Search Results (8,478)

Background: Infectious bronchitis virus (IBV) is a highly spreading, evolving virus that induces multiple manifestations, including respiratory, urinary, and reproductive symptoms, and presents a considerable risk to the Egyptian poultry sector. This study assessed various IBV vaccination protocols available in broiler populations comprising live attenuated vaccines such as IB Var II, 793/B (4/91), IB Primer, and H120 against the local novel IBV-GI-23.3 strain. Methods: Vaccines were administered to eight groups of SPF chicks at 1 day only or 1 + 14 days of age. Birds were challenged via the oculo-nasal route at 28 days of age using 10⁶ EID₅₀/0.2 mL/chick with the NewValley-1-EGYIBV-GI23.3-2023 local strain. Ciliostasis activity and the scores for histopathological lesions were evaluated at 7 days post-challenge (DPC). Virus shedding was monitored at 3, 5, and 7 DPC using the real-time RT-PCR method. Results: The ciliostasis test indicated that the vaccinated groups receiving the IB Primer + 4/91 vaccine regime at 1 day only or 1 + 14 days of age received the highest level of protection (65%, 68%, respectively). Similarly, administration of IB Primer + IB Var II at 1 + 14 days of age demonstrated substantial protection (63%). Conversely, administering the H120 + 4/91 vaccination protocol at days 1 and 14 resulted in a moderate level of protection (53%). Tracheal IBV shedding quantification and subsequent histopathological signs of trachea, proventriculus, bursa, and kidney degenerative changes were significantly lower in the vaccinated groups (especially the IB Primer + 4/91 vaccine regime at 1 day only or 1 + 14 days) than in the positive control groups. Conclusions: The heterologous combined IB Primer + 4/91 program demonstrated the most significant protective efficacy against the IBV field challenge strains compared with other vaccines in broiler chickens. Full article

In this study, we successfully cloned the fluorescent proteins eGFP and DsRed in-frame with the antimicrobial peptide epinecidin-1 (FIFHIIKGLFHAGKMIHGLV) at the N-terminal. The cloning strategy involved inserting the fluorescent reporters into the expression vector, followed by screening for positive clones through visual fluorescence detection and molecular validation. The visually identified fluorescent colonies were confirmed as positive by PCR and plasmid migration assays, indicating successful cloning. This fusion of fluorescent reporters with a short antimicrobial peptide enables real-time visualization and monitoring of the peptide’s mechanism of action on membranes and within cells, both in vivo and in vitro. The fusion of eGFP and DsRed to epinecidin-1 did not impair the expression or fluorescence of the reporter protein. Full article

Galactic cosmic ray (GCR) radiation is a major barrier to human space exploration beyond Earth’s magnetic field protection. Mesenchymal stem cells (MSCs) are found in all organs and play a critical role in repair and regeneration of tissue. We engineered bone marrow-derived MSCs and evaluated their response to ionizing radiation exposure. Epidermal growth factor receptor (EGFR) expression by certain types of cancers has been shown to induce radioresistance. In this study, we tested the feasibility of transfecting MSCs to overexpress EGFR (eMSC-EGFR) and their capacity to tolerate and recover from X-ray exposure. Quantitative real-time PCR (qRT-PCR) and immunoblotting results confirmed the efficient transfection of EGFR into MSCs and EGFR protein production. eMSC-EGFR maintained characteristics of human MSCs as outlined by the International Society for Cell & Gene Therapy. Then, engineered MSCs were exposed to various dose rates of X-ray (1–20 Gy) to assess the potential radioprotective role of EGFR overexpression in MSCs. Post-irradiation analysis included evaluation of morphology, cell proliferation, viability, tumorigenic potential, and DNA damage. eMSC-EGFR showed signs of radioresistance compared to naïve MSCs when assessing relative proliferation one week following exposure to 1–8 Gy X-rays, and significantly lower DNA damage content 24 h after exposure to 4 Gy. We establish for the first time the efficient generation of EGFR overexpressing MSCs as a model for enhancing the human body to tolerate and recover from moderate dose radiation injury in long-term manned space travel. Full article

Mosquito-borne viruses like West Nile virus (WNV) and Usutu virus (USUV) present growing public health concerns, especially with climate change and expanding vector ranges. This study describes the development and validation of a duplex Real-Time RT-PCR assay targeting β-actin (ACTB) mRNA as an endogenous control and a conserved 92 bp region shared by WNV and USUV genomes. Degenerate primers for ACTB ensure RNA extraction quality and PCR performance while enabling simultaneous detection of both viruses. A total of 1002 mosquito pools collected in Piedmont, Italy, during the 2024 vector season under the National Surveillance Plan for Arboviruses (PNA), were tested. The assay showed 100% accuracy—ACTB mRNA was detected in all pools, and six pools tested positive for WNV or USUV (three each). Diagnostic specificity was confirmed on 40 horse and bovine serum samples. Sanger sequencing confirmed ACTB identity across multiple mosquito species. The assay also demonstrated reproducibility across different operators and thermocyclers. The limit of detection (LOD) evaluation showed that the assay is capable of detecting viral RNA at very low concentrations, confirming its high analytical sensitivity. The duplex RT-PCR here developed is a reliable, sensitive, and specific tool for arbovirus surveillance, combining pathogen detection with internal quality control of RNA extraction and amplification, thus improving early warning and rapid response to mosquito-borne disease threats. Full article

Soybean (Glycine max) is a vital oilseed and economic crop in China, often constrained by drought, salinity, and biotic stresses. In this study, we identified a soybean bZIP transcription factor, GmbZIP59, whose expression is upregulated by salt, drought, ethylene (ETH), methyl jasmonate (MeJA), and abscisic acid (ABA). Overexpression of GmbZIP59 in Arabidopsis (OE-13 and OE-20, two independent Arabidopsis transgenic lines) exhibited enhanced resistance to Sclerotinia sclerotiorum (S. sclerotiorum), improved tolerance to salt stress, and increased sensitivity to phytohormones. Overexpression of GmbZIP59 in rice (OE-1 and OE-2, two independent rice transgenic lines) improved tolerance to salt and drought stresses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that elevated expression of stress-related genes occurred in transgenic lines under adverse conditions. Furthermore, chromatin immunoprecipitation-qPCR (ChIP-qPCR) assays confirmed that GmbZIP59 directly binds to the promoters of ETH, MeJA, and ABA, responsive genes associated with stress responses. These findings demonstrate that GmbZIP59 acts as a positive regulator of biotic and abiotic stress tolerance in soybean. Full article

In recent decades, the problem of resistant strains, which present resistance to different types of antimicrobials, has increased. Klebsiella pneumoniae is one of the most important species that exhibits an acquired resistance phenotype to at least one agent in three or more classes of antimicrobials and is thus characterized as a multidrug-resistant bacterium (MDR). 98 nosocomial strains of K. pneumoniae were isolated during the pre-COVID-19 period, and more specifically, from February 2015 to March 2019, were analyzed for the detection of class A, D, and B carbapenemase genes. The existence of KPC, OXA-48 like, IMP, VIM, and NDM carbapenemases has been examined. The immunochromatography showed that NDM carbapenemases are more frequently detected in the samples, reaching a percentage of 30.7%, while correspondingly the percentage for VIM carbapenemases was 7.68% among the strains with resistant phenotypes. No strain with carbapenemase IMP was found. Real-time multiplex polymerase chain reaction (PCR) showed, in contrast to immunochromatography kits, that a high percentage of bacterial isolates (94.26%) carry NDM and VIM carbapenemase genes, while no IMP carbapenemase genes were detected. Regarding the KPC enzymes, the immunochromatography kits showed that KPC positive strains are reaching 53.1%, and OXA-48 positive strains are reaching 3.1% among the strains with resistant phenotypes. Real-time multiplex polymerase chain reaction revealed a much higher percentage of 89.6% KPC positive isolates and a percentage of 14.6% OXA-48 carbapenemase producers. The aforementioned results indicate the dominance of the Multiplex Real-Time PCR as a “gold standard” method. This study could not fully support the usefulness of rapid immunochromatographic tests as a fast and useful diagnostic tool in the laboratory daily routine, as per the results of previous studies. Thus, more studies need to be conducted in this field to introduce these rapid tests safely into the daily laboratory workflow as a screening tool. Additionally, this study underlines the predominance of KPC enzymes from clinical isolates of ICUs and a significant shift over the OXA-48 like enzymes that are not limited to the ICU environment. Full article

HSV-1 is associated with oral lesions and non-sexual transmission; HSV-2 is primarily transmitted through sexual contact and causes genital infections. Understanding the epidemiological dynamics of both viruses is essential for guiding targeted public-health responses. We conducted a retrospective analysis of 177,599 clinical specimens collected between September 2018 and December 2022 from patients with symptoms suggestive of sexually transmitted infections (STIs) at healthcare institutions across South Korea. HSV-1 and HSV-2 were identified using a real-time PCR assay; positivity rates were stratified by age, sex, specimen type, and year of testing. The overall positivity rate was 0.26% for HSV-1 and 1.60% for HSV-2. HSV-1 was most prevalent among individuals aged <19 years; HSV-2 showed the highest positivity in females aged 20–29 years, declining with age thereafter. HSV-2 positivity was significantly higher in females than in males. A significant decline in HSV-2 positivity was observed over the 5-year study period, while HSV-1 positivity remained stable. This nationwide PCR-based study reveals distinct age- and sex-related epidemiological patterns of HSV-1 and HSV-2. The findings support the need for age-specific and gender-specific STI screening strategies and health education programs. The declining trend in HSV-2 infection may reflect the impact of recent public-health initiatives. Full article

(1) Background: This study investigated the presence of human papillomavirus (HPV), HPV genotypes, Epstein–Barr virus (EBV), cytomegalovirus (CMV) and herpes simplex virus (HSV) in patients with Head and Neck Cancer (HNC) at both molecular and serological levels. (2) Methods Fifty patients with histopathologically confirmed HNC who were admitted to the Department of Otorhinolaryngology, Istanbul Faculty of Medicine. Viral DNA was detected using quantitative real-time PCR, and serological IgM and IgG antibodies were analyzed using the CMIA method; (3) Results: In blood samples, CMV and HSV DNA were not detected, whereas EBV DNA was identified in 2% and HPV DNA in 4% of patients. In tumor tissues, CMV DNA was detected in 8%, EBV DNA in 10%, and HPV DNA in 6%; HSV DNA is 6%. HPV genotypes 18, 45, and 69 were found in tissue samples. Serologically, IgG positivity for CMV, EBV, and HSV-1 exceeded 90%, whereas IgM positivity was low and not statistically significant; (4) Conclusions: HPV, EBV, and CMV DNA were detected at low frequencies in patients with HNC, while HSV DNA was absent. These findings underline the need for larger multi-center studies and support the consideration of routine viral screening, particularly for HPV, in specific tumor subtypes. Full article

Intestinal organoids possess self-organizing capacity and recapitulate essential features of intestinal architecture and function, making them powerful models for investigating development, disease mechanisms, pharmacological testing, and host–microbe interactions. Although standardized protocols for chicken intestinal organoids have been established, a defined culture system for ducks has not been available. In this study, we optimized crypt isolation procedures and culture medium composition to establish a reproducible system tailored to duck intestinal stem cells. Among various digestive solutions, ethylene glycol tetraacetic acid (EGTA) achieved the highest crypt isolation efficacy and organoid survival. Suspension culture resulted in better survival, proliferation, and differentiation of intestinal stem cells than air–liquid interface and embedding methods (p < 0.05). Immunofluorescence and real-time PCR indicated the presence of multiple epithelial lineages, including stem cells, Paneth cells, enterocytes, goblet cells, and enteroendocrine cells. Media supplemented with CHIR99021 and LDN193189 (CL) supported growth comparable to that of media with EGF, Noggin, and R-spondin 1 (ENR). Duckling serum and specific factors, such as SB203580 and retinol, further improved organoid formation and promoted differentiation. While long-term passaging and expansion remain technically challenging, this work provides the first duck intestinal organoid model and lays the foundation for future applications in avian intestinal research, including nutrition, disease modeling, and intervention strategies. Full article

Background: Endometrial cancer is one of the most common female genital cancers and poses a significant clinical problem due to its increasing incidence and variable prognosis depending on the stage of the disease. The development of EC is largely dependent on interactions with the immune system, including immune checkpoints (ICPs) such as PD-1, PD-L1, and PD-L2. The aim of our study was to evaluate the PD-1/PD-L1/PD-L2 pathway in EC and its clinical significance. Methods: The analysis was performed by flow cytometry on myeloid and plasmacytoid dendritic cells and monocytes (MO) in peripheral blood (PB). The concentration of sPD-1, sPD-L1, and sPD-L2 in plasma was determined by ELISA. Additionally, PD-L1 and PD-L2 gene expression levels in tumor tissue (TT) were assessed using real-time polymerase chain reaction (qPCR). The obtained results were correlated with clinical data of EC patients. Results: Patients with EC had lower percentages of PD-L1-positive MO and pDCs, as well as PD-L2-positive MO and mDCs, compared with the control group. We observed accumulation of sPD-1 and lower levels of sPD-L1 and sPD-L2 in EC patients compared to the control group, with sPD-L2 correlating with PD-L2 gene expression level in the TT. Conclusions: The study results indicate a difference in the distribution of mDCs, pDCs, and MO with PD-L1/PD-L2 expression in EC patients. Reduced percentages of MO and DCs expressing PD-L1 and PD-L2, altered concentrations of soluble forms of these IPCs, and correlations with gene expression in TT suggest that dysregulation of this pathway may influence disease progression. Furthermore, the relationships between immunological parameters and clinical features such as BMI and FIGO stages suggest the potential use of these factors as diagnostic and prognostic biomarkers and the possibility of incorporating them into future therapeutic strategies. However, further studies are necessary to validate this hypothesis. Full article

Accurate diagnosis of adenoviral conjunctivitis is critical for timely treatment and infection control. However, conventional lateral flow kits often lack sufficient sensitivity, especially in mild cases. This multicenter prospective study evaluated the diagnostic performance of silver-amplified lateral flow kits (SA-LFKs) compared with non-silver-amplified kits (NSA-LFKs), using real-time polymerase chain reaction (qPCR) as the reference standard. Tear samples from 200 patients with suspected adenoviral conjunctivitis were collected across four clinics and analyzed for sensitivity, specificity, and genotype coverage. The SA-LFK demonstrated significantly higher sensitivity (86.0%) than the NSA-LFK (72.0%) (p < 0.001), with both kits showing high specificity (100% and 98.1%, respectively). Pooled analysis revealed that sensitivity was significantly lower in mild-to-moderate cases than in severe cases for both kits, suggesting that clinical severity influences detection performance. Across all severity levels, the SA-LFK consistently demonstrated higher sensitivity than the NSA-LFK, including in mild-to-moderate cases (77.8% vs. 59.3%, p = 0.004), supporting its superior diagnostic performance. The SA-LFK showed robust performance across eight identified adenovirus genotypes and maintained higher positivity rates even at lower viral loads. These findings support the clinical utility of SA-LFKs for early diagnosis and outbreak containment in diverse settings. Full article

Preeclampsia (PE) is a major cause of maternal and perinatal morbidity, and early prediction is critical for timely intervention. This study aimed to identify predictive biomarkers for PE through transcriptomic analysis of second-trimester amniotic fluid supernatant (AFS) collected prior to clinical symptom onset. AFS samples from women who later developed PE (n = 7) and matched controls (n = 7) underwent RNA sequencing to identify differentially expressed genes (DEGs). Candidate genes were validated by real-time PCR in HTR-8/SVneo cells exposed to fluid shear stress at 3, 10, and 20 dyn/cm² for 24 h, mimicking the hemodynamic environment of PE, and siRNA-mediated knockdown was used to assess effects on trophoblast migration and invasion. RNA sequencing revealed 19 DEGs, with 3 upregulated and 16 downregulated genes in the PE group. HOOK2 emerged as the most significantly upregulated gene. Four candidate genes, including HOOK2, CCDC160, CKB, and PARP15, were selected for further validation. HOOK2 mRNA expression significantly increased with higher shear stress levels, consistent with sequencing data. Knockdown of HOOK2 led to a significant increase in trophoblast invasion, while migration showed no significant change. These findings suggest that HOOK2 may serve as a promising early biomarker for PE by modulating trophoblast invasiveness under altered hemodynamic conditions, with potential to improve risk stratification and personalized monitoring in pregnancy. Full article

Although female (XX) and male (XY) placentas generally function the same, it is evident that there are sex-specific postnatal health outcomes following placental dysfunction and pregnancy complications. Although the underlying causes for these sex differences are unclear, it is postulated that differences in XX and XY placental function are involved due to sex chromosomes and/or sex steroids. Studies in breast and prostate cancer cells demonstrated a role for the citrullination enzyme peptidylarginine deiminase 2 (PAD2) in post-translational regulation of estrogen (ESR) and androgen receptor (AR) signaling. The goal of this study is to determine if PAD2 is present in mouse placentas and if XX versus XY differences exist in the relative level of PAD2. Fetuses and placentas were collected from three pregnant mice (C57BL6) at 14 days of gestation. Total RNA and protein were isolated from XX and XY placentas, and relative mRNA and protein were analyzed by real-time PCR and Western blot. AR and PAD2 levels were significantly higher in XY than in XX placentas. This study is the first to demonstrate XX and XY differences in PAD2 and AR in the placenta. It suggests a role for PAD2 regulation of androgen receptor signaling in the XY placenta. Full article

Mussels’ byssus and their adhesion ability play a crucial role in their attachment and artificial cultivation of mussels. In this study, transcriptomic and proteomic analyses were performed to identify byssogenesis-associated genes in the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819, seeking to advance our knowledge of the molecular basis of byssal secretion in mussels. Transcriptomic analysis identified 1742 and 1498 differentially expressed genes in the foot tissue of M. galloprovincialis at 9 h and 24 h post-byssal ablation, respectively. Meanwhile, proteomic analysis revealed 1254 and 484 differentially expressed proteins at the same two time points. Integrated analysis identified 121 genes differentially expressed at both transcript and protein levels. Among these genes, 44 were significantly upregulated, and they may constitute high-confidence gene sets associated with mussel byssogenesis. Notably, they included genes encoding tyrosinase-like protein, low affinity immunoglobulin epsilon Fc receptor, and O-methyltransferase MdmC. They were enriched in KEGG pathways, including metabolism of amino acids, lipid metabolism, nucleotide metabolism, and immune system. Quantitative real-time PCR was performed on seven selected genes, confirming that their expression patterns were consistent with those observed in transcriptomic and proteomic sequencing. This study provides novel data and insights for understanding the molecular basis involved in byssus development of M. galloprovincialis. Full article

Background: Genetic variability in the vitamin D receptor (VDR) may influence immune regulation and systemic inflammation, factors potentially relevant for outcomes in orthopedic surgery. This study explored the association of the VDR rs7975232 (ApaI) single nucleotide variation (SNV) with inflammatory biomarkers and health-related quality of life (HRQoL). Methods: The study included 292 orthopedic patients and 90 controls. Genotyping was performed using real-time PCR. Laboratory analyses comprised hematological parameters, C-reactive protein (CRP), and serum 25-hydroxyvitamin D₃ [25(OH)D₃]. HRQoL was assessed with the SF-36 questionnaire. Associations between genotype, inflammation, and HRQoL were examined using regression models adjusted for age and body mass index (BMI). Results: Genotype (p = 0.023) and allele (p = 0.007) distributions differed between patients and controls. In multivariable models, the CC genotype was associated with higher neutrophil counts (p = 0.029), whereas the AA genotype was associated with elevated CRP levels (p = 0.025). Genotypic variation was further associated with SF-36 scores, independently of age and BMI. Conclusions: The VDR rs7975232 SNV may be associated with baseline systemic inflammation and patient-reported quality of life in orthopedic surgery. Genotyping of this variant may complement conventional biomarkers in future research aimed at improving diagnostic precision. Moreover, it could contribute to innovation in laboratory diagnostics aimed at improving perioperative outcomes. Full article