Search Results (3,503)

Crimean–Congo hemorrhagic fever (CCHF) is a zoonotic viral disease endemic to parts of Africa, Asia and southeastern Europe. Bulgaria is one of the few European countries with the consistent annual reporting of human CCHF cases. This study provides a descriptive overview of 24 confirmed CCHF cases in Bulgaria between 2015 and 2024. Laboratory confirmation was performed by an enzyme-linked immunosorbent assay (ELISA) and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) testing. Common findings included fever, fatigue, gastrointestinal symptoms, thrombocytopenia, leukopenia, liver dysfunction and coagulopathy. Two fatal cases were recorded. Two samples collected in 2016 and 2024 were subjected to whole-genome sequencing. Phylogenetic analysis showed that both strains clustered within the Turkish branch of the Europe 1 genotype and shared high genetic similarity with previous Bulgarian strains, as well as strains from neighboring countries. These findings suggest the long-term persistence of a genetically stable viral lineage in the region. Continuous molecular and clinical surveillance is necessary to monitor the evolution and public health impact of CCHFV in endemic areas. Full article

Background/Objectives: Vitex agnus-castus has been traditionally used to treat hormonal disorders, and recent evidence suggests its potential anticancer properties. However, its effects on gastric cancer remain unclear. Methods: This study examined the cytotoxic, apoptotic, and anti-metastatic effects of hydroalcoholic Vitex agnus-castus seed extract in gastric cancer cells. Antioxidant capacity (DPPH, ABTS) and total phenolic and flavonoid contents were analyzed. Cytotoxicity was assessed using the MTT assay in HGC27, MKN45, and AGS gastric cancer cell lines and CCD-1072Sk fibroblasts. Apoptosis, mitochondrial membrane potential (MMP), and cell cycle changes were evaluated via Annexin V-FITC/PI, Rhodamine 123, and PI staining, respectively. RT-qPCR and gene enrichment analyses were conducted to investigate the molecular mechanisms. Apoptosis-related protein expression was analyzed through enzyme-linked immunosorbent assay (ELISA). Results: The extract exhibited high antioxidant activity and a significant phenolic content. It reduced cell viability in a dose-dependent manner in gastric cancer cells, while exerting low toxicity in fibroblasts. It significantly increased apoptosis, induced G0/G1-phase cell cycle arrest, upregulated pro-apoptotic genes (CASP3, CASP7, TP53, BCL2L11), and downregulated anti-apoptotic genes (XIAP, NOL3). Gene enrichment analysis highlighted pathways like apoptosis, necrosis, and cysteine endopeptidase activity. The extract also disrupted MMP, inhibited migration and spheroid formation, suppressed EMT markers (SNAIL, SLUG, TWIST1, N-CADHERIN), and upregulated E-CADHERIN. The expression of Caspase 3 and Bax proteins increased and Bcl2 protein decreased. Conclusions: These findings suggest that Vitex agnus-castus seed extract exerts strong anticancer effects in gastric cancer cells by promoting apoptosis, reducing proliferation, and inhibiting migration. Further studies are warranted to explore its clinical relevance. Full article

Background/Objectives: The diminished efficacy of azoles in treating fungal infections is attributed to the emergence of resistance among pathogenic fungi. Employing a synergistic approach with other compounds to enhance the antifungal activity of azoles has shown promise, yet the availability of clinically valuable adjuvants for azoles and allylamines remains limited. Studies have demonstrated that the human host environment provides multiple carbon sources, which can influence the susceptibility of C. albicans to antifungal agents. Therefore, a comprehensive investigation into the mechanisms by which carbon sources modulate the susceptibility of C. albicans to azoles may uncover a novel pathway for enhancing the antifungal efficacy of azoles. Methods: This study explored the impact of various carbon sources on the antifungal efficacy of azoles through methodologies including minimum inhibitory concentration (MIC) assessments, super-MIC growth (SMG) assays, disk diffusion tests, and spot assays. Additionally, the mechanism by which galactose augments the antifungal activity of azoles was investigated using a range of experimental approaches, such as gene knockout and overexpression techniques, quantitative real-time PCR (qRT-PCR), Western blot analysis, and cycloheximide (CHX) chase experiments. Results: This study observed that galactose enhances the efficacy of azoles against C. albicans by inhibiting the translation of Erg1. This results in the suppression of Erg1 protein levels and subsequent inhibition of ergosterol biosynthesis in C. albicans. Conclusions: In C. albicans, the translation of Erg1 is inhibited when galactose is utilized as a carbon source instead of glucose. This novel discovery of galactose’s inhibitory effect on Erg1 translation is expected to enhance the antifungal efficacy of azoles. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-stranded positive-sense RNA virus, poses a significant threat to global swine production. Despite the availability of modified live virus and inactivated vaccines, their limited efficacy and safety concerns highlight the urgent need for novel antiviral therapeutics. This study aimed to investigate the molecular mechanisms by which lycopene inhibits PRRSV replication. Initial assessments confirmed that lycopene did not adversely affect cellular viability, cell cycle progression, or apoptosis. Using fluorescence microscopy, flow cytometry, immunoblotting, quantitative real-time PCR (qRT-PCR), and viral titration assays, lycopene was shown to exhibit potent antiviral activity against PRRSV. Mechanistic studies revealed that lycopene suppresses reactive oxygen species (ROS) production, which is critical for PRRSV proliferation. Additionally, lycopene attenuated PRRSV-induced inflammatory responses, as demonstrated by immunoblotting, ELISA, and qRT-PCR assays. These findings suggest that lycopene inhibits PRRSV replication by modulating ROS levels and mitigating inflammation, offering a promising avenue for the development of antiviral therapeutics. This study provides new insights and strategies for combating PRRSV infections, emphasizing the potential of lycopene as a safe and effective antiviral agent. Full article

Honey bees are essential pollinators for the ecosystem and food crops. However, their health and survival face threats from both biotic and abiotic stresses. Fungi, microsporidia, and bacteria might significantly contribute to colony losses. Therefore, rapid and sensitive diagnostic tools are crucial for effective disease management. In this study, molecular assays were developed to quickly and efficiently detect the main honey bee pathogens: Nosema ceranae, Aspergillus flavus, Paenibacillus larvae, and Black queen cell virus. In this context, new primer pairs were designed for use in quantitative Real-time PCR (qPCR) reactions. Various protocols for extracting total nucleic acids from bee tissues were tested, indicating a CTAB-based protocol as the most efficient and cost-effective. Furthermore, excluding the head of the bee from the extraction, better results were obtained in terms of quantity and purity of extracted nucleic acids. These assays showed high specificity and sensitivity, detecting up to 250 fg of N. ceranae, 25 fg of P. larvae, and 2.5 pg of A. flavus DNA, and 5 pg of BQCV cDNA, without interference from bee DNA. These qPCR assays allowed pathogen detection within 3 h and at early stages of infection, supporting timely and efficient management interventions. Full article

Background/Objectives: Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal genetic disease caused by a silent mutation in the LMNA gene, leading to the production of progerin, a defective prelamin A variant. Progerin accumulation disrupts nuclear integrity, alters chromatin organization, and drives systemic cellular dysfunction. While autophagy and inflammation are key dysregulated pathways in HGPS, the role of microRNAs (miRNAs) in these processes remains poorly understood. Methods: We performed an extensive literature review to identify miRNAs involved in autophagy and inflammation. Through stem-loop RT-qPCR in aging HGPS and control fibroblast strains, we identified significant miRNAs and focused on the most prominent one, miR-181a-5p, for in-depth analysis. We validated our in vitro findings with miRNA expression studies in skin biopsies from an HGPS mouse model and conducted functional assays in human fibroblasts, including immunofluorescence staining, β-Galactosidase assay, qPCR, and Western blot analysis. Transfection studies were performed using an miR-181a-5p mimic and its inhibitor. Results: We identified miR-181a-5p as a critical regulator of premature senescence in HGPS. miR-181a-5p was significantly upregulated in HGPS fibroblasts and an HGPS mouse model, correlating with Sirtuin 1 (SIRT1) suppression and induction of senescence. Additionally, we demonstrated that TGFβ1 induced miR-181a-5p expression, linking inflammation to miRNA-mediated senescence. Inhibiting miR-181a-5p restored SIRT1 levels, increased proliferation, and alleviated senescence in HGPS fibroblasts, supporting its functional relevance in disease progression. Conclusions: These findings highlight the important role of miR-181a-5p in premature aging and suggest its potential as a therapeutic target for modulating senescence in progeroid syndromes. Full article

(1) The beach areas of Galveston, Texas, USA are heavily used for recreational activities and often experience elevated fecal indicator bacteria levels, representing a potential threat to ecosystem services, human health, and tourism-based economies that rely on suitable water quality. (2) During the span of 15 months (March 2022–May 2023), water samples that exceeded the U.S. Environmental Protection Agency-accepted alternative Beach Action Value (BAV) for enterococci of 104 MPN/100 mL were analyzed via microbial source tracking (MST) through quantitative polymerase chain reaction (qPCR) assays. The Bacteroides HF183 and DogBact as well as the Catellicoccus LeeSeaGull markers were used to detect human, dog, and gull fecal sources, respectively. The qPCR MST data were then utilized in a quantitative microbial risk assessment (QMRA) to assess human health risks. Additionally, samples collected in July and August 2022 were sequenced for 16S rRNA and matched with fecal sources through the Bayesian SourceTracker2 program. (3) Overall, 26% of the 110 samples with enterococci exceedances were positive for at least one of the MST markers. Gull was revealed to be the primary source of identified fecal contamination through qPCR and SourceTracker2. Human contamination was detected at very low levels (<1%), whereas dog contamination was found to co-occur with human contamination through qPCR. QMRA identified Campylobacter from canine sources as being the primary driver for human health risks for contact recreation for both adults and children. (4) These MST results coupled with QMRA provide important insight into water quality in Galveston that can inform future water quality and beach management decisions that prioritize public health risks. Full article

Via a One Health approach, this study concomitantly assessed the susceptibility of humans and dogs to Trypanosoma cruzi infections on three islands and in two mainland seashore areas of southern Brazil. Human serum samples were tested using an enzyme-linked immunosorbent assay (ELISA) to detect anti-T. cruzi antibodies, while dog serum samples were tested using indirect fluorescent antibodies in an immunofluorescence assay (IFA). Seropositive human and dog individuals were also tested using quantitative polymerase chain reaction (qPCR) in corresponding blood samples. Overall, 2/304 (0.6%) human and 1/292 dog samples tested seropositive for T. cruzi by ELISA and IFA, respectively, and these cases were also molecularly positive for T. cruzi by qPCR. Although a relatively low positivity rate was observed herein, these cases were likely autochthonous, and the individuals may have been infected as a consequence of isolated events of disturbance in the natural peridomicile areas nearby. Such a disturbance could come in the form of a fire or deforestation event, which can cause stress and parasitemia in wild reservoirs and, consequently, lead to positive triatomines. In conclusion, T. cruzi monitoring should always be conducted in suspicious areas to ensure a Chagas disease-free status over time. Further studies should also consider entomological and wildlife surveillance to fully capture the transmission and spread of T. cruzi on islands and in seashore mainland areas of Brazil and other endemic countries. Full article

Herbivore-induced plant volatiles (HIPVs) are well known for their roles in herbivore deterrence and attraction of natural enemies, but their direct impact on insect reproduction remains largely unexplored. In this study, we provide novel evidence that two representative HIPVs, 2-heptanol and α-cedrene, exert opposing effects on the reproduction of Chilo suppressalis, a major rice pest. While both volatiles repelled adults, α-cedrene unexpectedly enhanced oviposition, whereas 2-heptanol significantly suppressed egg laying. To examine these effects, we conducted oviposition assays, preoviposition and longevity tests, combined with qPCR and transcriptome analyses to explore underlying molecular responses. Mechanistically, α-cedrene upregulated Kr-h1, a gene linked to juvenile hormone signaling and vitellogenesis, promoting reproductive investment. Transcriptomic profiling revealed divergent molecular responses: α-cedrene activated reproductive pathways, whereas 2-heptanol induced stress- and immune-related genes, suggesting a trade-off between stress defense and reproduction. These findings demonstrate that HIPVs can exert compound-specific reproductive effects beyond repellency. This work fills a key knowledge gap and highlights the potential of HIPVs as precision tools in pest management strategies that exploit behavioral and physiological vulnerabilities beyond repellency. Full article

Resistance to cytarabine (Ara-C) remains a major obstacle to the successful treatment of acute myeloid leukemia (AML). Therefore, modulating Ara-C resistance is indispensable for improving clinical outcomes. We previously demonstrated that sodium caseinate (SC), a salt derived from casein, the principal milk protein, inhibits proliferation and modulates the expression of Ara-C resistance-related genes in chemoresistant cells. However, it remains unclear whether the combination of SC with antineoplastic agents enhances apoptosis, modulates chemoresistance-related genes, and prolongs the survival of tumor-bearing mice implanted with chemoresistant cells. Here, we investigated the effects of SC in combination with Ara-C or daunorubicin (DNR) on cell proliferation, apoptosis, the expression of chemoresistance-associated genes, and the survival of tumor-bearing mice. Crystal violet assays, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence, flow cytometry, and Kaplan–Meier survival curves were used to evaluate the effects of combinations in chemoresistant cells. We demonstrate that the IC₂₅ concentration of SC, when combined with antileukemic agents, increases the sensitivity of chemoresistant WEHI-CR50 cells to Ara-C by downregulating SIRT1 and MDR1, upregulating the expression of ENT1 and dCK, enhancing apoptosis, and prolonging the survival of WEHI-CR50 tumor-bearing mice. Our data suggest that SC in combination with antileukemic agents could be an effective adjuvant for Ara-C-resistant AML. Full article

With the increasing detection of artemisinin resistance to front-line antimalarials in Africa and notwithstanding the planned roll-out of RTS’S and R21 in Africa, the search for new vaccines with high efficacy remains an imperative. Towards this endeavour, we performed in silico screening to identify Plasmodium falciparum gametocyte stage genes that could be targets of protection or diagnosis. Through the analysis we identified a gene, Pf3D7_1105800, coding for a Plasmodium falciparum subtilisin-like domain-containing protein (PfSDP) and thus dubbed the gene Pfsdp. Genetic diversity assessment revealed the Pfsdp gene to be relatively conserved across continents with signs of directional selection. Using RT qPCR and Western blots, we observed that Pfsdp is expressed in all developmental stages of the parasite both at the transcript and protein level. Immunofluorescence assays found PfSDP protein co-localizing with PfMSP-1 and partially with Pfs48/45 at the asexual and sexual stages, respectively. Further, we demonstrated that anti-PfSDP peptide-specific antibodies inhibited erythrocyte invasion by 20–60% in a dose-dependent manner, suggesting that PfSDP protein might play a role in merozoite invasion. We also discovered that PfSDP protein is immunogenic in children from different endemic areas with antibody levels increasing from acute infection to day 7 post-treatment, followed by a gradual decay. The limited effect of antibodies on erythrocyte invasion could imply that it might be more involved in other processes in the development of the parasite. Full article

Carbapenem resistance is considered one of the greatest current threats to public health, particularly in the management of infections in clinical settings. Carbapenem resistance in bacteria is mainly due to mechanisms such as the production of carbapenemases (such as the imipenemase IMP, or other enzymes like VIM, NDM, and KPC), that can be detected by several laboratory tests, including immunochromatography and automated real-time PCR (qPCR). Methods: As part of local studies to monitor carbapenem-resistant bacteria in Costa Rica, two cases were initially identified with inconsistent IMP detection results. A possible gene drop-out in the automated qPCR test was suggested based on the negative result, contrasting with the positive result by immunochromatography and whole-genome sequencing. We hypothesized that molecular testing could be optimized through the development of tailored assays to improve the detection of IMP genes. Thus, using IMP gene sequences from the local isolates and regional sequences in databases, primers were redesigned to extend the detection of IMP alleles of regional relevance. Results: The tailored qPCR was applied to a local collection of 119 carbapenem-resistant isolates. The genomes of all 14 positive cases were sequenced, verifying the results of the custom qPCR, despite the negative results of the automated testing. Conclusions: Guided by whole-genome sequencing, it was possible to extend the molecular detection of IMP alleles circulating in Latin America using a tailored qPCR to overcome IMP gene drop-out and false-negative results in an automated qPCR. Full article

Background/Objectives: Saliva as a diagnostic medium for COVID-19 requires fewer resources to collect and is more readily adopted across a range of testers. Our study compared an Emergency Use Authorized direct saliva-to-RT-qPCR test against an FDA-authorized nasal swab RT-qPCR assay for participants who reported symptoms of respiratory infection. Methods: We analyzed 737 symptomatic participants who self-selected to test at either a community testing facility or a walk-in clinic due to respiratory symptoms and provided matched saliva and nasal swab samples. Samples were collected between March and September of 2023, both before and after the declared end of the public health emergency. Results: A total of 120 participants tested positive in at least one of the tests. For participants testing in the first 5 days of reported symptoms, the saliva test had a 94.0 positive percent agreement (PPA; 95% C.I. 88.9–99.1%) with the nasal test and a 99.0 negative percent agreement (NPA; 95% C.I. 98.1–99.9%). The viral load decreased beyond day 1 of reported symptoms for saliva testing. Viral load increased up to day 4 for nasal swabs and then decreased. The same number of discordant positive samples (five each) occurred for both tests within 5 days of symptoms onset. Conclusions: In the endemic phase of COVID-19 and for development of new tests, testing methods that are less invasive are more likely to be adopted. The results of saliva-based versus nasal swab PCR measurements relative to days of symptom onset are needed to optimize future testing strategies. Full article

Background/Objectives: Different risk factors are involved in the initiation and progression of melanoma. In particular, genetic and epigenetic pathways are involved in all stages of melanoma and are exploited in therapeutic approaches. This study investigated the role of circular RNA circ_0001591 in melanoma cell migration. Methods: Three different melanoma cell lines were transfected with siRNA targeting circ_0001591 and with mimic or inhibitor molecules for miR-20a-3p and miR-34a-5p. Gene and protein expression levels were analyzed by RT-qPCR and Western blot, respectively. Dual luciferase reporter assays were performed to confirm the direct interaction of miR-20a-3p and miR-34a-5p with circ_0001591, as well as with the 3’UTRs of AXL (for both miRNAs) and FOSL1 (miR-34a-5p only). Wound healing assays were conducted to assess cell migration velocity. Results: The silencing of circ_0001591 significantly reduces the migration ability of melanoma cell lines. This downregulation was associated with an increased expression of miR-20a-3p and miR-34a-5p. Dual luciferase reporter assays confirmed the direct binding of both miRNAs to circ_0001591, supporting its role as a molecular sponge. The same assays also verified that miR-20a-3p directly targets the 3’UTR of AXL, while miR-34a-5p binds the 3’UTRs of both AXL and FOSL1. Western blot analysis showed that the modulation of this axis affects the expression levels of the AXL and FRA1 oncoproteins. Conclusions: Our findings demonstrate that circ_0001591 promotes melanoma migration by sponging miR-20a-3p and miR-34a-5p, thereby indirectly modulating the expression of AXL and FRA1 oncoprotein. Further investigations of this new regulatory network are needed to better understand its role in melanoma progression and to support the development of targeted therapies. Full article

Background: Polymerase chain reaction (PCR) is highly sensitive and specific for the rapid diagnosis of invasive fungal disease (IFD) but is not yet widely implemented due to concerns regarding limited standardisation between assays, the lack of commercial options and the absence of clear guidance on interpreting results. Objectives and Methods: This review provides an update on technical and clinical aspects of PCR for the diagnosis of the most pertinent fungal pathogens, including Aspergillus, Candida, Pneumocystis jirovecii, Mucorales spp., and endemic mycoses. Summary: Recent meta-analyses have demonstrated that quantitative PCR (qPCR) offers high sensitivity for diagnosing IFD, surpassing conventional microscopy, culture and most serological tests. The reported specificity of qPCR is likely underestimated due to comparison with imperfect reference standards with variable sensitivity. Although the very low limit of detection of qPCR can generate false positive results due to procedural contamination or patient colonisation (particularly in pulmonary specimens), the rates are comparable to those observed for biomarker testing. When interpreting qPCR results, it is essential to consider the pre-test probability, determined by the patient population, host factors, clinical presentation and risk factors. For patients with low to moderate pre-test probability, the use of sensitive molecular tests, often in conjunction with serological testing or biomarkers, can effectively exclude IFD when all tests return negative results, reducing the need for empirical antifungal therapy. Conversely, for patients with high pre-test probability and clinical features of IFD, qPCR testing on invasive specimens from the site of infection (such as tissue or bronchoalveolar lavage fluid) can confidently rule in the disease. The development of next-generation sequencing methods to detect fungal infection has the potential to enhance the diagnosis of IFD, but standardisation and optimisation are essential, with improved accessibility underpinning clinical utility. Full article