain Safety Office (NÉBIH) as part of a Rabies monitoring programme from Hungary in 2024. We performed a Real-Time RT-PCR, followed by a CDV-specific amplicon-based sequencing method using Oxford Nanopore Technologies to obtain the complete genome. All golden jackal samples tested negative, while both oral and rectal samples of one red fox tested positive for viral RNA. From this positive sample, we were able to sequence a partial CDV genome. Based on phylogenetic analysis of the haemagglutinin gene, our CDV sequence was assigned to the Europe lineage, one of the endemic lineages in the continent, infecting both threatened and common animals. This finding highlights the ongoing presence of CDV in wildlife populations and illustrates the value of integrated monitoring systems. Full article

DNA polymerase beta (Polβ) is a 39 kDa, single polypeptide enzyme that possesses both gap tailoring and nucleotidyl transferase activity and is the key polymerase involved in base excision repair (BER) and the final steps of active gene demethylation. We demonstrated that residues in the mouse Polβ protein, L301 and V303, are critical for Polβ’s interaction with the BER scaffolding protein X-ray repair cross-complementing 1 (XRCC1), and mutation of these residues impairs Polβ’s ability to bind to XRCC1, negatively impacting BER complex assembly. We developed Polβ^{L301R-V303R/L301R-V303R} knock-in mice to explore how defects with this essential protein complex impact genome stability in the mouse. We found these mice to be viable and fertile yet exhibited a modest reduction in body weight. Here, we examined the protein and mRNA levels in tissues from wild-type (WT), heterozygous (HET), and homozygous (HOM) Polβ^{L301R-V303R/L301R-V303R} mice and the derived fibroblast cell lines. We show that HOM mice have significantly diminished Polβ protein levels, as compared to WT mice, in several tissues, yet Polβ mRNA levels were not significantly different, suggesting the decreased levels of Polβ protein could not be attributed to lower gene expression. Upon examination of Polβ stability in mouse ear fibroblasts derived from WT and HOM mice, results are consistent with human cell studies that the Polβ^L301R-V303R protein is unstable and undergoes proteasome-mediated degradation. Finally, we evaluated WT, and HOM, liver and brain genomic DNA samples for 5-methylcytosine/5-hydroxymethylcytosine (5mC/5hmC) levels by nanopore sequencing to investigate the impact of suppressed Polβ protein levels on active gene demethylation. As expected, we found tissue-specific trends in methylation, when comparing the brain and liver. However, we were unable to discern substantial differences in methylation levels between WT and HOM mice, suggesting that in the absence of external stressors, low Polβ levels do not impact methylation patterns. Full article

Solid-state nanopore sequencing, a key third-generation sequencing technology, offers considerable potential for genomics and diagnostics due to its long read lengths, real-time detection, and amplification-free operation. The technology identifies DNA sequences by measuring characteristic changes in ionic current as single-stranded DNA translocates through a nanoscale pore. However, its practical development faces challenges including limited spatiotemporal resolution, pore clogging from nonspecific adsorption, and significant electrical noise. This review systematically examines strategies developed to address these limitations. We discuss the use of ultrathin two-dimensional materials such as graphene and molybdenum disulfide to improve spatial resolution, and methods to modulate DNA translocation through optimized solution conditions, pore geometry, surface charge engineering, and bio-solid hybrid pore designs. Furthermore, we detail noise suppression strategies targeting key sources like thermal noise, 1/f noise, and dielectric noise. These approaches encompass careful material selection, surface coatings, innovations in chip and amplifier design, and machine learning–based signal processing. The review also outlines surface functionalization techniques that reduce clogging and enhance analytical specificity. While challenges remain, continued convergence of materials science, nanofabrication, and data science is advancing solid-state nanopore technology toward reliable, high-precision sequencing platforms, promising to significantly impact personalized medicine and biological research. Full article