Search Results (3,652)

Management of invasive species is especially difficult when the organisms involved are endophagous and their interactions complex. Such is the case with laurel wilt disease (LWD), a lethal vascular condition caused by Harringtonia lauricola, the fungal symbiont of the non-native redbay ambrosia beetle (RAB), Xyleborus glabratus Eichoff (Coleoptera: Curculionidae). LWD has caused extensive mortality of coastal redbay, Persea borbonia, and is expanding to utilize additional lauraceous hosts, including sassafras, Sassafras albidum. Current management has not been successful in preventing its spread, warranting investigation into additional techniques. RNA interference (RNAi) is a highly specific gene-silencing mechanism used for integrated pest management of crop pests and currently being investigated for use in forests. When targeting essential genes, RNAi can cause rapid insect mortality. Here we focus on RAB, identifying for the first time species-specific reference genes for quantitative real-time PCR (qPCR) and assessing mortality and gene expression after oral ingestion of double-stranded RNAs (dsRNAs) targeting essential genes (hsp, shi, and iap). Our study validates reference genes for expression analyses and shows significant mortality and changes in gene expression for all three target genes. Our research aims to contribute to the development of innovative management strategies for this invasive pest complex. Full article

Porcine deltacoronavirus (PDCoV) is an emerging pathogen that causes severe, often fatal, diarrhea in suckling piglets and has zoonotic potential. Its nonstructural protein 12 (Nsp12), functioning as the RNA-dependent RNA polymerase (RdRp), is a central component of the viral replication–transcription complex and a critical target for host antiviral mechanisms. Here, we identified eukaryotic elongation factor 1 gamma (eEF1G) as a host interactor of PDCoV Nsp12 by immunoprecipitation-coupled mass spectrometry in IPEC-J2 cells. This interaction was confirmed by co-immunoprecipitation, pull-down assays, and confocal microscopy. Functional analyses involving siRNA knockdown and overexpression of eEF1G, combined with viral titration, strand-specific real-time quantitative PCR, and RNA immunoprecipitation assays, demonstrated that eEF1G directly binds to Nsp12. Knockdown of eEF1G significantly enhanced viral replication and increased negative-stranded RNA synthesis, whereas overexpression did not affect viral proliferation. Furthermore, eEF1G was found to bind PDCoV genomic RNA and competitively disrupt the interaction between Nsp12 and viral RNA, thereby impairing RdRp activity. Our results indicate that eEF1G acts as a novel host restriction factor that inhibits PDCoV replication by competing with Nsp12 for genomic RNA binding, ultimately blocking negative-stranded RNA synthesis. This study unveils a new antiviral mechanism and highlights a potential target for developing interventions against PDCoV. Full article

Accurate differentiation between Coilia brachygnathus and Coilia nasus is imperative for the effective management of fisheries, the conservation of aquatic ecosystems, and the mitigation of commercial fraud. Current morphological identification remains challenging due to their high morphological similarity—particularly for processed samples—while conventional molecular methods often lack the speed or specificity required for field applications or high-throughput screening. In this study, a novel integrated approach was developed and validated, combining TaqMan quantitative real-time PCR (qPCR). for precise genotyping of C. brachygnathus and C. nasus with Recombinase Polymerase Amplification coupled with Lateral Flow Dipstick (RPA-LFD) for rapid on-site screening. First, species-specific RPA-LFD assays were designed to target the mitochondrial COI gene sequence. This enabled visual detection within 10 min at 37 °C, with a sensitivity of 10² copies/μL, and required no complex equipment. A dual TaqMan MGB qPCR assay was further developed by validating stable differentiating SNPs (chr21:3798155, C/T) between C. brachygnathus and C. nasus, using FAM/VIC dual-labeled MGB probes. Results showed that this assay could distinguish the two species in a single tube: for C. brachygnathus, Ct values in the FAM channel were significantly earlier than those in the VIC channel (ΔCt ≥ 1), with a FAM detection limit of 125 copies/reaction; for C. nasus, only VIC channel amplification was observed, with a detection limit as low as 12.5 copies/reaction. Validation with 171 known tissue samples demonstrated 100% concordance with expected species identities. This integrated approach effectively combines the high accuracy and quantitative capacity of TaqMan qPCR for confirmatory laboratory genotyping with the speed, simplicity, and portability of RPA-LFD for initial field or point-of-need screening. This reliable, efficient, and user-friendly technique provides a powerful tool for resource management, biodiversity monitoring, and ensuring the authenticity of high-quality C. brachygnathus and C. nasus. Full article

Background/Objectives: Selenoproteins play indispensable roles in embryonic development, with their dysregulation linked to various metabolic and neurological disorders. This study aims to systematically quantify the mRNA expression levels of all 24 selenoprotein genes in murine heart, brain, liver, and kidney tissues across embryonic (E8.5, E12.5, E18.5) and postnatal (P7, P30, P90) developmental stages, in order to elucidate the regulatory landscape of selenium metabolism during development. Methods: We collected tissues from mice at six developmental stages and performed RNA extraction followed by quantitative real-time PCR (qPCR) to measure the expression of all 24 selenoprotein genes. Data were normalized using the geometric mean of ActB and Gapdh, and statistical analyses were conducted using one-way ANOVA with Duncan’s post hoc test. Results: Our analysis reveals three principal findings: (1) Distinct expression patterns emerge among selenoprotein families—deiodinases (Dio1-3) and thioredoxin reductases (Txnrd1-3) exhibit limited embryonic expression (<20-fold changes), while glutathione peroxidases (Gpx1, Gpx3, Gpx4) and biosynthesis-related genes (Selenop, Msrb1) show substantial postnatal upregulation (up to 600-fold increases); (2) Selenoproteins essential for embryonic survival (Gpx4, Txnrd1, Txnrd2, Selenoi, Selenot) display expression profiles concordant with their essential developmental functions; (3) Selenop and Msrb1, involved in selenium transport and redox regulation, demonstrate early embryonic upregulation with further increases during postnatal development. Conclusions: These spatiotemporal expression patterns elucidate the regulatory landscape of selenium metabolism during development and provide mechanistic insights into the phenotypes associated with selenium deficiency. The findings offer valuable implications for human nutritional interventions and developmental health. Full article

Sugars are key metabolites influencing the flavor and quality of kiwifruit, with their accumulation in fruit relying on sugar transporters. Recently identified sugar transporters known as SWEETs play significant roles in modulating plant growth, development, and fruit ripening. However, the characteristics of SWEET genes in Actinidia eriantha remain poorly understood. In this study, a total of 26 AeSWEET genes were identified across 17 chromosomes. These genes encoded proteins ranging from 198 to 305 amino acids in length and contained 5 to 7 transmembrane helices. Both interspecific and intraspecific phylogenetic trees categorized AeSWEET proteins into four distinct clades. The motif and domain structures were conserved within each clade, although variations were observed in exon-intron organizations. One tandem and fourteen segmental duplication events were identified as primary drivers of the AeSWEET family expansion. Comparative syntenic mapping showed a closer homology of the AeSWEET family with that of dicotyledons compared to monocotyledons. Promoter cis-element analysis indicated the potential responses of AeSWEET genes to five phytohormones and seven environmental stressors. Quantitative real-time PCR analysis revealed tissue-specific expression profiles of AeSWEET genes, with two AeSWEET11 genes (AeSWEET11a and AeSWEET11b) showing significantly higher expression levels in fruit tissues. Their expressions were positively correlated with sucrose, fructose, and glucose contents throughout fruit development and ripening. Transient transformation tests in tobacco leaves verified the predominant localization of AeSWEET11a and AeSWEET11b to the plasma membrane. Functional assays in yeast mutants revealed that AeSWEET11a and AeSWEET11b both possessed sucrose and hexose transport activities. These findings highlight the potential of targeting AeSWEET11a and AeSWEET11b to enhance sugar accumulation in the fruit of A. eriantha, thereby providing a foundation for improving the flavor profile of commercial cultivars. Full article

Starvation in horses presents critical welfare, economic, and management challenges with underlying molecular mechanisms of metabolic modification and recovery left poorly defined. Prolonged caloric deprivation induces significant systemic shifts in carbohydrate, protein, and lipid metabolism, reflected in coordinated changes in tissue-specific gene expression. This review synthesizes current knowledge on equine metabolic responses to starvation, emphasizing pathways found through RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) studies. Molecular investigations using RNA-seq and RT-qPCR have provided insight into transcriptional reprogramming during starvation and subsequent refeeding. Shifts in gene expression reflect the metabolic transition from carbohydrate dependence to lipid use, suppression of anabolic signaling, and activation of proteolytic pathways. However, interpretation of these data requires caution, as factors such as post-mortem interval, tissue handling, and euthanasia methods particularly the use of sodium barbiturates can influence transcript stability and abundance, potentially confounding results. The literature shows that starvation-induced molecular changes are not uniform across tissues, with skeletal muscle, liver, and adipose tissue showing distinct transcriptional signatures and variable recovery patterns during refeeding. Cross-species comparisons with hibernation, caloric restriction, and cachexia models provide context for understanding these changes, though equine-specific studies remain limited. Identified gaps include the scarcity of longitudinal data, inconsistent tissue sampling protocols, and lack of standardized reference genes for transcriptomic analyses in horses. Addressing these limitations will improve the accuracy of molecular evaluations and enhance our ability to predict recovery trajectories. A more comprehensive understanding of systemic and tissue-specific responses to starvation will inform evidence-based rehabilitation strategies, reduce the risk of refeeding syndrome, and improve survival and welfare outcomes for affected horses. Full article

Salix suchowensis is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in Salix suchowensis under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH₄NO₃, NH₄⁺, NO₃⁻, and N deficiency). Five genes (UBQ1, UBQ3, 18S, H2A2, and H2B2) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified EF1α, EFβ, and αTUB as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene SsAMT1 and SsNRT2 confirmed the stability of EF1α, EFβ, and αTUB as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended EF1α + αTUB as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of EF1α and αTUB across nine willow genotypes highlighted their broader applicability within Salix species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants. Full article

The aim of our study is to determine the changes in the biomarkers CXCL-16, CCL-20, GDF-15, and CD8a, which play an immunological role in CHB patients according to viral load to determine their diagnostic potential and to investigate their relationships with hematological parameters and non-invasive fibrosis indices. Our study included 96 chronic hepatitis B patients and 30 healthy individuals as a control group. The patients were divided into three groups based on their serum HBV DNA levels: mild (0–10² IU/mL), moderate (10³–10⁵ IU/mL), and severe viral load (10⁶–10⁸ IU/mL). HBV DNA levels were determined by the real-time PCR (Anatolia, Istanbul, Turkey) method. CXCL-16, GDF-15, and CD8a levels in patient serum were quantitatively determined by the ELISA method (Elabscience, Wuhan, China), and CCL-20 levels were determined by the ELISA method BT LAB, Shanghai, China). ROC (Receiver Operating Characteristics) and HUM (Hypervolume Under Manifold) analyses were used to determine the diagnostic efficacy of the biomarkers. ROC analyses showed that GDF-15 (AUC = 0.920) and CCL-20 (AUC = 0.751) had “very good” and “good” diagnostic values, respectively, in predicting hepatitis B disease. HUM analyses revealed that all biomarkers have good potential when it comes to distinguishing the severity of the disease. This study has shown that the biomarkers GDF-15 and CCL-20 may be potential diagnostic biomarkers in detecting the presence of chronic hepatitis B, and the biomarkers CXCL-16, CCL-20, GDF-15, and CD8a may be potential diagnostic biomarkers in determining the severity of the disease. These findings suggest that these biomarkers, which can be measured by the simpler and more economical ELISA method, could be a supportive tool for the HBV DNA test. The clinical use of these biomarkers can be expanded with future prospective studies. Full article

EsBax (bcl-2 Associated X protein), a member of the bcl-2 family involved in the mitochondrial apoptosis pathway, plays a crucial role in immune response and defense in invertebrates. In this study, we successfully cloned the full-length cDNA of EsBax from the Chinese mitten crab (Eriocheir sinensis) and investigated its immune-related functions. The EsBax cDNA is 3374 bp in length, including a 1563 bp open reading frame (ORF) encoding 521 amino acids, a 142 bp 5′ untranslated region (UTR), and a 1699 bp 3′ UTR. The predicted EsBax protein has a molecular weight of 58.0786 kD, a theoretical isoelectric point of 7.28, and contains three conserved BH domains (BH1-BH3), and a transmembrane domain (TM). Amino acid sequence analysis revealed the highest sequence identity (99.42%) with E. sinensis. For the expression analysis, three biological replicates were performed for each tissue and treatment group. Real-time quantitative PCR showed that EsBax mRNA was ubiquitously expressed in all examined tissues, with the highest expression in the hepatopancreas, followed by hemocytes, intestine, gill, and the lowest in muscle. Upon stimulation with lipopolysaccharide (LPS), Aeromonas hydrophila (AH), or cycloheximide (CHX), EsBax expression increased and peaked at 24 h (LPS and CHX) or 48 h (A. hydrophila), then decreased. These results suggest that EsBax expression is dynamically responsive to exogenous stimulants (LPS, A. hydrophila, and CHX) in E. sinensis, implying a potential role of EsBax in the molecular events associated with pathogen-induced apoptosis in this species. Full article

Background: Head and neck squamous cell carcinoma (HNSCC) is a prevalent malignancy with poor clinical outcomes. microRNA-7-5p (miR-7-5p) has been described as both a tumour suppressor and an oncomiR depending on the tissue context, but its role in HNSCC remains unclear. This study aimed to clarify the clinical significance and biological function of miR-7-5p in HNSCC by integrating data from multiple sources. Methods: A systematic review of the literature was conducted to identify studies analysing miRNA expression in human head and neck tissues. A meta-analysis of individual patient data from Gene Expression Omnibus (GEO), ArrayExpress, and The Cancer Genome Atlas (TCGA) was performed to assess miR-7-5p expression in tumours and normal tissues, and its associations with clinical parameters and prognostic outcomes. Bioinformatics analyses were used to predict miR-7-5p target genes, classify hub genes, and perform gene ontology enrichment analysis. MicroRNA in situ hybridisation (miRNA ISH) and real-time quantitative PCR (RT-qPCR) were conducted on tissue samples, HNSCC cell lines, and an in vitro model of oral oncogenesis to validate miR-7-5p expression patterns. Results: miR-7-5p was significantly upregulated in tumours compared to normal tissues and associated with larger tumour size, HPV-negative status, poor disease-specific survival, and shorter progression-free intervals. Bioinformatics analysis highlighted miR-7-5p target genes enriched in pathways related to cell growth, survival, and tumourigenesis. Despite evidence supporting the anti-cancer role of exogenous miR-7-5p in preclinical models, the observed endogenous upregulation in tumours suggests that miR-7-5p expression may represent a compensatory or stress-responsive mechanism during tumourigenesis, rather than acting as a primary oncogenic driver. Conclusions: This study provides new insights into the complex role of miR-7-5p in HNSCC, supporting its potential as both a biomarker and a therapeutic target. Understanding the context-specific functions of miR-7-5p is essential for its development as an RNA-based therapeutic in HNSCC. Full article

Bovine leukemia virus (BLV), an oncogenic retrovirus of the genus Deltaretrovirus, causes enzootic bovine leukosis (EBL), the most prevalent neoplastic disease in cattle and a major source of economic loss. While BLV prevalence has been studied in commercial breeds, data on native Latin American cattle remain limited. This study assessed BLV infection and proviral load in 244 animals from six native breeds: Argentine Creole (CrAr), Patagonian Argentine Creole (CrArPat), Pampa Chaqueño Creole (CrPaCh), Bolivian Creole from Cochabamba (CrCoch), Saavedreño Creole (CrSaa), and Siboney (Sib), sampled across Argentina, Bolivia, Paraguay, and Cuba. BLV-CoCoMo-qPCR-2 assay detected BLV provirus in 76 animals (31.1%), with a mean load of 9923 copies per 10⁵ cells (range: 1–79,740). Infection rates varied significantly by breed (9.8% in CrAr to 83.8% in CrPaCh) and country (15.6% in Argentina to 83.8% in Paraguay) (p = 9.999 × 10⁻⁵). Among positives, 57.9% exhibited low proviral load (≤1000 copies), and 13.2% showed moderate levels (1001–9999), suggesting potential resistance to EBL progression. This is the first comprehensive report of BLV proviral load in Creole cattle across Latin America, offering novel epidemiological insights and highlighting the importance of native breeds in BLV surveillance. Full article

The present study aimed to identify genes encoding enzymes involved in the biotransformation of monoterpenoid (–)-isopulegol by Rhodococcus rhodochrous IEGM 1362. This strain is able to transform (–)-isopulegol with formation of two novel metabolites with promising antitumor and analeptic activities. Cell fractions of rhodococci and specific inhibitor of cytochrome P450-dependent oxygenase activity were used to establish the localization and type of biotransformation enzymes. The expression of nine CYP450 genes selected by bioinformatics analysis was analyzed by quantitative real-time PCR (qRT-PCR). Selection of optimal reference genes for normalization of qRT-PCR results was performed using BestKeeper, Normfinder, geNorm, Delta CT, and RefFinder algorithms. As a result of these studies, the role of CYP450 enzyme complexes in the biotransformation of (–)-isopulegol was confirmed, and their cytoplasmic localization was established. The genes encoding DNA gyrase subunit B (gyrB) and protein translocase subunit A (secA) were selected as the most stable reference genes. The induced expression of the gene encoding CYP450 hydroxylase in the presence of (–)-isopulegol was determined. The obtained data allow us to identify the specific CYP450 enzyme involved in (–)-isopulegol biotransformation by R. rhodochrous IEGM 1362 and lay the foundation for further studies of molecular and genetic mechanisms of monoterpenoid biotransformation. Full article

This study distinguished male and female individuals by wing morphology (males with long wings, females with short wings) and investigated sexual dimorphism in the chemosensory system of Blaptica dubia through integrated ultrastructural and transcriptomic analyses. Scanning electron microscopy (SEM) was used to characterize the type, number, and distribution of antennal sensilla, while Illumina HiSeq sequencing, Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) annotation, and Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) validation were employed to analyze sex-specific gene expression profiles. Both sexes exhibited Böhm’s bristles, chaetic, trichoid, and basiconic sensilla. Males showed significantly more chaetic sensilla on the pedicel and longer type I/II chaetic sensilla on the flagellum, whereas females had longer ST2 sensilla. Basiconic sensilla were predominantly flagellar-distributed and more abundant/longer in males. No sexual differences were observed in Böhm’s bristles. Transcriptomics revealed 5664 differentially expressed genes (DEGs) (2541 upregulated; 3123 downregulated), enriched in oxidation-reduction, extracellular space, lysosome, and glutathione metabolism. KEGG analysis identified five key pathways: lysosome, glutathione metabolism, cytochrome P450-mediated xenobiotic/drug metabolism, and ascorbate/aldarate metabolism. Among 11 chemosensory-related DEGs, chemosensory proteins (CSPs) and odorant binding proteins (OBPs) were downregulated in males, while gustatory receptors (GRs), olfactory receptors (Ors), and ionotropic receptors (IRs) were upregulated. These results demonstrate profound sexual dimorphism in both antennal sensilla morphology and chemosensory gene expression, suggesting divergent sex-specific chemical communication strategies in Blaptica dubia, with implications for understanding adaptive evolution in Blattodea. Full article

The grassland caterpillar Gynaephora qinghaiensis (Lepidoptera: Lymantriidae) is a dominant pest species in the alpine meadows of the Tibetan Plateau. Elucidating changes in key gene expression patterns will provide molecular insights into the adaptive evolutionary mechanisms of insects. Quantitative real-time PCR (qRT-PCR) is currently the predominant analytical methodology for assessing gene expression levels. However, variability among samples can compromise result reliability. Thus, selecting stably expressed reference genes for target gene normalization under diverse scenarios is critical. To date, suitable reference genes for G. qinghaiensis under varying experimental conditions have remained unidentified. In this study, the transcriptome data of G. qinghaiensis were obtained using the RNA-seq technique, and 13 candidate reference genes were selected. Four independent algorithms—ΔCt, geNorm, NormFinder, and BestKeeper—as well as a comprehensive online platform, RefFinder, were employed to evaluate the stability under six experimental conditions (tissues, developmental stages, sexes, temperatures, starvation, and insecticide treatments). Our findings identified the following optimal reference gene combinations for each experimental condition: RPS18, RPS15, and RPL19 for tissue samples; RPL19, RPS15, and RPL17 across developmental stages; RPS18 and RPS15 for different sexes; RPS8 and EF1-α under varying temperature conditions; RPL17 and RPL15 during starvation; and RPL19 and RPL17 following insecticide treatments. To validate the feasibility of the reference genes, we examined the expression of the target gene HSP60 in different tissues and under different temperatures. Our results established essential reference standards for qRT-PCR with G. qinghaiensis samples, laying the foundation for precise gene expression quantification in the future. Full article

Background: Colorectal cancer is the third most diagnosed cancer and a leading cause of cancer-related deaths worldwide. Early detection significantly improves patient outcomes, yet many cases are identified only at late stages. The high molecular and genetic heterogeneity of colorectal cancer presents major challenges in accurate diagnosis, prognosis, and therapeutic stratification. Recent advances in gene expression profiling offer new opportunities to discover genes that play a role in colorectal cancer carcinogenesis and may contribute to early diagnosis, prognosis prediction, and the identification of novel therapeutic targets. Methods: This study involved 142 samples: 84 primary tumor samples, 27 liver metastases, and 31 adjacent non-tumor tissues serving as controls. RNA sequencing was performed on a subset of tissues (12 liver metastases and 3 adjacent non-tumor tissues) using a targeted RNA panel covering 395 cancer-related genes. Data processing and differential gene expression analysis were carried out using the DRAGEN RNA and DRAGEN Differential Expression tools. The expression of six genes involved in hypoxia and epithelial-to-mesenchymal transition (EMT) pathways (SLC16A3, ANXA2, P4HA1, SPP1, KRT19, and LGALS3) identified as significantly differentially expressed was validated across the whole cohort via quantitative real-time PCR. The relative expression levels were determined using the ΔΔct method and log2FC, and compared between different groups based on the sample type; clinical parameters; and mutational status of the genes KRAS, PIK3CA, APC, SMAD4, and TP53. Results: Our results suggest that the expression of all the validated genes is significantly altered in metastases compared to non-tumor control samples (p < 0.05). The most pronounced change occurred for the genes P4HA1 and SPP1, whose expression was significantly increased in metastases compared to non-tumor and primary tumor samples, as well as between clinical stages of CRC (p < 0.001). Furthermore, all genes, except for LGALS3, exhibited significantly altered expression between non-tumor samples and samples in stage I of the disease, suggesting that they play a role in the early stages of carcinogenesis (p < 0.05). Additionally, the results suggest the mutational status of the KRAS gene did not significantly affect the expression of any of the validated genes, indicating that these genes are not involved in the carcinogenesis of KRAS-mutated CRC. Conclusions: Based on our results, the genes P4HA1 and SPP1 appear to play a role in the progression and metastasis of colorectal cancer and are candidate genes for further investigation as potential biomarkers in CRC. Full article