Search Results (28,752)

Lipid nanoparticles (LNPs) have become the cornerstone of nucleic acid delivery platforms, particularly in RNA-based vaccines and therapeutics. However, the conventional methods of LNP production, which are primarily reliant on microfluidic mixing of aqueous and organic solvent phases, pose limitations in terms of mRNA stability, residual organic contamination, scalability, cost, and environmental impact. These limitations prompted a renewed search for non-conventional strategies with the promise of improving mRNA-LNP encapsulation approaches. These emerging approaches aim to address key bottlenecks, including mRNA hydrolysis-driven degradation, high production losses, and complex downstream purification. Moreover, the ability to decouple LNP synthesis from mRNA encapsulation could enable streamlined, modular manufacturing workflows and customizable payload delivery, including single- or multiple-mRNA payloads, thereby expanding the therapeutic scope of LNPs. This review offers an early insight into the design principles and scalability potential of emerging non-conventional LNP encapsulation approaches, including solvent-free and microfluidics-free methodologies, and pre-built LNP workflows. We also examine trends in emerging LNP encapsulation tools, including high-shear mixing, sonication, membrane contraction, and other approaches. Finally, we extrapolate the suitability of the methods for scale-up approaches and their economic implications based on the process information. Full article

RNA–protein interactions (RPIs), mediated primarily by RNA-binding proteins (RBPs), are central to post-transcriptional gene regulation and govern RNA splicing, transport, localization, translation, and decay. Dysregulation of RBPs and their associated RNA networks contributes to diverse pathologies, including cancer, neurodegenerative disorders, and viral infections. However, profiling RPIs remains a challenge due to their inherent transience, low binding affinity, and shifting spatial dynamics. This review provides a comprehensive and systematic overview of current methodologies for investigating RPIs. We discuss RNA-centric and protein-centric strategies. In addition, imaging-based approaches are evaluated for their capacity to resolve spatial and temporal dynamics of RBP–RNA interactions in situ. We compare these methodologies in terms of resolution, sensitivity, specificity, and biological applicability, emphasizing the importance of integrative strategies for constructing high-resolution, context-dependent RPI maps in physiological and disease settings. Full article

Despite prolonged viral inhibition with combination antiretroviral therapy (ART), HIV-1 survives as genetically intact, replication-capable proviruses within durable CD4+ T-cell fractions, involving central memory, transitional memory, and stem cell-like memory populations, as well as within tissue-resident compartments including lymphoid follicles and gut-associated lymphoid tissue. Reservoir stability is preserved via clonal growth of infected cells and epigenetic processes that impose proviral transcriptional silencing. As a result, current therapeutic approaches seek to either directly alter proviral survival or to improve immune-driven elimination of infected cells. At the molecular level, investigational strategies such as CRISPR–Cas9 and CRISPR–Cas12 gene-editing systems are intended to remove or induce inactivating mutations inside embedded proviral DNA, as well as alter host entrance co-receptors such as CCR5 to provide cellular resistance to infection. In addition, pharmacologic latency regulation is being studied via histone deacetylase inhibitors, protein kinase C agonists, and bromodomain inhibitors to reverse latency, along with Tat inhibitors and other transcriptional repressors aimed to persistently silence proviral expression. Moreover, immunological techniques aim to counteract inefficient endogenous antiviral defenses. Broadly neutralizing antibodies with tailored Fc-driven effector functions are under examination for both neutralization and antibody-dependent cellular cytotoxicity. Therapeutic vaccine approaches seek to elevate polyfunctional HIV-specific CD8+ T-cell responses, while adoptive cellular approaches, involving CAR-T cells aiming HIV envelope epitopes, remain in early clinical research. Immune checkpoint blockade is also being investigated to reverse T-cell depletion inside reservoir-rich tissues. Nevertheless, the key obstacles continue to be the diverse reservoir composition, restricted tissue penetration, viral escape, and safety limitations. The molecular and translational obstacles that characterize attempts toward an HIV cure must be addressed through ongoing multidisciplinary research. Full article

HIV-1 protease (PR) is an essential enzyme in the viral life cycle and a primary target of antiretroviral therapies, particularly protease inhibitors (PIs). Understanding the dynamics of viral evolution and the factors governing the emergence or loss of resistance-associated mutations is critical for improving PI efficacy and managing drug resistance in HIV/AIDS treatment. In this study, we investigated the impact of three natural HIV-1 polymorphisms (T12A, L63Q, and H69N), whose prevalence varies depending on treatment status and viral subtype, on the structural stability and conformational dynamics of PR using molecular dynamics (MD) simulations. Three independent 500 ns MD simulations were performed for the native protease and each mutant system. Although none of the mutations disrupts the overall structural integrity of HIV-1 PR, they induce mutation-specific alterations in flexibility and residue interactions. In particular, T12A and H69N exhibit increased structural deviations, especially in the flap regions, along with enhanced conformational fluctuations. In contrast, the L63Q mutation shows a slight reduction in flap flexibility compared to both the native protease and the other mutants. Consistently, the fraction of time spent in open-flap conformations is higher for T12A and H69N and lower for L63Q relative to the native system. Moreover, mutations in the Fulcrum (T12A) and Cantilever (L63Q and H69N) regions do not disrupt the long-range network of correlated motions observed in the native protease, both inter- and intra-monomer, but instead increase the extent of correlated and anti-correlated motions in other regions of PR. Full article

The human virome, comprising eukaryotic viruses, bacteriophages, and viral genetic material, is a dynamic component of the microbiome with growing relevance in infectious diseases. This narrative review is structured to: (i) summarize the general composition of the human virome and methodological challenges, including the fraction of unclassified viral “dark matter”; (ii) describe virome alterations in chronic infections; and (iii) explore site-specific virome dynamics across respiratory, intestinal, and genito-urinary tracts in both chronic and acute infections. In chronic viral infections such as HIV, HBV, HCV, and HPV, a recurrent feature is the expansion of Anelloviridae—particularly torque teno virus—reflecting impaired immune surveillance rather than direct pathogenicity, suggesting their potential as surrogate biomarkers of immune competence. Evidence on virome changes in chronic bacterial and parasitic infections remains limited, highlighting a critical knowledge gap. Acute infections are associated with compartment-specific shifts in eukaryotic viruses and bacteriophage communities, often paralleling changes in bacterial populations and inflammatory responses, with implications for disease severity. Despite advances in metagenomic approaches, a substantial proportion of viral sequences remains unclassified, limiting functional interpretation. Nevertheless, virome profiling provides an ecosystem-level perspective, offering insights beyond single-pathogen detection and supporting emerging applications in diagnostics, immune monitoring, prognosis, and infectious disease surveillance. Full article

Background/Objectives: Chronic liver diseases, including metabolic dysfunction-associated steatohepatitis (MASH) and chronic viral hepatitis (CVH), are major global health concerns due to their potential progression to cirrhosis, liver failure, and hepatocellular carcinoma. Because liver biopsy, despite meeting the diagnostic gold standard, is invasive and associated with complications, non-invasive fibrosis assessment tools have been increasingly recommended in clinical practice. This study aimed to compare the diagnostic performance of several non-invasive fibrosis markers (ARR, APRI, FI, FIB-4, API, NFS, BARD) and transient elastography in detecting advanced liver fibrosis (F4) in patients with MASH and CVH. Methods: This retrospective study included 237 adult patients (77 MASH, 160 CVH) who underwent liver biopsy between 2017 and 2025 at the University Clinical Center of Serbia. CVH included chronic hepatitis B (CHB) and C (CHC). Patients were evaluated using serum fibrosis indices and TE, and results were compared to histological staging (F0–F4). ROC analysis assessed diagnostic performance. Results: Cirrhosis (F4) was more common in CVH than MASH (p < 0.001). In MASH, NFS (AUROC 0.931), FIB-4 (0.915), BARD (0.872), and APRI (0.878) showed high diagnostic accuracy for F4. In CHC, APRI (0.931), FIB-4 (0.863), and TE (0.938) had strong performance, while in CHB, TE (0.987) outperformed FIB-4 (0.821). Sensitivity and specificity varied by test and cohort, with TE consistently yielding the best results where available. Conclusions: Non-invasive methods, particularly NFS and FIB-4 for MASH and TE for CVH, effectively identify advanced fibrosis. Their application could significantly reduce the need for biopsy, especially in high-risk groups. TE demonstrated superior accuracy, but access limitations highlight the continued relevance of serum-based scores. Full article

Mosquitoes are recognized as the arthropod group with the greatest vectorial capacity, and the viruses they transmit constitute a significant concern in the context of global One Health. In addition, these insects act as hosts for a wide diversity of insect-specific viruses (ISVs), which exclusively infect arthropods. Expanding knowledge of ISVs is particularly relevant, given their potential influence on arbovirus replication and their role in elucidating the evolutionary processes that shape virus–vector interactions. In this study, we report the isolation and molecular analysis of three negeviruses associated with different mosquito species of the genera Culex, Coquillettidia, Mansonia, and Ochlerotatus, collected in Belém, Pará State, in the Brazilian Amazon: Loreto virus, Wallerfield virus, and a putative new species, designated Terra firme virus. Eleven pools exhibited cellular alterations consistent with cytopathic effects in invertebrate C6/36 cells but showed no evidence of replication in vertebrate Vero cells. Notably, simultaneous infections by two or three negeviruses were detected in some mosquito pools, indicating the occurrence of multiple viral infections within individual samples. Genomic analyses revealed that the isolated strains share conserved domains with previously described isolates from other countries. Phylogenetic inferences demonstrated that the investigated strains are classified within the clades Nelorpivirus and Sandewavirus. Taken together, these findings expand the currently known diversity of the negevirus group and contribute to a more comprehensive understanding of its host range and geographic distribution. Full article

Chemotherapy-induced mucositis (CIM) is a dose-limiting toxicity of cancer therapy that is mainly associated with mitochondrial dysfunction in epithelial cells. We investigated whether GV1001, a mitochondrial protective peptide from human telomerase reverse transcriptase (hTERT), attenuates 5-fluorouracil (5-FU)-induced mucositis in a murine model. 5-FU induced notable mortality, leukopenia, and mucositis in the gastrointestinal (GI) tract, including tongue, esophagus and small intestine. It promoted epithelial–mesenchymal transition (EMT), nuclear factor kappa-B (NF-κB) activation, systemic and mucosal inflammation, DNA damage, impaired cell proliferation, and apoptosis throughout the GI tract. GV1001 blocked 5-FU–associated mortality, significantly attenuated leukopenia, and notably prevented mucositis. GV1001 also suppressed 5-FU-induced DNA damage, EMT, loss of proliferative capacity, apoptosis, and NF-κB activation in mucosal epithelium. In normal human keratinocytes, 5-FU inhibited the cell proliferation, disrupted mitochondrial function, as evidenced by reduced mitochondrial membrane potential, increased reactive oxygen species (ROS) production, impaired electron transport chain (ETC) complex integrity, decreased ATP synthesis, and cytochrome c release into the cytosol. GV1001 markedly mitigated these 5-FU-induced mitochondrial defects. Taken together, GV1001 mitigates CIM by most likely preserving mitochondrial integrity and function, supporting its potential as a strategy to prevent cancer chemotherapy-associated mucosal injury in patients. Full article

Viruses are key regulators of marine microbial communities, yet their temporal dynamics in tropical intertidal sediments remain poorly characterized. We conducted a year-long metagenomic survey of sandy intertidal sediments in Sanya Bay (60 monthly samples from five sites) to examine viral taxonomy, community structure, lytic proteins, and auxiliary metabolic genes (AMGs). Within the classifiable fraction, the assemblages were consistently dominated by Assiduviridae. However, NMDS analysis revealed a significant overall seasonal shift, with October–December samples separating from the rest of the year. Co-occurrence network analysis identified five co-occurrence modules with distinct temporal patterns, alongside a concurrent decline in module abundance and lytic proteins in October. Functional annotation showed that cysteine and methionine metabolism, primarily driven by DNA methyltransferases, was identified as a highly represented AMG category among the annotated functions, while other pathways displayed seasonal variability. Collectively, these findings suggest that although characterized by a classifiable fraction dominated by Assiduviridae, the highly complex tropical intertidal viral communities undergo substantial seasonal reorganization in structure and functional potential. Full article

We performed a protein-docking study for eight DNA aptamers (SEQ1–SEQ8) against chicken Cluster of Differentiation 40 (chCD40), which were experimentally identified via SELEX in our previous study. In silico and molecular docking analyses were performed to predict and obtain the secondary and tertiary structures of the aptamers. Aptamers SEQ3 and SEQ4, which showed the best inhibitory effects, were selected and utilized to produce a DNA-based vaccine adjuvant using rolling circle amplification (RCA). These aptamers had been previously characterized via mass spectroscopy to determine their molecular weight and regions that could potentially interact with chCD40. In the present study, these results were corroborated and expanded. A series of free software methods, including Mfold v.1.0, 3dADN v.2.0, ClusPro v.2.0, Hdock v.1.0, and PLIP v.1.0, were used to determine the aptamers’ secondary and tertiary structures and docking interactions, as well as the specific residues involved in the interactions and their distances. The structures were used to explain and thus understand their effect on the binding, selectivity, and stability of the aptamers. The main objective of the study was to determine whether these aptamers could be used as vaccine adjuvants against viral and bacterial pathogens, specifically chicken avian influenza. The docking results were in good agreement with the experimental and biological results. The procedure employed in this study could be an easy and effective tool for exploring the potential of the new technology of systematic evolution of ligands by exponential enrichment (SELEX) in the preparation of aptamers to control viral and bacterial infections as well as diseases, such as cancer and Alzheimer’s. Full article

Bovine viral diarrhea virus (BVDV) is a major cattle pathogen associated with significant economic losses worldwide. In Brazil, the high genetic diversity of circulating strains represents an additional challenge for disease control. To update the molecular epidemiology of BVDV in southern Brazil, 16,198 bovine serum samples collected in 2020 through a national surveillance program were screened for pestivirus RNA by RT-qPCR. Forty-nine samples (0.36%) were positive and subjected to partial sequencing of the 5′UTR and N^pro regions. Phylogenetic analysis identified BVDV-1a (25/49; 51%), BVDV-1b (1/49; 2%), BVDV-1d (7/49; 14%), and BVDV-2b (16/49; 33%), with no detection of HoBiPeV. When compared descriptively with data from 2010 in the same region, BVDV-1a remained the most frequent subgenotype, while BVDV-2b also represented a substantial proportion of detections, contrasting with other regions worldwide. Although the two datasets are not directly comparable, and no statistically significant differences were observed, these findings provide an updated overview of circulating BVDV subgenotypes in Rio Grande do Sul. The absence of HoBiPeV contrasts with reports from other regions of Brazil and suggests a distinct regional pattern of pestivirus circulation. Overall, the results reinforce the importance of continuous genomic surveillance to monitor changes in viral diversity and support control strategies in cattle populations. Full article

Human parainfluenza virus type 4 (hPIV4) remains poorly characterized compared with other hPIV serotypes and information on its genomic diversity is particularly limited for Russia and Eastern Europe. In this study, we report the first complete genome sequences of hPIV4 isolates from Russia and place them in the context of global hPIV4 genetic diversity. Eight hPIV4 viruses were isolated in cell culture from respiratory samples collected from hospitalized children in Saint Petersburg between 2017/2018 and 2023/2024. Complete viral genomes were recovered using a metagenomic whole-genome amplification approach based on SMART-9N technology. Phylogenetic analysis of 178 complete hPIV4 genomes showed clear separation into hPIV4a (n = 132) and hPIV4b (n = 46) subtypes. Based on genetic distance approach, hPIV4a formed two major clusters, with the dominant cluster B subdivided into four subclusters (B1–B4); and subcluster B4 further resolved into four genetic lineages. All Russian isolates belonged to the subcluster B4 and were distributed among multiple co-circulating lineages. In contrast, hPIV4b genomes segregated into three distinct clusters, reflecting structured genetic diversity within the subtype. Collectively, this study provides, to the best of our knowledge, the first p-distance-based framework for hPIV4 whole-genome classification and contributes new complete genome sequences for an underrepresented region. Full article

The m⁶A RNA methylation pathway plays a critical role in host antiviral defense. Host cells employ m⁶A readers such as YTHDF2 to regulate viral RNA fate through diverse mechanisms, including degradation, translational control, and immune recognition. However, we found that YTHDF2 is essential for SARS-CoV-2 replication, suggesting that a virus may exploit this host machinery to its advantage. Through integrative RNA-proteome analysis, we identified the SARS-CoV-2 nucleocapsid (N) transcript as the most heavily m⁶A-modified viral transcript and a direct interactor of YTHDF2. The N protein forms a complex with YTHDF2 in the cytoplasm and redirects this host RNA decay machinery toward host antiviral transcripts. N suppresses ISG15, IFIT1, MX1 and pro-inflammatory cytokines in a largely YTHDF2-dependent manner, an effect that is lost in YTHDF2-knockout cells. These findings reveal a viral immune evasion strategy wherein a viral protein actively hijacks an m⁶A reader to silence antiviral gene expression, establishing the N-YTHDF2 axis as a therapeutic target against SARS-CoV-2 and other coronaviruses. Full article

Peste des Petits Ruminants (PPR) is a highly contagious viral disease of small ruminants and remains a major threat to food security and rural livelihoods across Africa, the Middle East, and Asia. In the Mediterranean, uneven outbreak reporting and intense spatial clustering hinder the identification of regions where environmental and anthropogenic conditions favor disease occurrence. This study applied an interpretable machine-learning ecological niche modeling framework to characterize PPR spatial suitability in North Africa. A merged outbreak dataset (n = 744) was compiled from the Food and Agriculture Organization (FAO) EMPRES-i and the World Animal Health Information System (WAHIS) databases for 2005–2026. Outbreak locations were linked to environmental and anthropogenic predictors, spatially thinned, and paired with randomly sampled pseudo-absences at a 1:1 ratio. After correlation-based screening and Boruta feature selection, four classifiers were compared under five-fold spatial block cross-validation: a generalized linear model (GLM), a support vector machine (SVM), Random Forest (RF), and extreme gradient boosting (XGBoost). All models showed good discriminatory performance. Random Forest (RF) and extreme gradient boosting (XGBoost) yielded the highest area under the receiver operating characteristic curve value (AUC = 0.94). Random Forest achieved the highest specificity, XGBoost achieved the highest sensitivity, and the support vector machine showed the most even sensitivity–specificity tradeoff among the machine-learning classifiers. Sheep density, mean diurnal temperature range, temperature seasonality, and human population density were consistently the dominant drivers. Predicted PPR suitability based on reported outbreaks was concentrated along the North African coastal belt and low across most arid inland regions. These findings suggest that passive surveillance is likely to be most informative in coastal production systems where host density, environmental suitability, and reporting opportunity overlap. At the same time, areas of lower reported-outbreak suitability should not be interpreted as disease-free and may require complementary active surveillance approaches. Full article

Syphilis and HIV are sexually transmitted disease (STDs) that interact synergistically. However, data on HIV–syphilis co-infection in Romania remain limited. We conducted a retrospective cohort study at a single Romanian HIV/AIDS Day Clinic, including 439 adult people living with HIV (PLWH) monitored between 2020 and 2025. Demographic, epidemiological, clinical, and laboratory data were collected, including HIV staging and syphilis history. Syphilis co-infection was identified in 81 patients (18.5%), and 61.5% met criteria for AIDS. Viral suppression was achieved in 82.2%, and 78.4% achieved CD4 counts >350 cells/mm³. Male sex, urban residence, unmarried status, sexual HIV transmission, genital condyloma, and other STIs were independently associated with syphilis. First episodes of syphilis were predominantly secondary (61%), neurosyphilis was present in 5%, and serofast evolution occurred in 12%, more frequently after reinfection. Among deceased patients, 20.9% had a history of syphilis, but co-infection was not significantly associated with mortality. Nine of 28 patients lost to follow-up had prior syphilis, suggesting a potential impact on retention in care. These findings indicate that HIV–syphilis co-infection is increasingly prevalent in Romania, driven primarily by behavioral factors, and highlight the need for targeted STD screening and prevention strategies among high-risk PLWH. Full article