Search Results (16,285)

Leptospirosis remains a public health concern in the Philippines. Conventional diagnostic methods, including the microscopic agglutination test (MAT) and qPCR, are routinely used for outbreak response and surveillance. However, these methods often yield discordant results due to cross-reactivity, limited sensitivity, or lack of species-level resolution. To address these diagnostic gaps, this study optimized the Boonsilp 16S rRNA PCR assay and applied Sanger sequencing for accurate species identification of Leptospira in 92 archived DNA samples collected between 2018 and 2020. The sensitivity and specificity of the optimized assay were compared with those of MAT and qPCR. Species-level identification was confirmed via sequencing, and a phylogenetic tree was constructed. Among the 92 samples, 46 (50.0%) tested positive by qPCR, 39 (42.4%) by MAT, and 67 (72.8%) by at least one of the two methods. The optimized Boonsilp assay detected Leptospira in 23 samples (25.0%), of which 22 were also qPCR positive. Twenty-one samples were confirmed as L. interrogans, one as L. borgpetersenii, and one as an unclassified Leptospira species. One sample undetected by both MAT and qPCR tested positive using the optimized assay. Compared to the composite reference, the Boonsilp assay showed 32.8% sensitivity and 96.0% specificity. Phylogenetic analysis revealed multiple L. interrogans strains, including those closely related to reference sequences of Copenhageni, Manilae, and Canicola. While the optimized Boonsilp PCR assay demonstrates diagnostic value as an adjunct molecular tool to qPCR and MAT supporting species-level identification during outbreak surveillance, this warrants further validation in freshly isolated DNA samples. Full article

Burkholderia pseudomallei is a saprophytic environmental bacterium and the causative agent of melioidosis, a serious opportunistic infection in tropical regions, including northern Australia. Infection occurs following environmental exposure via percutaneous inoculation, ingestion, or inhalation; however, the environmental reservoirs and transmission pathways responsible for human disease remain poorly defined. Groundwater has been implicated as a potential source of infection, but the factors influencing the persistence and mobility of B. pseudomallei in surface waters in North Queensland are not well understood. Water samples were collected from a groundwater-connected seasonal creek in Townsville, North Queensland, over a 12-month period encompassing wet and dry seasons. Samples were cultured on Ashdown agar and confirmed as B. pseudomallei by qPCR. Multi-locus sequence typing (MLST) was performed using targeted allele sequencing on the Oxford Nanopore MinION platform. Eighteen of 59 water samples were culture-positive for B. pseudomallei. Detection occurred exclusively in turbid, flowing water following ≥30 mm of rainfall and was observed in both wet and dry seasons. MLST of 48 isolates identified 18 sequence types, including 12 novel types. Six sequence types matched previously reported Townsville clinical isolates. These findings indicate that groundwater from a connected urban creek may function as a mobile reservoir for clinically relevant B. pseudomallei strains under specific hydrological and climatic conditions, highlighting rainfall-driven processes as key drivers of environmental exposure risk. Full article

Chronic alcohol consumption exacerbates kidney injury, particularly in individuals with hepatitis B virus (HBV) infection. This study investigated the protective effects of boric acid supplementation against alcohol-induced renal damage in HBV transgenic mice. HBV transgenic mice were divided into four groups: control (C), boric acid (B), alcohol (A), and alcohol + boric acid (A + B). Renal injury was evaluated using H&E, PAS, TUNEL, and desmin staining. The expression of caspase-3, cytochrome c, and APAF-1 was analyzed by qRT-PCR. Biochemical analyses included BUN, creatinine, oxidative stress markers (ROS, MDA, TOS, OSI), total antioxidant status, and antioxidant enzyme activities (SOD, CAT, GPx). Histopathological findings showed activated parietal epithelial cells in all groups, indicating renal injury. Alcohol significantly increased tubular damage, podocyte desmin expression, apoptosis, cytochrome c and APAF-1 mRNA levels, and oxidative stress markers, while reducing antioxidant enzyme activities and BUN levels compared with controls. Boric acid supplementation significantly mitigated alcohol-induced tubular injury, apoptosis, oxidative stress, and serum creatinine levels, and improved BUN values. Boric acid treatment alone also alleviated glomerular and tubular injury and reduced tubular apoptosis compared with HBV control mice. Overall, boric acid exerts renoprotective effects in HBV-transgenic mice subjected to chronic alcohol exposure by inhibiting oxidative stress, apoptosis, and podocyte injury. Full article

Aspergillus flavus contaminates food commodities and produces carcinogenic aflatoxins. Pamamycin, a macrodiolide antibiotic from Streptomyces alboflavus TD-1, shows potent antifungal activity, yet its action against A. flavus and efficacy in complex food matrices largely remains unknown. Here, pamamycin was purified and evaluated using in vitro assays together with a peanut kernel model. Pamamycin reduced colony formation of A. flavus on PDA in a concentration-dependent manner, with near-complete inhibition at 4.0 mg/L on surface-treated PDA plates. Microscopy revealed progressive deformation and collapse of conidia and hyphae. Pamamycin increased membrane permeability, as indicated by elevated extracellular nucleic acid leakage, and impaired cell envelope integrity, as reflected by alkaline phosphatase release. In addition, pamamycin reduced Rh123-associated fluorescence, indicating an apparent dissipation of mitochondrial membrane potential under the tested conditions. Notably, at pamamycin concentrations of ≥0.5 mg/L, AFB1 accumulation was markedly reduced and fell below the limit of detection (LOD). This suppression was accompanied by distinct transcriptional changes in the aflatoxin regulatory network. RT–qPCR showed concentration-dependent repression of the pathway-specific regulators aflR and aflS, whereas the global regulator veA displayed a biphasic response with transient upregulation at lower concentrations. Notably, at 0.5 mg/L, multiple structural genes (aflC, aflD, aflK, aflP, and aflQ) were reduced to near-background transcript levels, coinciding with the loss of detectable AFB1. In inoculated peanut kernels incubated under high-humidity conditions, pamamycin significantly reduced fungal colonization and decreased AFB1 accumulation by >99%. Transcriptomic analysis of cultures treated with 0.5 mg/L pamamycin further revealed extensive transcriptional reprogramming, with enrichment of pathways related to branched-chain amino acid biosynthesis, central carbon metabolism, and ABC transporters. Collectively, pamamycin inhibits A. flavus through combined disruption of cell envelope integrity, apparent mitochondrial potential collapse, and broad suppression of the aflatoxin biosynthetic pathway, supporting its potential utility for mitigating aflatoxin contamination in peanut kernels, pending further safety evaluation. Full article

Developing novel anabolic agents for bone regeneration remains a clinical priority. Polydeoxyribonucleotide (PDRN) exhibits tissue-regenerative properties, but its direct cellular effects on bone remodeling remain unclear. This in vitro study investigated PDRN’s effects on osteoblast (MC3T3-E1) and osteoclast (primary bone marrow-derived macrophages) differentiation. We evaluated metabolic activity, gene/protein expression, and specific differentiation markers using MTS, qRT-PCR, Western blotting, and functional assays (ALP, Alizarin Red S, TRAP, pit formation). In osteoblasts, PDRN dose-dependently modulated metabolic activity while upregulating the early transcription factor Runx2. PDRN significantly enhanced osteoblast differentiation, evidenced by increased ALP activity, elevated mineralized matrix deposition, and robust upregulation of osteocalcin and Runx2. Conversely, PDRN exhibited no direct effect on osteoclast precursor metabolic activity, differentiation, or resorptive function. These findings support a working hypothesis in which PDRN selectively promotes osteoblast differentiation without directly affecting osteoclastogenesis. While further pharmacological investigations are required to definitively elucidate the specific purinergic receptor mechanisms, our results highlight PDRN as a promising candidate anabolic agent for bone regeneration. Full article

A liver fluke, Clonorchis sinensis is a representative fish-borne parasite infecting humans, and sensitive detection in fish hosts or aquatic environments is important for monitoring infection sources in endemic areas. Conventional diagnostic methods based on microscopy or conventional PCR often show limited sensitivity, particularly under low-parasite conditions. In this study, we developed a high-sensitive and species-specific molecular marker and established a real-time PCR (qPCR)-based diagnostic method targeting metacercariae isolated from freshwater fish, representing the transmission stage of C. sinensis. Primers and a hydrolysis probe targeting the mitochondrially encoded cytochrome c oxidase 1 (COI) gene were designed, and all primer combinations produced stable amplifications with single melt curves in C. sinensis-positive samples. Among them, one combination was finally selected as the optimal marker due to its high specificity, including validation against mixed trematode samples to confirm species-specific detection. The qPCR assay showed excellent linearity (R² = 0.998), with a detection limit of 10¹ copies per reaction and a quantification limit of 10² copies per reaction. In addition, the assay successfully detected C. sinensis DNA in environmental water samples spiked with metacercariae, demonstrating its applicability to aquatic samples for environmental surveillance purposes. Compared with conventional PCR, the developed qPCR method in this study exhibited markedly improved sensitivity in fish-derived samples. Overall, this qPCR assay provides a robust diagnostic tool for laboratory analysis and has potential utility for environmental DNA-based monitoring of clonorchiasis risk areas within a One Health framework. Full article

This study employed integrated transcriptomics and proteomics analysis to investigate the potential role of methionine (Met) in regulating the proliferation, differentiation, and lipid deposition of yak intramuscular preadipocytes (YIMA). Five Met concentrations (0, 0.05, 0.5, 5, and 50 mM) were used to establish the Met model of YIMA. The results of Bodipy, Oil Red O, EdU staining, and qPCR showed that the appropriate Met (0.05, 0.5, and 5 mM) supplementation significantly promoted the proliferation and adipogenic differentiation of YIMA, whereas excessive Met (50 mM) markedly inhibited these processes. To further evaluate the mechanism of Met on YIMA, cells supplemented with 0 mM (control, CON), 0.5 mM (moderate) and 50 mM (excessive) Met were selected for the transcriptomic and proteomic analyses. The results showed that moderate Met treatment primarily enriched pathways related to extracellular matrix–receptor interaction and the PI3K/AKT signaling pathway, while excessive Met significantly enriched processes involving abnormal methylation, DNA damage, and metabolic stress. Functional validation experiments further confirmed that Met regulates YIMA proliferation and differentiation by upregulating p-Akt protein expression and activating the PI3K/AKT pathway. These findings provide molecular insights that support improving yak meat quality and IMF content through dietary Met supplementation. Full article

Amplification efficiency is one of the key parameters in quantitative real-time PCR, as it directly influences the accuracy of both absolute and relative quantification. Amplification efficiency, the fold increase per cycle, is affected by oligonucleotide design, reaction chemistry, sample and template properties, and instrument performance. Consequently, it differs between samples, assays and experimental runs. Although methods for estimating the amplification efficiency have been available for more than two decades, most published qPCR studies continue to assume equal and ideal efficiency across assays. This simplifying assumption introduces efficiency- and expression-dependent error into relative expression and fold-change analyses, contributing to the poor reproducibility observed in many PCR-based studies. This review examines the role of the amplification efficiency in qPCR quantification; the consequences of ignoring, assuming or misapplying efficiency; and the practical implications for data interpretation. The review also considers the development of efficiency estimation models and their implications for contemporary analytical approaches, including emerging data-driven methods for amplification curve analyses. Full article

Rationale: Radium-223 dichloride (²²³RaCl₂) is an FDA-approved alpha-emitting radiopharmaceutical that targets bone metastases in metastatic castration-resistant prostate cancer (mCRPC). This study investigates the therapeutic and immunological effects of combining ²²³RaCl₂ with immune checkpoint inhibitors (ICIs) in a clinically relevant, immunocompetent murine model of prostate cancer bone metastasis. Methods: Luciferase-expressing MyC-CaP prostate cancer cells were implanted intratibially into FVB mice to establish bone metastases. Mice were treated with escalating doses of ²²³RaCl₂ (0.04–0.27 µCi) alone or a single dose combined with anti-CTLA-4 and anti-PD-L1 ICIs. Tumor growth was monitored using bioluminescence imaging. Micro-CT, alpha camera imaging, histology, and qPCR were used to assess bone remodeling, radiopharmaceutical distribution, immune infiltration, and gene expression. Ex vivo biodistribution and blood analyses quantified tissue uptake and toxicity. Results: Escalating doses of ²²³RaCl₂ did not significantly inhibit tumor growth or improve survival. Biodistribution and imaging showed preferential localization of ²²³RaCl₂ to tumor-adjacent bone, with minimal signal in isolated tumor tissue. Immunohistochemistry revealed increased CD4⁺ and CD8α⁺ T-cell infiltration in regions of high γH2AX expression, indicating localized immune modulation. However, combination therapy with ICIs did not enhance tumor control or immune infiltration beyond monotherapy. qPCR demonstrated significant upregulation of Mhc1 only in the combination group, suggesting localized immune activation. Toxicity profiles remained acceptable. Conclusions: ²²³RaCl₂ localizes primarily to bone surfaces, limiting direct cytotoxic and immunomodulatory effects within the tumor microenvironment. While combination with ICIs did not improve efficacy, these findings provide a platform for studying spatial dose distribution and support future development of tumor-targeted alpha therapies to potentiate immunotherapy in mCRPC. Full article

Teosinte branched1/cycloidea/proliferating cell factor (TCP) transcription factors are key regulators of plant growth and stress adaptation. However, their evolutionary history and functional divergence in rye (Secale cereale L.) remain unclear. Here, 26 ScTCP genes were identified from the reference rye genome. Phylogenetic and collinearity analyses with six representative cereals (Secale cereale, H. vulgare, O. sativa, T. aestivum, Z. mays, and A. tauschii Coss) revealed that segmental duplication, rather than tandem repetition, drove ScTCP expansion, with ScTCP2 located in a conserved syntenic block shared across the Poaceae family. Promoter analysis identified numerous hormone- and stress-responsive cis-elements, while a predicted protein–protein interaction network indicated extensive cross-talk with ERF and MYB transcription factors. Expression profiling of 12 representative ScTCP genes using qRT-PCR across different organs, developmental stages, six abiotic stress conditions, and three hormone treatments showed that ScTCP6 plays an important role in rye development and in responses to hormonal signals and abiotic stresses. Therefore, this study provides the first genome-wide characterization of the TCP gene family in rye and contributes to a broader understanding of the evolution and functional diversification of the TCP superfamily in higher plants. Full article

Background/Objectives: Bone metastases are a common complication of breast cancer. In our previous study, we reported that extracellular vesicles released by osteotropic human (MDA-MB-231) and murine (4T1) breast cancer cells disrupt bone homeostasis by enhancing osteoclast differentiation and impairing osteoblast function. Based on these findings, we investigated whether microRNAs contained within tumour-derived EVs could mediate these bone-altering effects. Methods: MDA-MB-231- and 4T1-EVs were tagged with the RNA-specific fluorophore SYTORNA and employed to treat mouse primary bone marrow macrophages (BMMs) and osteoblasts (OBs). We also performed RNAseq on MDA-MB-231- and 4T1-EVs to assess their miRNAs content. Finally, we evaluated the effect of selected miRNA-mimics on OBs, BMMs and HUVEC cells. Results: Fluorescence microscopy demonstrated EV-RNAs shuttling to recipient cells, while RNA sequencing on MDA-MB-231- and 4T1-EVs revealed that, of the top 20 expressed miRNAs, 10 were common. Among them, we first focused on the following four: miR-26a-5p, miR-24-3p, miR-29a-3p, and miR-29b-3p, which were linked to bone biology. We confirmed their presence in MDA-MB-231-/4T1-EVs by qPCR. Then, we evaluated their EV-mediated shuttling to BMMs and OBs using affinity tags. Among all the conditions tested, miR-29a and miR-29b were the best-shuttled miRNAs, with efficiency between 50–100% in both OBs and BMMs, both for MDA-MB-231- and 4T1-EVs. Finally, to test whether miR-29a and miR-29b could have a functional role in bone cells, OBs were transfected with miR-29a and 29b-mimics, discovering that this treatment reduced collagen1α1 and 1α2 mRNA as well as the OBs’ mineralisation ability, while the same miRNA mimics were found to have no effect on osteoclastogenesis or on in vitro angiogenesis. Conclusions: MDA-MB-231- and 4T1-EVs shuttle miRNAs to bone cells, which likely contributes to OBs’ activity impairment. Full article

Enteric diseases are a significant challenge for the poultry industry, causing substantial economic losses and affecting productivity. Turkey astrovirus (TAstV) types 1 and 2 and avian nephritis virus (ANV) are recognized as viral pathogens contributing with enteric diseases in turkeys, particularly in young poults. These viruses, part of the Astroviridae family, are small, round, non-enveloped, positive-sense RNA viruses with high prevalence in turkey flocks. Despite their importance, they had not been identified in Ecuador until now. This study presents the first detection and molecular characterization of TAstV-1, TAstV-2, and ANV in Ecuadorian turkeys using RT-qPCR assays based on SYBR Green, developed and optimized for high sensitivity and specificity. Two hundred intestinal samples were collected from turkeys with enteric disorders, along with fifty cloacal swabs from apparently healthy turkeys in Pichincha Province. The RT-qPCR assays developed demonstrated a limit of detection of one copy of viral genetic material and high repeatability, with inter and intra-assay coefficients of variation below 1%. Based on these tests, TAstV was detected in 93% of turkey samples with gastroenteritis, and none of the samples of the healthy group tested positive, with ANV being the most prevalent, followed by TAstV-2 and TAstV-1. Phylogenetic analysis of the partial ORF1b gene confirmed the genetic relationships between Ecuadorian strains and those from other countries, highlighting possible routes of introduction and evolution of the virus. Co-infections with TAstV-2 and ANV were common, while single infections were predominantly caused by ANV. These findings underscore the critical need for surveillance and biosecurity measures to control the spread of these viruses within Ecuador’s poultry industry. This study provides valuable insights on astrovirus presence in Ecuadorian turkey flocks and establishes robust diagnostic tools for monitoring and managing turkey astrovirus infections. Full article

This study’s aim was to clarify the regulatory roles of the MKI67 rs11016073 and APOB rs1367117 polymorphisms in the relationship between blood Pb levels and cancer risk. Blood Pb concentrations were measured using inductively coupled plasma mass spectrometry, and genotyping was performed by real-time PCR with TaqMan probes. Cancer incidence was assessed during a mean follow-up of six years and two months. During follow-up, 210 incident cancers were diagnosed among 2782 women. Pb exposure was categorized into quartiles (Q1: <9.44 µg/L; Q2: 9.44–12.58 µg/L; Q3: 12.59–17.16 µg/L; Q4: >17.16 µg µg/L). The association between Pb levels and cancer risk was strongly genotype dependent. Women carrying APOB non-GG and MKI67 non-AA genotypes exhibited the lowest breast cancer risk at the highest Pb levels (Q4), whereas carriers of APOB GG and MKI67 AA showed the lowest risk at the lowest Pb levels (Q1). Age-stratified analyses further demonstrated genotype-specific differences in optimal Pb exposure ranges, particularly for breast cancer. Cancer risk associated with Pb exposure is not uniform but depends on genetic background. These findings identify genotype-specific optimal blood Pb levels and suggest that incorporation of MKI67 and APOB genotyping may improve risk stratification and interpretation of non-linear Pb–cancer associations. Full article

Post-infectious Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic disease with unresolved pathophysiology and limited diagnostic options. Extracellular vesicles (EVs) carry disease-specific protein and miRNA signatures and may enable improved disease profiling. We aimed to identify novel protein and miRNA markers as potential biomarkers in plasma EVs from female ME/CFS patients, including post-COVID-19 ME/CFS and post-infectious ME/CFS of other origins, compared with healthy controls. EVs were isolated from plasma by size-exclusion chromatography and characterized for number, size, morphology, and surface marker expression. Flow cytometry showed that small EVs strongly expressed tetraspanins, with only minor differences between ME/CFS patients and healthy donors. Proteomic profiling of EVs from ME/CFS patients identified altered cargo proteins, including hemoglobin subunit alpha and insulin-like growth factor-binding protein acid labile subunit compared with healthy controls (n ≤ 10/cohort). Small RNA sequencing followed by qPCR revealed significant downregulation of hsa-let-7b-5p in EVs from post-COVID-19 ME/CFS patients (n = 12) versus healthy controls (n = 15). Reduced hsa-let-7b-5p expression correlated with impaired physical functioning and increased fatigue, pain, and immune activation. These findings indicate that EV cargo differences, particularly hemoglobin subunit alpha and insulin-like growth factor-binding protein acid labile subunit, as well as hsa-let-7b-5p, represent promising candidates for ME/CFS diagnosis and patient stratification. Full article

Among various ethanologenic microorganisms, thermotolerant Zymomonas mobilis has emerged as a promising candidate for industrial ethanol production at elevated temperatures. However, the comparative fermentation efficiency and the underlying molecular mechanisms driving thermotolerance in newly developed strains remain largely unexplored, hindering their industrial application. In this study, the recently developed thermotolerant strains Z. mobilis 200M and Z. mobilis PYK exhibited critical high temperatures for growth approximately 2.0 and 2.5 °C higher than the wild-type, respectively. While 40 °C represents severe heat stress that completely inhibits the growth of the wild-type, the thermotolerant strains remained viable, exhibiting significantly shorter cell lengths under these conditions. This study provides the first evidence of their superior multi-stress tolerance toward heat, ethanol, acetic acid, formic acid, and H₂O₂. Furthermore, the thermotolerant strains exhibited significantly higher ethanol fermentation efficiencies than the wild-type. At 40 °C, Z. mobilis 200M produced approximately 5.8-fold and 3.0-fold more ethanol than the wild-type after 24 and 48 h, respectively, while Z. mobilis PYK yielded 6.4-fold and 3.1-fold increases. Novel transcriptional insights via RT-qPCR revealed the simultaneous overexpression of genes involved in ethanol production, protein quality control, and signal transduction, particularly during the exponential phase under heat stress. Collectively, these findings bridge the gap between strain development and molecular understanding, suggesting that the coordinated upregulation of these genetic pathways enhances the adaptive capacity and fermentation efficiency of these thermotolerant strains during sustained growth at 40 °C. Full article