Search Results (476)

Monkeypox virus, a zoonotic DNA virus belonging to the Orthopoxvirus genus, has emerged as a global health issue because of its fast spread to 104 nations over six continents. In the current study, an immunoinformatics pipeline was used to design a multiepitope-based prophylactic vaccine targeting the A35R glycoprotein and E8L membrane proteins of the monkeypox virus. Selected target proteins were surface-exposed, non-homologous to the human proteome, and essential for viral pathogenesis. B-cell and T-cell (MHC-I and MHC-II) epitopes with high antigenicity (>0.5), non-allergenicity, non-toxicity, and highly soluble in water with strong affinity towards innate and adaptive receptors, were prioritized. Shortlisted epitopes were combined to design the final vaccine utilizing an adjuvant (50S ribosomal L7/L12) and appropriate linkers for improved immunogenicity. Population coverage analysis showed wide HLA representation with 83.57% (MHC-I) and 88.8% (MHC-II) global coverage, including 89.6% for West Africa and 87.3% for Central Africa. Docking analysis of the vaccine construct with the TLR-4 receptor revealed stable interactions (−695.6 kcal/mol). Molecular dynamics simulations and binding free energies further confirmed structural stability. Immune simulations predicted strong activation of both humoral and cellular immune responses. These results indicate that the designed multiepitope vaccine construct is a viable option for additional experimental validation against the monkeypox virus. Full article

Background/Objectives: Influenza, RSV, and SARS-CoV-2 co-circulate and evolve under immune and therapeutic pressures, complicating decision-making for both vaccine formulation and antiviral use. Fragmented, pathogen-specific sequencing approaches limit cross-virus comparability. Methods: We applied a standardized, multiplexed, amplicon-based next-generation sequencing (NGS) workflow to 34 diagnostic specimens (Ct < 35) positive for influenza A/B, RSV-A/B, or SARS-CoV-2. Sequencing libraries were generated and run on an Illumina MiSeq platform (2 × 250 bp). Although the wet-lab workflow is standardized across pathogens, consensus generation and annotation utilized two different analysis environments: Geneious Prime for influenza and MicrobioChek for RSV and SARS-CoV-2. Quality metrics included genome breadth and depth of coverage. Results: Near-complete genomes (mean coverage ≥98%) were recovered for all samples. Influenza A(H1N1)pdm09 sequences clustered in clade 6B.1A; A(H3N2) clustered in subclade 3C.2a1b.2a.2; and influenza B belonged to the Victoria lineage V1A.3a.2. RSV sequences were assigned to Nextclade clades A.D.5.1, A.D.1.10, A.D.2.1, and A.D.3 (RSV-A) and to B.D.4.1.3 and B.D.E.1 (RSV-B), consistent with the ON1 (RSV-A) and BA (RSV-B) genotypes prevalent in recent seasons. Clinically relevant mutations included changes in the influenza HA site and neuraminidase substitutions, RSV F-protein polymorphisms, and spike protein substitutions associated with recent Omicron sublineages (L455F/S, F456L) in SARS-CoV-2. Conclusions: A unified amplicon–NGS approach yields harmonized genomic data across respiratory viruses, enabling timely detection of antigenic drift and resistance markers while supporting integrated, cross-pathogen surveillance. Full article

Equine Infectious Anemia (EIA) is a retroviral disease of equids, for which there is no vaccine particularly adapted to American viral strains. In this work we searched for possible epitope regions for the surface proteins gp90 and gp45, rationally employing the latest available bioinformatics tools that constitute the state of the art in the field. We selected eight regions that contain numerous overlapping epitopes that have a high coverage amongst American viral strains and designed a chimeric envelope protein with those proteins fused in tandem as a novel vaccine candidate. In silico predictors were used to analyze chimeric protein physicochemical and immunogenic properties, as well as its allergenicity and toxicity. Protein structure was predicted and validated, and its ability to trigger cytotoxic immune responses was predicted by molecular docking to ELA alleles. The proposed sequence is predicted to be highly immunogenic and sets the base for a novel EIAV vaccine that could be used to protect against several American field strains. Full article

Silica particles are promising multifunctional drug delivery platforms; however, when in contact with blood or other biological fluids, proteins rapidly adsorb to their surface, forming the protein corona that modulates their biological interactions. In this study, silica microparticles were coated with lipid bilayers using two approaches: the lipid film hydration method and the on-particle solvent-assisted lipid coating (OPSALC) technique. We investigated how phospholipids with varying charges (zwitterionic, anionic, and cationic) and membrane phase influence coating formation and protein corona adsorption. The coating coverage and aggregation were characterized by fluorescence microscopy. The lipid film hydration method enabled coating with a broad range of lipids, but was highly dependent on the membrane phase and electrostatic interactions between lipid head group and particle surface. Pure anionic coatings were not achievable with this method; however, when combining the OPSALC method with a pre-silanization step, fully anionic coatings of silica microparticles were successfully obtained. Assessment by SDS-PAGE revealed differences in protein corona profiles modulated by the lipid compositions on the particles’ coatings. Overall, this study highlights the dependence of coating formation and protein corona composition on the phospholipid coatings’ properties. Full article

Biogas slurry is rich in nitrogen, phosphorus and bioactive substances, making it an effective material for restoring degraded grasslands. Against this background, we conducted a field experiment in Zhenglan Banner, Xilingol League, Inner Mongolia Autonomous Region, China, from 2024 to 2025, to study the short-term effects of biogas slurry fertilizer on vegetation characteristics and above- and belowground plant traits. The experiment comprised three treatments: a water control (CK), 50% diluted biogas slurry (BS_50%), and full-strength biogas slurry (BS_100%). All treatments were applied at a rate of 300 m³·ha⁻¹, with CK receiving an equivalent volume of water. The biogas slurry contained 0.11% nitrogen (N), 0.07% phosphorus (P₂O₅), and 0.09% potassium (K₂O). Results showed that, compared with the control, biogas slurry application increased plant height, coverage, and biomass by 8.04–54.00%, 5.48–17.76%, and 18.40–96.01% in the first year, respectively. Plant crude protein and crude fat also increased by 7.33–31.17% and 21.54–30.00%. In the second year, the increases were 26.41–50.22%, 6.16–20.55%, and 13.91–52.42% for plant height, coverage, and biomass and 4.46–28.27% and 14.24–19.89% for crude protein and crude fat, respectively. The carbon, nitrogen and isotope indices of leaves and roots also increased simultaneously. Biogas slurry application altered plant community composition, BS_50% transiently increased plant family richness, BS_100% exerted persistent inhibitory effects, and species diversity across all fertilization treatments showed a recovery trend in the second year. Principal component analysis and redundancy analysis showed that treatment groups were clearly separated in 2024 but overlapped substantially in 2025. Root δ¹³C and root δ¹⁵N were key indicators distinguishing vegetation community characteristics. The results of this study confirmed that the application of biogas slurry fertilizer could actively improve the vegetation recovery of degraded grasslands. It provided reference support for the resource utilization of biogas slurry fertilizer and the sustainable management of grassland ecosystems. Full article

Fish consumption provides nutritional benefits but can also contribute to exposures to bioaccumulative contaminants, requiring guidance that integrates both dimensions. We conducted a deterministic pilot risk–benefit assessment of Latvian inland lake fish using pooled samples stratified by lakes and species. Risks were characterized for methylmercury, estimated from total mercury, and for Σ4 PFAS (PFOS, PFOA, PFNA, PFHxS) by calculating weekly intakes under three consumption scenarios (150, 300, and 450 g/week) for a 70 kg adult and comparing them to health-based guidance values. Benefits were quantified as weekly contributions of EPA + DHA, iodine, and protein relative to reference intakes, combined into a nutritional index and integrated with risk using a benefit–risk quotient (BRQ). The primary decision outputs were safe weekly consumption amounts (g/week) and the contaminant limiting factor. Across lake-species groups, mercury was the dominant constraint on safe consumption for most predatory fish, while PFAS limited selected groups with lower mercury burdens. EPA + DHA provided the strongest differentiating benefit signal between groups, whereas iodine contribution was limited because measurements were left-censored and constant after limit of quantification (LOQ) handling. This pilot demonstrates an interpretable framework for generating lake- and species-specific consumption guidance that can be updated as monitoring coverage expands. Full article

Untargeted metabolomics faces significant challenges in standardization due to variability introduced by sample preparation and analytical workflows. We systematically evaluated the impact of biological matrices, extraction protocols, and chromatographic configurations to establish a mechanism-informed framework aimed at improving reproducibility in large-scale clinical and epidemiological studies. Three extraction protocols were compared using an in-house pooled heparin plasma: monophasic protein precipitation with isopropanol (IPA), methanol:acetonitrile (MeOH:ACN), and a modified Matyash biphasic method. The most reproducible protocol was then applied to four blood matrices. Samples were analysed using untargeted metabolomics on hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) HPLC columns, with mass spectrometry data processed using Compound Discoverer. Both IPA and MeOH:ACN extractions achieved over 80% of features with coefficient of variation (CV%) ≤ 30% for both RP and HILIC, whereas the Matyash method showed higher variability, with a larger proportion of metabolites exhibiting CV% > 30%. Across matrices, RP chromatography detected over 80% of metabolites with CV% < 30%, while HILIC showed higher variability, with at least 20% of metabolites above this threshold. Among matrices, serum and heparin plasma outperformed EDTA and citrate in reproducibility. We propose a standardized workflow in which monophasic extractions combined with RP chromatography maximize reproducibility and metabolite coverage, minimizing methodological artefacts and providing a reliable framework for robust biological discovery in large-scale untargeted metabolomics studies. Full article

The genus Blunervirus comprises plant viruses that infect a diverse range of plants, but no blunervirus has been reported infecting elm trees (Ulmus parvifolia) in China to date. Using high-throughput sequencing and reverse-transcription PCR assays, a novel blunervirus, tentatively named elm blunervirus 1 (ElmBlV1), was identified from a symptomatic elm plant (Ulmus parvifolia) in China. The genome of ElmBlV1 harbors canonical molecular features of blunerviruses and comprises six RNA segments (RNAs1–6), with RNA5 and 6 being two additional genomic components not reported in known blunerviruses. Sequence analyses revealed amino acid (aa) identity of ElmBlV1 proteins ranging from 25.9% (polyprotein encoded by RNA1) to 64.2% (movement protein encoded by RNA4) relative to reported blunerviruses and include five orphan open reading frames. Phylogenetically, ElmBlV1 is most closely related to blueberry necrotic ring blotch virus. Furthermore, ElmBlV1 P37 localizes to both plasmodesmata and the nucleus. Additionally, the RNA reads mapping revealed high read coverage was observed on RNAs3–4 for this virus. To our knowledge, this is the first report of a blunervirus infecting an elm tree in China. Our results enrich the diversity of known viruses in the genus of Blunervirus and expand our understanding of their genomic characteristics and molecular biology. Full article

Lactation is a dynamic process characterised by a production peak at 6–8 weeks, followed by a steady decline. To understand the molecular drivers of these phases and the influence of production systems, this study aims to provide a transcriptomic characterisation of bovine milk somatic cells (BMSCs) in Holstein (HO), Simmental (SM), Simmental × Holstein crossbreed (SM × HO), and Podolica (POD) cows at 60 and 120 days in milk (DIM). Total RNA was sequenced at high coverage, and differential expression and functional enrichment analyses were performed. While a core set of milk protein and fatty acid genes was identified, breed-specific analysis showed SM × HO had the highest variation (677 differentially expressed genes, DEGs). Genes upregulated at 120 DIM involved mitochondrial metabolism and oxidative phosphorylation, while downregulated genes were associated with nuclear transcriptional regulation. At 60 DIM, SM × HO vs. HO showed 66 DEGs, with upregulated genes linked to chromatin remodelling and immune regulation. Comparing production systems, 28 DEGs between POD and HO/SM highlighted differences in mitochondrial activity and transcriptional regulation. This study bridges a knowledge gap by profiling the milk transcriptome of unexplored cattle breeds, providing novel insights into the molecular regulation of lactation. Full article

Background: Low-volume trapping columns are essential for sample enrichment, desalting, and injection profile focusing on nano-LC–MS-based proteomics. They enable higher sample loading, improve chromatographic performance, and protect the analytical column by removing salts and contaminants. Recently, monolithic trap columns with micropillar architecture have emerged as alternatives to conventionally packed traps. This study compares the performance of a packed and a micropillar monolithic trap column for the analysis of tryptic peptides. Methods: A tryptic digest of HeLa cell lysate was analyzed under identical LC–MS conditions using both trap types. Peptides were detected at 214 nm and analyzed by nano-ESI on a Q Exactive Plus Orbitrap. Data were searched against the human UniProt database (February 2023) using FragPipe v20.0, and statistical evaluation of MaxLFQ intensities was performed in Perseus using Welch’s t-test and clustering analysis. Results: Over 2500 proteins were identified with both setups. The packed trap column yielded more total peptides, particularly those with post-translational modifications and higher hydrophilicity, whereas the monolithic column favored peptides of intermediate hydrophobicity. Chromatographic profiles confirmed a slight reduction in the trapping efficiency of hydrophilic peptides by the monolithic trap. Conclusions: Trap column design significantly influences peptide recovery and proteome coverage. Full article

Riverine ecosystems represent critical nodes in the global carbon cycle, where the mechanistic role of viruses in modulating eukaryotic carbon cycling remains underexplored, particularly across heterogeneous landscapes. Here, we applied metatranscriptomics to dissect how multi-scale environmental factors and viral gene activity jointly regulate the spatial transcription of carbon cycling genes in riverine eukaryotic communities along the Yongding River, China. Our analyses reveal pronounced spatial heterogeneity in both viral gene expression—notably major capsid proteins of large eukaryotic DNA viruses—and carbon fixation, conversion, and metabolism pathways, peaking in agriculturally impacted plain regions. Multivariate statistics and network analyses demonstrate that land use enhances viral gene activity, serving as biological amplifiers that modulate host carbon metabolism and transformation. Structural equation modeling further identifies a cascade in which cropland coverage elevates viral gene expression, ultimately driving a 1.8-fold increase in TCA cycle gene transcription in plain regions, whereas nitrogen loading at the site scale suppresses viral activity and carbon fixation. Phylogenetic analysis corroborates that virus–host specificity underpins these spatial patterns. Collectively, these findings advance a new model in which viruses act as key intermediaries, transmitting multiscale environmental signals to shape riverine carbon cycling. Our study highlights the urgency of incorporating viral ecology into predictive frameworks of riverine biogeochemical cycling under accelerating environmental change. Full article

Exogenous genes are generally expressed by integration into the chromosomes of Pichia pastoris. However, systematic studies on the chromosomal position effect are lacking, and locations that are conducive to the high expression of foreign genes are rarely reported. In this study, a genomic random insertion mutagenesis library for P. pastoris was successfully constructed using the piggyBac (PB) transposon system. Through sequencing, the sequence TTAA was identified as the major recognition site of the PB transposon, which exhibited relatively high coverage on P. pastoris chromosomes, making it a valuable tool for studying position effect variegation in P. pastoris. Using the enhanced green fluorescent protein gene (eGFP) as a reporter, two libraries including low-expression positions and high-expression positions were obtained by flow cytometry. The low-expression sites were mainly located upstream of ORFs around the promoter region and downstream near the terminator region, while the high-expression sites were predominantly located at the gene interior. KEGG and GO analyses showed that genes in high-expression positions were significantly enriched in the ATP-dependent chromatin remodeling and histone binding pathways, and genes in low-expression positions were significantly enriched in the MAPK signaling pathway, autophagy, mitochondrial autophagy, ABC transporters, and the arginine synthesis pathway. This study has clarified the genome-wide landscape of position effect variegation in P. pastoris. Additionally, it has provided novel insights into high-throughput screening strategies for strains with high exogenous gene expression. Full article

Efficient and reproducible protein extraction is a critical prerequisite for high-quality proteomic and glycoproteomic analyses. In this study, four commonly used lysis buffers, sodium dodecyl sulfate (SDS), guanidine hydrochloride (GuHCl), urea (UA), and mammalian protein extraction reagent (MPER), were systematically evaluated within an integrated proteomic and N-glycoproteomic workflow. Using HeLa and HEK293T cells as model systems, we assessed buffer performance in terms of protein and intact N-glycopeptide identification depth, quantitative reproducibility, subcellular coverage, and glycan type distribution. Across both cell lines, SDS consistently achieved the deepest proteome and N-glycoproteome coverage, yielding the highest numbers of identified proteins, N-glycopeptides, glycoproteins, and glycosylation sites. Quantitative analysis demonstrated that SDS provided superior reproducibility, with approximately 85% of quantified proteins exhibiting coefficients of variation below 5%. Subcellular localization analysis at the global proteome level showed that SDS enabled more comprehensive extraction of proteins from multiple cellular compartments, including the nucleus, cytoplasm, mitochondria, and plasma membrane, indicating reduced extraction bias toward specific subcellular regions. Consistently, subcellular localization analysis of identified glycoproteins revealed enhanced coverage of membrane-associated compartments, particularly the plasma membrane, endoplasmic reticulum, Golgi apparatus, and lysosome. In addition, the analysis of glycan type classification for intact N-glycopeptides revealed that the SDS lysis buffer demonstrated the most comprehensive identification capability for glycopeptides with multiple glycosylation modifications in both cell lines. MPER and UA showed a highly consistent distribution across various glycosylation types, whereas the guanidine hydrochloride method was comparatively least effective. Overall, these results establish SDS as a robust lysis buffer for comprehensive, reproducible, and quantitatively stable proteomic and N-glycoproteomic analyses, providing practical guidance for buffer selection in quantitative glycosylation-focused studies. Full article

Although RNA sequencing (RNA-seq) enables rapid transcriptome profiling, functional annotation of fungal transcriptomes remains challenging. Existing tools prioritize broad taxonomic coverage, and reference genomes are scarce for non-model species. This study aimed to develop a fungal-specific functional annotation workflow to support rapid and accurate functional analyses downstream of RNA-seq, independent of reference genome availability. To evaluate the workflow, RNA-seq data from 57 samples of Lentinula edodes strain H600 (shiitake mushroom) were retrieved, along with full-length transcript sequencing (Iso-Seq) data and corresponding RNA-seq data from 20 samples of Phakopsora pachyrhizi (Asian soybean rust) from public databases. The workflow successfully annotated over 96% of protein-coding transcripts and demonstrated applicability to Iso-Seq data. Functional enrichment analyses revealed higher-resolution functional detection than existing annotation tools. Furthermore, integrating homology searches against fungal-specific databases with expression pattern-based annotations highlighted the workflow’s utility for target identification in genome editing and other applications. Overall, the results of this study highlight the potential of the developed workflow in facilitating the discovery of functionally important transcripts and their translation into biotechnological applications. Full article

Background/Objectives: Brucella is a major global One Health threat, causing an estimated 2.1 million human infections and substantial livestock losses annually, with no vaccine currently available for humans, underscoring the urgent need for a safe and effective vaccine. Methods: Employing a reverse vaccinology approach, a novel 175-mer multiepitope vaccine (Mvax) targeting Brucella FrpB was computationally designed in this study, incorporating two B-cell, two MHC class I (MHC-I), and three MHC class II (MHC-II) epitopes selected for their high predicted antigenicity, safety, and IFN-γ-inducing potential. Human β-defensin-3 (hBD3) was fused to the N-terminus as an adjuvant, followed by comprehensive in silico evaluation of the construct. Results: Population coverage analysis predicted 99.59% global MHC class I/II coverage for selected epitopes. In silico analyses predicted that Mvax has high solubility (Protein-SOL score: 0.808), a high antigenicity score (VaxiJen: 1.06), and a negative GRAVY index (−0.881), indicating favorable predicted physicochemical characteristics. iMODS, CABS-Flex 3, and molecular dynamics simulations suggested theoretical stability trends for the modeled vaccine complexes. C-ImmSim immune simulations further predicted elevated Th1 cell populations and associated cytokines (IL-12, IFN-γ, IL-2) following both single and multiple simulated Mvax exposures. Conclusions: The computational analyses described here provide a theoretical modeling basis for an antivirulence multi-epitope vaccine design against human brucellosis, with predicted metrics and simulated immune responses requiring empirical validation. Full article