Search Results (844)

The yellowfin seabream (Acanthopagrus latus) is an important aquaculture species, yet endocrine gene regulation during practical fasting and feeding schedules remains poorly understood. Here, we identified and characterized two duplicated proglucagon genes (Gcga and Gcgb) and examined tissue distribution of expression and transcriptional responses to feeding-related challenges. Sequence and phylogenetic analyses confirmed that Gcga and Gcgb cluster with teleost proglucagon paralogs and contain conserved peptide domains. Both genes were broadly expressed, with the strongest relative qRT-PCR signal detected in brain and fin, while other tissues (including intestine, gill, stomach, and liver) showed comparatively low but detectable expression. Because the liver is a central metabolic organ and displayed reproducible feeding-dependent regulation, we further quantified hepatic transcription under two paradigms. In a short-term starvation–refeeding trial, hepatic Gcga was significantly suppressed during fasting and rebounded after refeeding, whereas Gcgb showed a distinct, weaker response. In an acute peri-feeding assay, hepatic Gcga and Gcgb displayed rapid but differential regulation around meal time, and Gcgb expression differed between feeding and non-feeding groups. Together, these results support transcriptional divergence between the two proglucagon paralogs in nutritional regulation within a liver-focused metabolic-response model. Our findings provide baseline molecular information for A. latus and offer endocrine insights relevant to evaluating feeding strategies in aquaculture. Full article

In recent years, pigeon rotavirus A (PiRVA) infection, an important emerging disease, has posed a major threat to the healthy development of the pigeon industry and public health. Therefore, developing an accurate, rapid and convenient detection method for this virus is vital for monitoring and early diagnosis of the disease. In this study, on the basis of the ORF sequence characteristics of the PiRVA VP6 gene, crRNA and reverse transcription recombinase-aided amplification (RT-RAA) primers were designed. On the basis of the CRISPR/Cas12a system, for the first time, the RT-RAA-CRISPR/Cas12a rapid detection method of PiRVA was established by combining RT-RAA and lateral flow strips. This method could specifically detect PiRVA, and there was no cross-reaction with other common viruses originating from pigeons. The minimum detection limit was 16.8 copies/μL, and the results of the intrabatch and interbatch repeated tests were consistent. Moreover, the method established in this study and the previously established common PCR method were used to analyse 56 clinical tissue samples from racing pigeons and domestic pigeons collected in 2025. The positive rates of racing pigeon and domestic pigeon samples detected by PCR were 17.6% and 12.8%, respectively, and the positive rates of racing pigeon and meat pigeon samples detected by the RT-RAA-CRISPR/Cas12a method were 23.5% and 17.9%, respectively, indicating that PiRVA infection occurs in both racing pigeon and domestic pigeon populations in China. In summary, the PiRVA RT-RAA-CRISPR/Cas12a detection method established in this study has good specificity, sensitivity, and reproducibility, and allows visualization of the results, which can be used for field applications. This study provides technical support for epidemiological surveillance and etiological research on PiRVA. Full article

Approximately 2–4% of all breast cancer cases are inflammatory breast cancer (IBC), an extremely rare and severe subtype of the disease. Current therapies, including chemotherapy, surgery, and radiotherapy, remain insufficient, underscoring the need for novel therapeutic approaches. IBC exhibits elevated basal endoplasmic reticulum (ER) stress, suggesting a potential vulnerability. We recently developed ERX-41, a small molecule that exacerbates ER stress in cancer cells by inhibiting the endoplasmic reticulum-localized function of Lysosomal acid lipase A (LIPA). Here, we evaluated the therapeutic potential of ERX-41 in IBC models. ERX-41 markedly reduced the viability of IBC cells and significantly impaired clonogenic survival while promoting apoptosis. The specificity of ERX-41 was confirmed using LIPA-knockdown and LIPA-knockout cells. RT-PCR-based assays revealed rapid induction of XBP1 splicing within 6 h of treatment, and Western blot analyses demonstrated activation of ER stress markers including CHOP, PERK, and ATF4. In KPL4 xenografts, ERX-41 treatment significantly decreased tumor volume, accompanied by reduced proliferation and increased ER stress marker expression by IHC. Collectively, these findings identify LIPA as a therapeutically actionable vulnerability in IBC and establish ERX-41 as a potential drug for IBC. Full article

Clusters of acute flaccid paralysis (AFP) caused by oral vaccine-derived poliovirus (VDPV) in 2022 and sporadic outbreaks in New York and Gaza highlight the ongoing risk of polio, alongside the persistent global threat posed by wild-type poliovirus. This study aims to develop and validate a quantitative reverse transcription PCR (RT-qPCR) panel that employs different primer–probe sets to simultaneously detect vaccine and wild-type poliovirus (WPV) in wastewater. Using an inactivated poliovirus vaccine (IPV) and engineered DNA fragments (eDNAf), the qPCR master mix (MM) performance, assay specificity, sensitivity (limit of detection, LOD), and recovery from IPV-spiked wastewater were evaluated. Compared with two-step RT-qPCR and qScript MM, one-step RT-qPCR with TaqMan MM improved sensitivity for the following polioviruses (PV): Sabin 1 in IPV and the eDNAf of Sabin 1, 2, and 3; WPV1 and WPV3; and poliovirus type 2 (any serotype 2). The LOD for Sabin 1 in IPV was 2.49 copies/PCR, while LODs for eDNAf of polio targets ranged from 1.06 to 3.12 copies/PCR. Sabin 1 recovery from IPV-spiked wastewater ranged from 10.26% to 57.27%. The RT-qPCR panel for poliovirus exhibited good specificity and sensitivity, with moderate viral recovery, enabling rapid implementation of wastewater monitoring for PV as needed. Full article

Molecular point-of-care testing (POCT) for respiratory infections has undergone remarkable advancement over the past two decades, driven by technological innovation and urgent clinical needs highlighted by the COVID-19 pandemic. This comprehensive systematic review was conducted following PRISMA 2020 guidelines, synthesizing evidence from 254 peer-reviewed studies published between 2006 and 2026, with detailed analysis of the 30 most relevant papers selected through a rigorous four-stage screening process. The review examines the evolution of molecular POCT technologies, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-based detection systems. Key findings demonstrate that modern molecular POCT platforms achieve diagnostic performance comparable to laboratory-based testing, with sensitivities ranging from 88% to 100% and specificities from 98% to 100%, while delivering results in 15 to 80 min. These technologies enable rapid, accurate detection of major respiratory pathogens, including SARS-CoV-2, influenza A/B, respiratory syncytial virus (RSV), and atypical bacteria. The integration of microfluidic systems, portable devices, and smartphone-based analysis has expanded access to testing in resource-limited settings, emergency departments, and wearable platforms. This review provides critical insights for clinicians, researchers, and policymakers regarding the current state, clinical applications, and future directions of molecular POCT for respiratory infections. Full article

Exosomes are nanoscale extracellular vesicles that carry disease-associated microRNAs (miRNAs) and represent promising biomarkers for cancer diagnosis. Triple-negative breast cancer (TNBC) lacks well-defined molecular markers, necessitating sensitive and integrable analytical approaches for TNBC-related exosomal miRNAs. In this study, exosomes were isolated from MDA-MB-231 TNBC cells using a paddle screw-based system designed to enhance mass transfer through active rotation, providing a mechanically driven isolation strategy that is compatible with miniaturized and microfluidic platforms. This dynamic isolation process enabled rapid and efficient exosome recovery within a short processing time. Three TNBC-associated miRNAs encapsulated in the isolated exosomes were quantitatively analyzed using polyadenylation tailing (poly(A) tailing) and specific bidirectional extension sequence-based assays combined with reverse transcription quantitative real-time PCR (RT-qPCR). The bidirectional extension (BDE) assay generated highly specific PCR templates, leading to improved amplification specificity and reduced background signals. The RT-qPCR analysis exhibited high sensitivity, wide dynamic range, and good reproducibility for all target miRNAs. Overall, these results demonstrate that the integration of a paddle screw-based exosome isolation module with an extension-based nucleic acid detection strategy provides a scalable and biosensor-compatible analytical framework for profiling TNBC-associated exosomal miRNAs, with potential applications in microfluidic liquid biopsy platforms and exosome-based cancer diagnostics. Full article

Salt stress severely constrains wheat growth and yield by inducing osmotic imbalance, ion toxicity, and excessive accumulation of reactive oxygen species (ROS). Although salt-tolerant cultivars can adapt through rapid signaling transduction and maintenance of cellular homeostasis, the underlying dynamic regulatory networks remain insufficiently characterized. In this study, we reanalyzed publicly available time-series RNA-seq data (0, 1, 3, 6, 12, and 24 h) from the salt-tolerant wheat cultivar Xiaoyan22 under salt stress and constructed a time-series-based co-expression network using weighted gene co-expression network analysis (WGCNA). Multiple gene modules were identified, among which the black module showed significant positive correlations with both salt treatment (treatment_bin) and stress duration (time_h). This module displayed a progressively increasing eigengene expression pattern throughout the stress period. Gene significance (GS) was positively correlated with module membership (MM), facilitating the identification of highly connected hub genes within this module. Functional enrichment analysis indicated that genes in the black module were primarily associated with DNA replication and genome stability maintenance, RNA metabolic regulation, phenylpropanoid metabolism, and cuticle/suberin/wax biosynthesis. Physiological analysis further revealed enhanced activities of superoxide (SOD), peroxide (POD), and catalase (CAT), enhanced accumulation of proline and soluble sugars, and a time-dependent increase in MDA under salt stress. qRT-PCR confirmed significant induction of candidate genes, including a ZAR1-like receptor kinase, Remorin, and NETWORKED 1D. Collectively, these findings integrate co-expression network inference with physiological and molecular validation, providing candidate regulators and pathways for understanding salt tolerance and supporting future molecular breeding efforts. Full article

Background: During the COVID-19 pandemic, Thailand prioritized hemodialysis patients for vaccination. Due to limited supply, heterologous regimens were used. This study evaluates the mortality rate and risk factors in hemodialysis patients who received heterologous versus homologous vaccine regimens. Methods: We retrospectively reviewed data of hemodialysis patients in Thailand from January 2021 to December 2022, using data from the Department of Medical Sciences, Ministry of Public Health, and Thailand Renal Replacement Therapy Registry. Mortality was defined as death within 30 days of a positive RT-PCR or rapid antigen test for SARS-CoV-2. Multivariate logistic regression was used to identify mortality risk factors. Results: The associated risks of mortality in hemodialysis patients with COVID-19 were female sex, age ≥ 50 years, diabetes, and BMI ≥ 25.0 kg/m². Regarding vaccination regimens, the inactivated–Viral vector–mRNA regimen was associated with lower mortality compared with the mRNA–mRNA regimen (OR 0.29, 95% CI 0.08–0.99). In contrast, no vaccination (OR 16.95, 95% CI 7.86–36.54) and single-dose vaccination with inactivated vaccine (OR 17.54, 95% CI 7.01–43.88) or Viral vector vaccine (OR 20.74, 95% CI 9.38–45.86) were associated with markedly higher mortality risk. Conclusion: The inactivated–Viral vector–mRNA vaccine regimen was associated with a decreased mortality risk among this population. Full article

Background/Objectives: Next to malaria, respiratory viruses, particularly respiratory syncytial virus (RSV), are responsible for the hospitalization and death of thousands of young children each year in sub-Saharan Africa. During peak seasons, conducting separate tests is time-consuming and distressing. This underscores the need for efficient, rapid multiplexed diagnostic tools. This study aimed to evaluate the clinical performance of a lateral flow assay (LFA) based antigen combo rapid diagnostic test (ML Ag Combo RDT, manufactured by MobiLab) that detects RSV, influenza viruses A and B (Flu A/B), and SARS-CoV-2. Methods: The Allplex panel 1 rRT-qPCR assay was used as a reference assay to evaluate the clinical performance of the LFA Ag Combo RDT in pediatric hospital settings. It was performed using 470 nasopharyngeal swab (NPS) specimens from hospitalized children under two years of age with respiratory symptoms. Results: Based on the comparative analysis of the testing results for 470 NPS, the ML Ag Combo RDT demonstrated high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 90.06%, 98.38%, 93.67, and 97.39% for RSV, and 30%, 100%, 100%, and 95.43 for Flu A/B, respectively. Agreement with the Allplex panle1 1 rRT-qPCR was strong (κ = 0.90 for RSV) and moderate (κ = 0.45 for Flu A/B), with overall accuracies of 96.63% for RSV and 95.5 for Flu A/B. This was further supported by ROC analysis for aggregated data (RSV and, Flu A/B) with an AUC value of 0.925. As expected, in samples with high viral loads (Ct < 20), the Ag Combo RDT achieved 100% sensitivity for RSV and Flu A/B. Sensitivity declined slightly at lower viral loads (Ct > 35). Conclusions: The ML Ag Combo RDT demonstrates high specificity and diagnostic accuracy for the detection of RSV and Flu A/B in pediatric hospital settings where timely diagnosis is critical. Full article

As a tropical fruit of considerable economic importance, passionfruit (Passiflora edulis Sims) is extensively cultivated in the tropical and subtropical regions of China; however, the widespread incidence of viral diseases has significantly hampered the safety of its production. Rapid, sensitive, and visual detection of plant viruses is essential to effectively prevent and manage these viral diseases. In this study, we developed a visual detection system (displaying under blue light or UV) utilizing reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with CRISPR/Cas12a to detect three viruses harmful to passionfruit production: telosma mosaic virus (TelMV), East Asian passiflora virus (EAPV), and passiflora mottle virus (PaMoV). Within this system, the optimal primer concentration for the RT-RAA reaction was determined to be 0.4 μM for all three viruses, with an optimal reaction temperature of 37 °C. The optimal reaction times were established as 20 min for TelMV, 15 min for EAPV, and 30 min for PaMoV. The entire detection process could be completed within 30 min without the need for sophisticated equipment or instruments. For TelMV and EAPV, the detection system demonstrated the capability to detect samples at a dilution of 10⁶, representing an approximately 10⁴-fold improvement over RT-PCR, while for PaMoV, it could identify samples at a dilution of 10⁶, representing an approximately 10²-fold improvement over traditional RT-PCR methods. These results confirm the successful development of the CRISPR/Cas12a-based detection systems. Subsequently, the system was applied for in situ detection of the three target viruses in field settings, yielding results that were fully consistent with laboratory-based RT-PCR assays, a consistency which underscores the system’s strong potential for field application in detecting important crop viruses. Full article

Citrus viroid V (CVd-V), a member of the species Apscaviroid epsiloncitri (family Pospiviroidae), is a graft-transmitted pathogen that spreads through infected propagation material and has been reported in several countries. Recently, it has been detected for the first time in Italy. Although CVd-V is generally considered asymptomatic, foliar and stem symptoms have been observed in Etrog citron. In several citrus species, mixed infections of CVd-V with other viroids give synergistic effects that cause more severe symptoms. To evaluate its spread, a specific and sensitive RT-qPCR assay was developed. The assay specificity, sensitivity, and performance were compared with a conventional end-point RT-PCR, showing a higher sensitivity and no cross-reactivity with other citrus viroids. Furthermore, the assay was evaluated using two RNA extraction procedures: total RNA extraction with commercial kits and a rapid crude extract preparation using a simple extraction buffer. Moreover, RNA extracts of 111 samples, collected from commercial citrus orchards across the main Sicilian citrus-producing areas and from old varieties maintained in germplasm collection, were analyzed. The RT-qPCR revealed an overall CVd-V incidence of 8.1%. Notably, the crude extract preparation showed a sensitivity comparable with conventional total RNA extraction, with substantial savings in cost and processing time. This finding paves the way for using the developed assay with portable qPCR instruments directly in the field, as well as for routine surveillance analyses in citrus production systems. Full article

Low-temperature stress is a major factor limiting the development of the chrysanthemum industry. Chrysanthemum indicum L., wild germplasm with strong cold tolerance within the genus, is an ideal material for mining cold resistance genes. Through preliminary transcriptome analysis of C. indicum under low-temperature stress (PRJNA1391062), we found that multiple R2R3-MYB family members were significantly differentially expressed (|log₂FC| ≥ 1, p < 0.05), suggesting that this family may play important roles in cold stress responses. Within the C. indicum genome, we identified 63 R2R3-MYB members (CiMYBs) through HMMER and BLAST searches combined with domain validation. Phylogenetic analysis classified these genes into 19 subgroups, with most key nodes supported by bootstrap values > 80%. Promoter cis-element analysis revealed enrichment of elements related to light responsiveness, hormone signaling, and stress responses, including 41 low-temperature responsive elements distributed across 28 genes and 32 drought-induced MYB-binding sites present in 23 genes. Synteny analysis identified 13 duplicated gene pairs within the C. indicum genome and 41 collinear gene pairs between C. indicum and Arabidopsis thaliana L. Transcriptome data under low-temperature stress showed that 22 of the 63 CiMYB members were differentially expressed under 4 °C acclimation and −4 °C freezing stress, and they could be classified into three response patterns: acute stress-responsive (rapid upregulation upon initial stress), acclimation-induced (significant activation after 4 °C acclimation), and freezing-suppressed (downregulation after −4 °C freezing). Six differentially expressed genes were randomly selected for RT-qPCR validation, and the results showed consistent trends with the transcriptome data. This study provides a comprehensive identification of R2R3-MYB family members in C. indicum and reveals their expression divergence under low-temperature stress, offering candidate gene resources for deciphering the cold adaptation mechanisms of C. indicum and breeding new cold-resistant chrysanthemum cultivars. Full article

During ischemia, endothelial cell integrity is compromised, as a consequence, blood barrier homeostasis is disrupted. Therefore, the structural and functional preservation of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightforward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 h of oxygen–glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hypoxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. Cell viability decreased by approximately 33% following OGD, while CHOP expression increased ~4-fold, indicating significant ER stress induction. The model enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress resolution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to the mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approaches that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress-modulating interventions aimed at preserving endothelial barrier function, in various ischemic contexts. Full article

With the rapid development of intensive aquaculture, unreasonable stocking density has become a major factor restricting the healthy growth of grass carp (Ctenopharyngodon idella). This study aimed to evaluate the effects of three stocking densities (0.57, 1.13, and 2.27 kg/m³) on the growth performance, stress response, antioxidant capacity, and immunity of grass carp. Grass carp with an initial body weight of 81.76 ± 17.69 g were randomly assigned to three groups with three replicates. After 75 days of cultivation, we randomly sampled and measured their growth performance. Reagent kits were used to detect serum biochemical indicators, kidney immune enzyme activity, and liver antioxidant indicators in each treatment group. The expression of spleen immune-related genes was detected using real-time quantitative PCR (RT-qPCR). Results showed that the final body weight, weight gain rate, specific growth rate, and condition factor were significantly higher in the medium-stocking-density group (p < 0.05). High stocking density significantly increased serum cortisol, glucose, transaminases, creatinine, and urea nitrogen, and decreased cholesterol and triglyceride levels (p < 0.05). For immune parameters, the activities of immunoglobulin M (IgM), lysozyme (LZM), antimicrobial peptide (AMP), alkaline phosphatase (AKP), and acid phosphatase (ACP) in the kidneys decreased with increasing density. The mRNA levels of IL-1β, IL-6, TNF-α, and IL-10 in the spleen were significantly upregulated, while IgM was downregulated in the high-density group (p < 0.05). Regarding antioxidant capacity, hepatic total antioxidant capacity (T-AOC), catalase (CAT), and glutathione (GSH) levels increased initially and then decreased with increasing density, while malondialdehyde (MDA) content increased continuously. Collectively, these findings suggest that high stocking density induces growth inhibition, oxidative stress, and immune dysfunction in grass carp. The medium density of 1.13 kg/m³ was found to be optimal for the growth and physiological health of grass carp in this study, providing a scientific basis for the optimization of intensive farming strategies. Full article

Feline infectious peritonitis (FIP) is a highly lethal disease caused by feline infectious peritonitis virus (FIPV), which poses significant diagnostic challenges in clinical practice. FIPV is divided into two serotypes (serotype I and serotype II) based on distinct serological responses driven by substantial sequence divergence in the spike (S) protein. Serotype I predominates in Europe and North America, whereas serotype II is more common in Asia. In this study, we developed a triplex reverse transcription quantitative PCR (RT-qPCR) assay for simultaneous detection and serotyping of FIPV. Primers and TaqMan probes were designed to target the conserved nucleocapsid (N) gene and serotype-specific regions within the S gene. After systematic optimization of reaction conditions, the final assay employed an annealing temperature of 64 °C and optimized primer–probe concentrations. The assay exhibited excellent linearity (R² > 0.99 for all targets), with amplification efficiencies ranging from 97.39% to 109.97%. No cross-reactivity was observed with other common feline pathogens, confirming high specificity. The limit of detection was as low as 0.5 copies/µL, and intra-assay repeatability showed coefficients of variation below 2.1%. Clinical validation using 63 feline samples revealed an overall FIPV positivity rate of 21.63%, with serotype II (17.46%) markedly more prevalent than serotype I (3.17%). Collectively, this triplex RT-qPCR assay demonstrates high sensitivity, exceptional specificity, and robust reproducibility, making it a valuable tool for rapid clinical diagnosis through the simultaneous detection of feline coronavirus (FCoV) and serotyping of FIPV. Full article