Search Results (805)

Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host–virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5′-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5′-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development. Full article

Background and Objectives: Helicobacter pylori (H. pylori) infection remains one of the most common chronic bacterial infections worldwide and is associated with a wide range of gastrointestinal disorders, including gastritis, peptic ulcer disease, and gastric cancer. Increasing rates of antibiotic resistance, particularly to clarithromycin and fluoroquinolones, represent a major therapeutic challenge. The objective of this study was to determine the prevalence of resistance-associated mutations in H. pylori-positive gastric biopsy samples from western Romania. Materials and Methods: We conducted a prospective study from January to December 2024, enrolling 138 patients undergoing gastroscopy. Biopsies were collected from the gastric antrum, and H. pylori infection was confirmed using the rapid urease test (RUT). Positive samples were further analyzed with the GenoType HelicoDR assay to detect mutations in the 23S rRNA gene (clarithromycin resistance) and gyrA gene (fluoroquinolone resistance). Clinical, demographic, and endoscopic data were also collected. Results:H. pylori infection was confirmed in 41.3% of the patients (57), of whom 63.2% (36) were treatment-naïve and 36.8% (21) had prior eradication therapy. Among treatment-naïve patients, clarithromycin resistance was identified in 19.4%, whereas previously treated patients showed a markedly higher resistance rate of 47.6% (p = 0.018). All clarithromycin-resistant cases carried the A2147G (23S MUT3) mutation. Fluoroquinolone resistance was present in 13.9% of naïve patients and increased to 23.8% in those with prior eradication therapy, with resistance linked to gyrA mutations at codons 87 (N87K) and 91 (D91 variants). Combined resistance to both antibiotics was observed only in a subset of previously treated patients. Conclusions: Primary resistance to clarithromycin in western Romania exceeds the 15% threshold defined by international guidelines, making clarithromycin-based triple therapy unsuitable as an empirical first-line option. The findings support the use of bismuth quadruple therapy as the preferred empirical regimen in this region. Also, molecular testing proved effective for rapid identification of resistance-associated mutations. Full article

Background: Advances in American mink (Neogale vison) genomics have identified candidate genes linked to disease resistance, including RNF165, which is involved in immune response and virus–host interactions. Objectives: This study aimed to characterize genetic variation within the RNF165 (Ring Finger Protein 165) gene in farmed and wild mink to identify polymorphisms potentially associated with resistance to Aleutian mink disease virus (AMDV). Methods: Two groups of mink were analyzed: farmed animals from Latvia (n = 50) and wild animals (n = 20) from Poland. The presence of viral DNA was verified by PCR amplification targeting the VP2 and NS1 viral protein genes. To assess polymorphism within RNF165, primers spanning eight exons were designed, optimized, and applied in PCR amplification. PCR products were sequenced and subjected to bioinformatic analysis. Results: Two single nucleotide polymorphisms were identified: a synonymous substitution at c.G141A and a missense mutation c.G596A (p.R199K). Both variants were present in farmed and wild populations; however, the G allele at position c.141 was nearly twice as frequent in farmed mink compared to wild mink (p < 0.05). In silico functional prediction (SNAP2) indicated that the p.R199K mutation may moderately affect RNF165 protein function. Conclusions: Owing to its high conservation and role in immune regulation, RNF165 may serve as a promising candidate gene for molecular selection in breeding programs aimed at enhancing resistance to Aleutian mink disease (AMD). Full article

Protein kinase R (PKR) is an antiviral protein that is involved in molecular “arms races” with viral antagonists. As a result, some PKR inhibitors, including the vaccinia virus (VACV) protein K3 and its orthologs from other poxviruses only inhibit PKRs of selected species. We previously reported contrasting inhibition patterns of human, sheep, and cow PKRs by VACV K3 and the sheeppox virus (SPPV) K3 ortholog, SPPV 011. Here we show that the differential sensitivities of cow and sheep PKRs to VACV K3 were mediated by only two residues in PKR helix αG. In contrast, SPPV 011 sensitivities were governed by additional residues and regions. Analysis of the PKR sensitivities from 20 mammalian species to VACV K3 and SPPV 011 revealed four different sensitivity patterns: some PKRs were inhibited by only one K3 ortholog, as previously reported, whereas other PKRs were either resistant or sensitive to both inhibitors. Furthermore, we characterized a residue (K45) in VACV K3 that is involved in the species-specific inhibition of PKR. Mutating this residue increased the inhibition of sheep but not human PKR, whereas it decreased the inhibition of mouse PKR, highlighting that a single mutation in a viral protein can result in distinct species-dependent inhibition changes. Full article

Tsetse flies are major vectors of trypanosomes in Sub-Saharan Africa, posing risks to livestock and human health. This study investigated the diversity, distribution, and infection rates of tsetse species, as well as the genetic diversity of drug resistance-associated trypanosome strains in Kenya. Flies were collected from Kwale, Taita-Taveta, Kajiado, Narok, and Turkana counties between November 2024 and February 2025. DNA analyses targeting rRNA and transporter genes (TbAT/P2, E6M6, DMT, TcoAde2) identified infections and resistance-associated mutations among 4693 sampled flies. Apparent density was highest in Kwale (101.52 flies/trap/day) and lowest in Turkana (1.18). Species distribution varied by county, with Kwale dominated by G. pallidipes, G. austeni, and G. brevipalpis; Taita-Taveta G. pallidipes, and G. brevipalpis; Kajiado G. pallidipes and G. longipennis; Narok G. pallidipes, G. brevipalpis, G. swynnertoni, and G. longipennis; and Turkana only G. pallidipes. Trypanosoma congolense was most prevalent, especially in Kwale, while T. brucei was common in Kajiado and Kwale. G. brevipalpis, G. austeni, and G. pallidipes showed higher infection risks. Drug resistance-associated T. congolense strains were found in Kwale and Taita-Taveta, with TcoAde2 and E6M6 gene diversity linked to Kenyan isolates. These findings highlight the need for targeted control of high-risk tsetse species and drug-resistant trypanosomes in Kenya. Full article

Background: Diffuse large B-cell lymphoma (DLBCL) is a biologically heterogeneous malignancy, with various outcomes despite significant advances in therapeutic options. Current conventional prognostic tools, e.g., the International Prognostic Index (IPI), lack sufficient precision at an individual patient level. However, artificial intelligence (AI), including machine learning (ML) and deep learning (DL), can enable specialists to navigate complex datasets, with the final aim of improving prognostic models for DLBCL. Objectives: This scoping review aims to systematically map the current literature regarding the use of AI/ML techniques in DLBCL outcome prediction and risk stratification. We categorized studies by data modality and computational approach to identify key trends, knowledge gaps, and opportunities for their translation into current practice. Methods: We conducted a structured search of the PubMed/MEDLINE, Scopus, and Cochrane Library databases through July 2025 using terms related to DLBCL, prognosis, and AI/ML. Eligible studies included original papers applying AI/ML to predict survival outcomes, classify risk groups, or identify prognostic subtypes. Studies were categorized based on input modality: clinical, positron emission tomography/computed tomography (PET/CT) imaging, histopathology, transcriptomics, genomics, circulating tumor DNA (ctDNA), and multi-omics data. Narrative synthesis was performed in line with PRISMA-ScR guidelines. Results: From the 215 records screened, 91 studies met the inclusion criteria. Group-wise we report the following categories: clinical risk features (n = 8), PET/CT imaging (n = 30), CT (n = 1), digital pathology (n = 3), conventional histopathology (n = 2), gene expression profiling (n = 19), specific mutational signatures (n = 18), ctDNA (n = 3), microRNA (n = 2), and multi-omics integration (n = 5). The most common techniques reported amongst the papers included ensemble learning, convolutional neural networks (CNNs), and LASSO-based Cox models. Several AI techniques demonstrated superior predictive performance over IPI, with area under the curve (AUC) values frequently exceeding 0.80. Multi-omics models and ctDNA-based predictors showed strong potential for clinical translation, a perspective worth considering in further studies. Conclusions: AI/ML methods are increasingly used in DLBCL to improve prognostic accuracy by leveraging data types with diverse inputs. These approaches allow an enhanced stratification, superior to traditional indices, and support the early identification of high-risk patients, earlier guidance for therapy tailoring, and early trial enrollment for flagged cases. Future investigations should focus on external validation and improvement of model interpretability, with tangible perspectives of integration into real-world workflows and translation from bench to bedside. Full article

Mpox, a zoonotic viral disease, has emerged as a global concern due to outbreaks in both endemic and non-endemic regions in 2022. Rodents, including African squirrels and Gambian pouched rats, are suspected key reservoirs, with human infections occurring through direct contact with infected animals or bushmeat consumption. Previously confined to rural Africa, mpox has spread via international travel and the exotic pet trade. Human-to-human transmission occurs mainly via respiratory droplets and direct contact with bodily fluids or lesions. The virus has a double-stranded DNA genome within a lipid envelope. Despite lower mutation rates in DNA viruses, mpox has developed mutations, particularly in genes like F8L, G9R, and F13L, facilitating viral replication and immune evasion. The virus targets immune cells such as monocytes and macrophages, weakening host defenses and prolonging infection. Immunocompromised individuals are at higher risk of severe complications. Although generally self-limiting, severe cases may require antiviral treatment. This article briefly summarizes the therapeutic and preventive strategies, and public health measures to combat zoonotic threats. Full article

The Diamondback moth (DBM), Plutella xylostella (L.), the most significant worldwide pest of Brassica crops, is notorious for resistance to a diverse number of insecticides. Field populations bioassayed in Georgia and Florida, USA, in 2018 were resistant to chlorantraniliprole but susceptible to cyantraniliprole. Subsequently, populations assayed in 2021, 2022, and 2023 were cross-resistant to both diamides. We used NextSeq analysis of the ryanodine receptor PxRyR, the target of diamides, to quantify target site mutations associated with resistance. Three populations sampled in 2018 had a high prevalence (75.0–98.3% of total reads) of the G4946E mutation, associated with resistance to chlorantraniliprole, and additionally, in one population, a very low (2.7%) prevalence of another mutation, I4790K, was associated with diamide cross-resistance. Populations sampled in 2021 had a decreased prevalence of G4946E (0.7 and 8.4%) and increased prevalence of I4790K (9.3 and 18.0%). The G4946E allele was almost absent (0.2% to 3.9%) in populations sampled in 2022 and 2023, while I4790K was present at frequencies from 34.1% to 84.0%. These data suggest a remarkable shift in PxRyR target site mutations, replacing G4946E with I4790K, which occurred between 2018 and 2022, and were associated with the recent occurrence of cross-resistance in DBM populations in the Southeastern USA. Full article

Background: Cutaneous melanoma (CM) poses significant diagnostic challenges due to its biological heterogeneity and the subjective interpretation of histopathologic criteria. While early and accurate diagnosis remains critical for patient outcomes, conventional pathology is limited by interobserver variability and diagnostic ambiguity, especially in borderline lesions. Objective: This narrative review explores the integration of digital pathology (DP) and artificial intelligence (AI)—including deep learning (DL), machine learning (ML), and interpretable models—into the histopathologic workflow for CM diagnosis. Methods: We systematically searched PubMed, Scopus, and Web of Science (2013–2025) for studies using whole slide imaging (WSI) and AI to assist melanoma diagnosis. We categorized findings across five domains: WSI-based classification models, feature extraction (e.g., mitoses, ulceration), spatial modeling and TIL analysis, molecular prediction (e.g., BRAF mutation), and interpretable pipelines based on nuclei morphology. Results: We included 87 studies with diverse AI methodologies. Convolutional neural networks (CNNs) achieved diagnostic accuracy comparable to expert dermatopathologists. U-Net and Mask R-CNN models enabled robust detection of critical histologic features, while nuclei-level analyses offered explainable classification strategies. Spatial and morphometric modeling allowed quantification of tumor–immune interactions, and select models inferred molecular alterations directly from H&E slides. However, generalizability remains limited due to small, homogeneous datasets and lack of external validation. Conclusions: AI-enhanced digital pathology holds transformative potential in CM diagnosis, offering accuracy, reproducibility, and interpretability. Yet, clinical integration requires multicentric validation, standardized protocols, and attention to workflow, ethical, and medico-legal challenges. Future developments, including multimodal AI and integration into molecular tumor boards, may redefine diagnostic precision in melanoma. Full article

Introduction. Over the past two decades, the αGal (Galα1–3Galβ1–4GlcNAc–R) epitope, a carbohydrate found in many non-primate mammals, has gained significant relevance in medicine due to its association with an increasing number of allergic reactions to animal-derived foods, drugs, and medical devices. Due to a mutated gene coding for α1,3-galactosyltransferase (α1–3GT), humans lack αGal and, therefore, naturally produce anti-α-Gal antibodies (IgM, IgA, and IgG), especially in the context of a xenotransplantation, which can lead to extreme immunological reactivity, including hyperacute rejection of the transplant. Recently, these uncontrollable immune reactions have driven demand for more accurate procedures to better detect αGal in animal-derived foods or bioprosthetics. The currently most widely used α-Gal-specific monoclonal antibody is an IgM antibody (clone M86), developed in Ggta1 KO mice and isolated from hybridoma tissue culture. As the IgM isotype has limited purification properties, specificity, and sensitivity, we aimed to produce a novel IgG antibody with high affinity and extensive applicability. Methods. An experimental murine IgG1 anti-αGal antibody (IgG-αGalomab) was developed by immunization of Ggta1 knockout (KO) mice, and its affinity was evaluated using ELISA, Western blot, flow cytometry, and immunohistochemistry/immunofluorescence. Results. Compared to IgM-M86, IgG-αGalomab demonstrated ~1200-fold higher binding potency and lower cross-reactivity with competitive molecules, i.e., bovine serum albumin, galactobiose, and lactose. Unlike IgM-M86, IgG-αGalomab showed an increasing affinity over time in the binding tests performed on xenogeneic tissues. Notably, high-affinity for αGal was detected by Western blot at high dilution [1:200,000] of IgG-αGalomab compared to IgM-M86 [1:1000]. By flow cytometry, specificity and dose-dependent response were confirmed using in vitro cultures of porcine and human fibroblasts. Finally, in immunofluorescence and immunohistochemistry analysis, αGal was demonstrated to be detectable by IgG-αGalomab at a dilution of [1:1000], while IgM-M86 was demonstrated to be detectable at [1:100]. Conclusions. Altogether, our newly developed antibody showed high sensitivity and specificity for α-Gal in various applications. Based on its potential binding capacity, IgG-αGalomab could have important applications in precision medicine for predicting, treating, and preventing immune-mediated phenomena of patients in different medical areas. Full article

Wheat blast, caused by Pyricularia oryzae Triticum lineage (PoTl), is one of the most destructive and significant fungal diseases affecting wheat crops. The stability of the G143A mutation in the cytB gene, which confers resistance to Quinone outside inhibitor fungicides (QoIs) in PoTl isolates, has not been extensively studied. This study was conducted to evaluate the stability of fungicide resistance, fitness, and competitive ability of the QoI-resistant (R) PoTl isolates group over nine and five consecutive transfer cycles in vitro and in vivo, respectively, without fungicide exposure. No changes in azoxystrobin sensitivity were observed in either the QoI-resistant or sensitive (S) PoTl isolate groups after the successive transfer cycles in vitro and in vivo. The mycelial growth of the QoI-R PoTl isolate group remained stable, while the conidial germination capacity increased over time. For the QoI-resistant isolates, leaf and head disease, conidial production, and the latent period on wheat leaves did not change between the first and fifth infection cycles. In each transfer cycle, the highest levels of leaf and head disease, as well as the largest quantities of conidia collected from wheat leaves, were observed in isolate mixtures. Also, the G143A mutation responsible for QoI resistance remained stable after five transfer cycles of the QoI-resistant (0S:100R) isolate on wheat leaves. Our findings indicate that the G143A mutation remains stable, and there are adaptive benefits in QoI-R PoTl isolates. We discuss the ecological implications of the wheat blast population’s adaptation and PoTl QoIs resistance stability in wheat-cropping areas in Brazil. Full article

Background/Objectives: Allele-specific genome editing using the CRISPR–Cas9 system is crucial for achieving precise therapeutic interventions in dominant inherited diseases that are otherwise difficult to treat with conventional approaches. However, Cas9–guide RNA (gRNA) complexes often tolerate single-base mismatches in target sequences, making it challenging to discriminate between wild-type and mutant alleles differing by only one nucleotide. Although previous studies have attempted to improve specificity by introducing mismatches into gRNAs, none has systematically investigated the impact of different mismatch types and positions on editing outcomes. In this study, we developed an effective strategy to enhance specificity and minimize off-target effects by deliberately introducing mismatches into gRNAs and comprehensively evaluating their editing performance. Results: We established an efficient strategy for the selective editing of mutant alleles that reduces Cas9 sequence tolerance and enhances specificity through the intentional introduction of mismatches into gRNAs. The efficacy of this approach was demonstrated by successful allele-specific editing of cancer-associated heterozygous point mutations in EGFR L858R and KRAS G12V, while minimizing editing of the corresponding wild-type alleles. Conclusion: Compared with perfectly matched gRNAs, the strategic incorporation of mismatches into gRNAs enhanced editing specificity for single-base mutant alleles. Our findings substantially improve the precision and safety of CRISPR-based genome editing for cancer therapy, particularly in cases involving mutant alleles. Full article

Background/Objectives: Molnupiravir (MOV) and nirmatrelvir (NMV) are antiviral drugs that were FDA-approved under the emergency use authorization (EUA) for coronavirus disease-2019 (COVID-19) treatment. MOV and NMV target the viral RNA-dependent RNA polymerase and main protease, respectively. Paxlovid is a combination of NMV and ritonavir (RTV), an inhibitor of the human cytochrome P450-3A4 (hCYP3A4). In this study, the structural consequences in the hCYP3A4 caused by MOV-induced mutations (MIM) were evaluated using in silico tools. Methods: MOV-induced mutations (MIM) were inserted into all the possible hotspots in the active site region of the hCYP3A4 gene, and mutant protein models were built. Structural changes in the heme-porphyrin ring of hCYP3A4 were analyzed in the presence and absence of substrates/inhibitors, including RTV. Molecular dynamics (MD) simulations were performed to analyze the effect of MIM-induced structural changes in hCYP3A4 on drug binding. Results: MD simulations confirm that MIMs, R375G and R440G in hCYP3A4 severely affect the heme-porphyrin ring stability by causing a tilt that in turn affects RTV binding, suggesting a possible inefficiency in the function of hCYP3A4. Similar results were seen for amlodipine, atorvastatin, sildenafil and warfarin, which are substrates of hCYP3A4. Conclusions: The current in silico studies indicate that hCYP3A4 containing MIMs can create complications in the treatment of COVID-19 patients, particularly with co-morbidities due to its functional inefficiency. Hence, clinicians must be vigilant when using MOV in combination with other drugs. Further in vitro studies focused on hCYP3A4 containing MIMs are currently in progress to support our current in silico findings. Full article

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic diseases with complex aetiology involving genetic, immunological, and environmental factors and intestinal microbiota disorders. Mutations in genes such as NOD2, ATG16L1, IRGM, TLR4, and IL23R disrupt the functioning of the intestinal barrier and the immune response, increasing susceptibility to chronic inflammation. Recent studies indicate that interactions between diet, gene expression, and epigenetic mechanisms play a key role in modulating the course of IBD, e.g., DNA methylation, histone modifications, and microRNA activity. The use of bioactive dietary components in combination with epigenome modulation is a promising tool in the treatment of IBD, enabling the reduction in chronic inflammation, improving intestinal barrier function, and supporting the immune response. Full article

KRAS mutations occur in over 90% of pancreatic ductal adenocarcinomas (PDACs), most commonly at codon 12, but the clinical implications of codon-specific subtypes in advanced disease remain unclear. We retrospectively analyzed 269 patients with advanced PDAC who underwent next-generation sequencing between 2020 and 2024 at a single tertiary cancer center. Clinical features, co-mutations, treatment outcomes, and survival were evaluated. KRAS mutations were detected in 92% of patients, most frequently G12V (36%) and G12D (34%), followed by G12R (10%) and non-G12 variants (9%). TP53 was the most frequent co-mutation (67%) and was significantly enriched in G12D tumors compared with wild type (74.2% vs. 31.8%). Mutations in homologous recombination and DNA damage response genes were more common in non-G12 and wild-type tumors, although not statistically significant. Serum CA 19-9 was elevated in most G12-mutant tumors, whereas approximately 40% of non-G12 and wild-type patients showed normal levels despite advanced disease. No significant survival differences were observed among KRAS subtypes in the overall or treated cohorts. However, patients with G12V mutations achieved significantly longer survival with fluorouracil-based than gemcitabine-based chemotherapy. These findings suggest that, while KRAS subtyping alone lacks prognostic value, the G12V subtype may inform chemotherapy selection and warrants further prospective validation. Full article