Search Results (1,391)

Diarrhea in young ruminants is a global issue and causes significant economic losses worldwide. In addition to common pathogens like rotavirus, coronavirus, and astrovirus, new viruses can be identified through unbiased next-generation sequencing (NGS) techniques. Here, we report the initial identification of a hunnivirus from a one-month-old goat with diarrhea using shotgun metagenomic NGS. A complete hunnivirus genome was recovered. Phylogenetic tree analysis revealed that this goat hunnivirus was more closely related to cattle hunnivirus than to small ruminant hunnivirus strains, suggesting a prior cross-species transmission event. The genome was used to design primers/probes for the conserved 3D^pol RdRP gene for real-time RT-PCR to screen banked ruminant fecal samples. Screening of 144 ruminant fecal samples showed that 9 of 38 goat, 22 of 96 cattle, and 0 of 8 sheep samples were positive for hunnivirus. Sequencing of the 3D^po region was performed on selected positive samples and revealed two lineages of hunnivirus circulating in North America. Our study highlights the importance of further investigation and monitoring of fecal samples using unbiased metagenomic tools to identify potential pathogens or co-infections in ruminants. Full article

Background/Objectives: miR5200 is miRNA unique to Poaceae plants. Induced under short-day conditions, it modulates flowering time by regulating the florigen FT gene expression. However, to date, the genetic locus responsible for mature miR5200 formation remains experimentally unvalidated, and its biological function in abiotic stress responses remains unknown. This has hindered systematic elucidation of miR5200’s physiological role and molecular mechanisms. Methods: This study utilized wheat as the research material. First, through bioinformatics analysis at the genomic level, 13 potential candidate tae-miR5200 gene loci were screened. Subsequently, the authenticity of these gene loci was systematically validated by combining tobacco transient transfection-based GUS staining assay and quantitative real-time PCR (qRT-PCR) to detect expression levels. Building upon this foundation, the expression patterns of tae-miR5200 under abiotic stresses such as low temperature, drought, and salinity, as well as SA, ABA, IAA, GA₃, and MeJA treatments, were further investigated. Results: Experimental validation confirmed that 7 out of 13 potential gene loci are authentic and functional, and tae-miR5200 exhibited specific expression changes under different types of abiotic stress. Conclusions: This study confirms the authenticity of tae-miR5200 gene loci, effectively eliminating interference from bioinformatics-predicted false-positive loci in subsequent functional studies. It provides an experimental foundation for further investigation into the molecular mechanisms of tae-miR5200 in wheat responses to abiotic stress. Full article

The formation of plant flower buds is regulated by various genes occurring upstream in floral induction pathways. However, the precise regulatory roles and underlying molecular mechanisms of these pathways in Camellia flower bud formation remain unclear. This study investigated the annual periodicity pattern of flower bud formation in two Camellia species exhibiting distinct flowering phenotypes: Camellia azalea, which initiates flower buds continuously throughout the year, and Camellia japonica, which forms buds only between May and July. C. azalea helps address the lack of summer-flowering representatives within the Camellia genus. Elucidating its unique molecular mechanism of flowering regulation provides valuable guidance for breeding new cultivars with summer blooming traits. Comparative transcriptome analysis of mature leaves sampled annually from the two Camellia species revealed the highest number of differentially expressed genes (DEGs) in C. azalea between May and December, whereas in C. japonica, the peak number of DEGs occurred between June and December. Gene ontology analysis indicated that the most enriched category in the transcriptomes of both species was oxidoreductase activity, which was followed by cofactor binding in C. azalea, whereas in C. japonica, it was cellular amino acid metabolic process. Flowering-related genes were identified from the transcriptome database, yielding 248 transcripts in C. azalea and 257 in C. japonica. The transcriptome analysis also revealed that C. azalea lacks certain floral inhibitory pathways that are present in C. japonica, such as the photoperiod pathway genes including GI2, FKF1, and COL14 and the thermosensitive pathway gene SVP. The reliability of the transcriptome results was further validated by quantitative real-time PCR (qRT-PCR) analysis. These results suggest that differences in upstream regulatory mechanisms within the floral induction pathways of C. azalea and C. japonica may underlie the species-specific patterns in the annual distribution of flower bud formation. Full article

Background: Previous studies have indicated an association between endoplasmic reticulum stress (ERS) and the formation of kidney stones. To further investigate this mechanism, this research sought to identify key genes linked to ERS in calcium oxalate (CaOx) kidney stones. Methods: Key cells with the highest ERS-related gene (ERSRG) scores were identified through single-cell analysis. These key cells were then categorized into high- and low-score groups based on their average ERSRG scores. To identify key genes, we analyzed the intersection of key ERSRGs and differentially expressed genes (DEGs) within key cells, focusing on genes demonstrating significant expression differences between control and CaOx kidney stone samples. A nomogram was constructed using these key genes to predict the risk of CaOx kidney stones. Gene set enrichment analysis (GSEA) was further performed to explore the functions of these key genes in the disease. Additionally, secondary clustering analysis was conducted on key cells to identify subtypes and evaluate the expression of key genes within these subtypes. Finally, the identified key genes were validated using quantitative real-time PCR (qRT-PCR) and Western blot analysis on cultured HK-2 cells, which were exposed with 2 mM CaOx for 24 h at 37 °C with 5% CO₂ or incubated with regular culture medium. Results: Endothelial cells were identified as key cells, and nine key genes were pinpointed in CaOx kidney stones: ACSL4, PTK2, DUSP4, MMP7, PHLDB2, TGM2, PPT1, SPARCL1, and LTF. The nomogram developed from these key genes demonstrated robust predictive ability for CaOx kidney stones risk. Additionally, GSEA revealed that olfactory transduction was enriched by key genes except PTK2. Secondary clustering analysis identified four key cell subtypes within endothelial cells, with LTF, MMP7, and SPARCL1 showing significantly differential expression between control and CaOx kidney stones groups across all key cell subtypes. qRT-PCR and Western blot analyses revealed that, compared to the control group, CaOx-exposed HK-2 cells exhibited significantly increased expression of ACSL4, MMP7, TGM2, PPT1, and LTF (p < 0.05), while showing significantly decreased expression of PTK2, DUSP4, SPARCL1, and PHLDB2 (p < 0.05). Conclusions: This study identified key genes associated with ERS in CaOx kidney stones through single-cell and transcriptomic analysis. The discovery of these genes provides new insights into the treatment of CaOx kidney stones and offers valuable references for subsequent research. Future research should focus on elucidating the precise roles of these candidate genes in CaOx stone pathogenesis to assess their potential for therapeutic intervention. Full article

Rural tropical regions face escalating threats from zoonotic AIV and dengue virus but lack sewered infrastructure for conventional wastewater surveillance. We implemented surface water-based surveillance (SWBS) in peri-urban Dhaka (Bangladesh) and Ruili (China) from July to November 2023 and coupled it with machine learning-enhanced digital epidemiology. Reverse transcription quantitative PCR (RT-qPCR) was employed to detect the M gene of AIV and to subtype H1, H5, H7, H9, and H10 in surface water. Wild bird feces (n = 40) were collected within 3 km of positive sites to source-track AIV. For the dengue virus, a serogroup-specific RT-qPCR assay targeting the CprM gene was used. Genomic sequencing of AIV and dengue virus was performed to elucidate phylogenetic relationships with local clinical strains. Clinical data related to dengue fever were also collected for correlation analysis. Meanwhile, 13 dengue-related keyword search volumes were harvested daily from Google, Bing and Baidu for four cities to reveal the relationship between dengue epidemics and the web search index. AIV H5 was detected in Dhaka city from week 38, peaking at week 39, while dengue virus was persistently detected from week 29 to week 45, aligning with clinical trends. Time-series cross-correlation analysis revealed that variations in surface water viral load led clinical case reports by approximately two weeks (max CCF = 0.572 at lag −2). In Ruili city, dengue virus was detected from week 32 to week 44. To sharpen sensitivity, 383 weekly web search series for 13 dengue keywords from four countries were screened; random-forest and XGBoost models retained five symptom queries that generated a composite index explaining 79% of variance in dengue RNA levels in an independent Ruili test set (n = 24) and reduced superfluous sampling by 35%. Phylogenetic analysis verified identity between water-derived and patient-derived DENV-2, confirming local transmission. The study demonstrates that AIV SWBS is optimally integrated with wild bird sampling for source attribution, whereas dengue SWBS achieves maximal efficiency when combined with real-time web search monitoring, providing tailored, low-cost early-warning modules for resource-constrained tropical settings. 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

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

Human rhinovirus (HRV) is the most common cause of upper respiratory tract infections and can cause substantial morbidity in children. Because its clinical features are nonspecific, differentiation from influenza virus and respiratory syncytial virus is often difficult, underscoring the diagnostic importance of real-time reverse transcriptase polymerase chain reaction (Real-Time RT-PCR)-based detection. This study aimed to characterize long-term epidemiological patterns of HRV in the Republic of Korea and assess their clinical and public health implications. We retrospectively analyzed 23,284 nasopharyngeal swab specimens collected between 2007 and 2024 from outpatients and inpatients presenting with influenza-like illness at a tertiary care hospital. HRV RNA was detected by Real-Time RT-PCR, and positivity rates were compared by year, month, and age group. Annual detection peaked in 2015 (31.3%) and 2016 (28.6%), then dropped sharply during the COVID-19 pandemic (2020–2021, 4.2–11.0%) and remained low through 2024. Seasonally, rates were highest in July (24.4%) and September (24.1%) and lowest in January (6.9%). Age-specific analysis showed peak positivity in children (26.1%) and infants (20.3%), with lower rates in adults (3.9%) and older adults (3.3%). These findings underscore the diagnostic value of HRV detection and provide evidence for pediatric-focused prevention, outbreak preparedness, and climate-informed surveillance strategies. Full article

Chronic rhinosinusitis without nasal polyps (CRSsNP) is a chronic inflammatory disease that lacks a clear pathogenesis/pathophysiology. While large studies focused on elucidating the pathophysiology of CRS with NPs (CRSwNP), this study aimed to use a systemic evaluation approach to identify the redox gene expression profile, its association with oxidative damage in CRSsNP, and the differences between CRSsNP and -wNP. The expression of 84 redox genes was analyzed using real-time PCR array in control and CRSsNP nasal mucosae. Changes in the mRNA and protein levels of these redox differentially expressed genes (DEGs) were verified using a customized real-time PCR array, RT-PCR, and Western blotting in an additional 18 patients. 4-Hydroxynonenal (lipid peroxidation) and 3-nitrotyrosine (protein nitrosylation) expression, representing oxidative stress (OxS) and nitrosative stress (NsS) status, were examined using immunohistochemistry. We found 27 DEGs (24 upregulated and 3 downregulated) in CRSsNP. AKR1C2, GCLM, GPX2, NOS2, and NQO1 were upregulated and LPO was downregulated more than 4-fold. These changes led to a substantial increase in OxS in CRSsNP nasal mucosa. In a comparison of the currently identified 27 DEGs with the 23 previously reported CRSwNP genes, there were 16 unique redox DEGs expressed between CRSsNP and -wNP. A String protein interaction network analysis revealed that CRSsNP possessed “an adaptive antioxidant defense signature”, while CRSwNP showed “a pro-inflammatory and -oxidant pathway”. Collectively, we systemically performed transcriptomic analysis to profile OxS-related genes in CRSsNP and highlighted the unique redox gene sets and pathway differences between CRSsNP and -wNP. Full article

Background/Objectives: Aseptic loosening (AL) is among the most common causes of late failure following total hip arthroplasty (THA), often necessitating complex revision surgery. Current diagnostic tools, mainly based on clinical and radiological findings, are primarily able to identify advanced changes of periprosthetic osteolysis (PPOL). Therefore, early detection of AL remains a challenge. Circulating microRNAs (miRNAs) have emerged as promising, minimally invasive biomarkers in musculoskeletal disorders. This study investigates the expression of inflammation-related miRNAs let-7i-5p, let-7e-5p, miR-15a-5p, miR-30a-3p and miR-130a-3p in patients with confirmed AL after THA to evaluate their potential role in AL. Methods: AL patients undergoing revision were compared with asymptomatic post-THA individuals and controls with degenerative osteoarthritis. Preoperative, peripheral blood samples were collected; total RNA was extracted; and quantitative real-time PCR (qRT-PCR) was performed to quantify miRNA expression. The relative expression of miRNAs was calculated using the 2–ΔΔCt method after proper normalization of Ct values. Statistical analysis assessed differences between groups. Results: The under investigation miRNAs exhibited distinct expression patterns. Several targets demonstrated significant downregulation in AL patients, suggesting a potential link to inflammatory and osteolytic pathways like Toll-like receptor 4 (TLR4)–Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, NLRP3 inflammasome activation and macrophage polarization. Conclusions: The observed alterations in circulating miRNAs support their capability as biomarkers for early detection of AL following THA. Larger cohorts could facilitate translation into routine clinical diagnostics. Full article

Background/Objectives: A comparative study of silver (Ag), titanium nitride (TiN), zirconium nitride (ZrN), and copper (Cu) coatings on titanium (Ti) disks, considering the specifications of a microporous skin- and bone-integrated titanium pylon (SBIP), was performed to assess their biocompatibility, osseointegration, and mechanical properties. Methods: To assess cytotoxicity and biocompatibility, Ti disks with various metal coatings were co-cultured with FetMSCs and MG-63 cells for 1, 3, 7, and 14 days and subsequently evaluated using a cell viability assay, as supported by SEM and confocal microscopy studies. The antimicrobial activity of the selected four materials coating the implants was tested against S. aureus by mounting Ti disks onto the surface of LB agar dishes spread with a bacterial suspension and measuring the diameter of the growth inhibition zones. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) analysis of the relative gene expression of biomarkers that are associated with extracellular matrix components (fibronectin, vitronectin, type I collagen) and cell adhesion (α2, α5, αV integrins), as well as of osteogenic markers (osteopontin, osteonectin, TGF-β1, SMAD), was performed during the 14-day follow-up period. Additionally, the activity of matrix metalloproteinases (MMP-1, -2, -8, -9) was assessed. Results: All samples with metal coatings, except the copper coating, demonstrated a good cytotoxicity profile, as evidenced by the presence of a cellular monolayer on the sample surface on the 14th day of the follow-up period (as shown by SEM and inverted confocal microscopy). All metal coatings enhanced MMP activity, as well as cellular adhesion and osteogenic marker expression; however, TiN showed the highest values of these parameters. Significant inhibition of bacterial growth was observed only in the Ag-coated Ti disks, and it persisted for over 35 days. Conclusions: The silver-based coating, due to its high antibacterial activity, low cytotoxicity, and biointegrative capacity, can be recommended as the coating of choice for microporous titanium implants for further preclinical studies. Full article

GRAS family plays a critical role in plant growth and stress responses. In this study, we identified 47 GRAS (DlGRAS) genes in the longan genome and conducted a comprehensive bioinformatics analysis of these genes. RNA-seq analysis revealed that the expression of these DlGRAS genes differed during early SE and across various longan tissues. The quantitative real-time PCR (qRT-PCR) results indicated that the DlGRAS genes exhibited differential expression during the early SE of longan, with most of them showing high expression at the globular embryo (GE) stage. Under GA₃ treatment, the transcript levels of DlGRAS12/15 decreased significantly. In contrast, exogenous ABA promoted the expression of DlGRAS6/10/23, indicating that DlGRAS genes are responsive to hormones. Compared with CaMV35S-driven GUS expression, the promoters of DlGRAS10/22 increased GUS expression, GA₃ and ABA treatments enhanced promoter activity. DlGRAS10/22 were located in the nucleus. Overexpression of DlGRAS10/22 in longan SE significantly promoted the transcription levels of SE-related genes, including DlGID1, DlGA20ox2, DlLEC1, DlFUS3, DlABI3 and DlLEC2. Therefore, DlGRAS may be involved in the early morphogenesis of longan SE through the hormone signaling pathway. Full article

(1) Background: The BMP/GDF (Bone Morphogenetic Protein/Growth Differentiation Factor) subfamily (Decapentaplegic-Vg1-related, DVR) within the transforming growth factor beta (TGF-β) superfamily plays critical roles in governing biological developmental processes and physiological functions. (2) Methods: In this study, we systematically investigated the DVR gene family in hexaploid Qihe gibel carp (Carassius gibelio var. Qihe) through comprehensive genomic identification, phylogenetic analysis, chromosome mapping, and cis-regulatory element prediction. The experimental design for gene expression analysis involved collecting samples from multiple tissues (brain, muscle, liver, kidney, etc.) and different developmental stages (20, 45, and 60 days post hatching, dph) to examine the expression patterns of four GDF8 genes using quantitative real-time PCR (qRT-PCR). (3) Results: We identified 50 DVR members in Qihe gibel carp. Phylogenetic analysis classified the 50 DVR family members into 20 distinct protein types, with 29 BMPs (Bone Morphogenetic Proteins) and 21 GDFs (Growth Differentiation Factors) identified. All 50 DVR proteins of Qihe gibel carp have similar TGF-β domains except for four BMP1 proteins. Chromosomal localization revealed widespread distribution of DVR members across 36 chromosomes, a pattern potentially linked to the hexaploid genome of Qihe gibel carp. Genes within the same subgroup exhibited conserved intron–exon architectures and similar intron numbers; syntenic conservation within subgroups may reflect functional constraints after polyploidization, implying evolutionary pressure to maintain functional domains. Through spatiotemporal expression profiling, we uncovered functional divergence among four GDF8 (myostatin) paralogs: GDF8-1 and GDF8-2 were predominantly expressed in brain and muscle tissues (dorsal and caudal), while GDF8-3 and GDF8-4 showed hepatic, cerebral, and renal specificity. Intriguingly, all paralogs exhibited a gradual upregulation during late development (20–60 days post hatching, dph), with peak expression staggered between 45 dph (GDF8-1/2) and 60 dph (GDF8-3/4). (4) Conclusions: These findings suggest that GDF8 plays a critical regulatory role in the growth and development of Qihe gibel carp. Collectively, these results provide a foundation for further investigations into the functional roles of the DVR gene family during the ontogenetic development of this species. Full article

Salt-tolerance improvement of tomatoes is largely a task of modern selection and plant molecular genetics because of cultivation on dry and irrigated lands under salt stress. To reveal the salt resistance gene, we need quantitative real-time polymerase chain reaction (qRT-PCR) normalization through reference genes analysis. Sometimes, housekeeping gene expression changes in response to various stress factors, especially salinity. In this manuscript, we evaluated expression changes of elongation factor 1α X53043.1 (EF1α), actin BT013707.1 (ACT), ubiquitin NM_001346406.1 (UBI), nuclear transcript factor XM_026030313.2 (NFT-Y), β-tubulin NM_001247878.2 (TUB), glyceraldehyde-3 phosphate dehydrogenase NM_001247874.2 (GAPDH), phosphatase 2A catalytic subunit NM_001247587.2 (PP2a), and phosphoglycerate kinase XM_004243920.4 (PGK) in salt-sensitive Solanum lycopersicum L. YaLF line and salt tolerance Rekordsmen cv. under 100 mM NaCl. We also suggested potential correlations between relative water content (RWC), ion accumulation, and reference gene expression in tomato genotypes with contrasting salinity tolerance. We used geNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder algorithms to establish a set of the most reliable tomato candidate genes. The most stable genes for YaLF tomatoes were ACT, UBI, TUB, and PP2a. Despite differences in ranks, the NFT-Y was present in Rekordsmen’s stable set. Full article

Background/Objectives: Breast cancer remains one of the leading causes of cancer-related mortality in women worldwide, highlighting the urgent need for effective and less toxic therapeutic strategies. Thymoquinone (TQ), a bioactive phytochemical derived from Nigella sativa, possesses antioxidant and anticancer activities. Methotrexate (MTX), a widely used folate antagonist, is an established chemotherapeutic agent but is limited by toxicity and resistance. This study aimed to investigate the potential synergistic effects of TQ and MTX in estrogen receptor-positive MCF-7 breast cancer cells. Methods: MCF-7 cells were exposed to TQ (0–100 μM), MTX (0–10 μM), and their combinations for 24–72 h. Cell viability was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and drug interactions were evaluated using the Chou–Talalay method. Apoptosis was quantified by Annexin V/Propidium Iodide (PI) flow cytometry, and cell cycle distribution was analyzed by PI staining. Intracellular reactive oxygen species (ROS) generation was measured using a 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) assay, while antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT)) activities were quantified spectrophotometrically. Gene expression of Bax, Bcl-2, NF-κB, MMP-2, and MMP-9 was determined by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: TQ and MTX each reduced cell viability in a dose- and time-dependent manner, while combination treatment significantly enhanced cytotoxicity compared with single agents (p < 0.01). Combination Index (CI) values < 1 confirmed a synergistic interaction, particularly at 50 μM TQ + 5 μM MTX and 100 μM TQ + 10 μM MTX. Combination therapy increased total apoptosis up to 83.6%, markedly elevated the Bax/Bcl-2 ratio, and enhanced caspase-3 activation. Cell cycle analysis revealed pronounced G2/M arrest. ROS levels increased approximately six-fold, accompanied by significant suppression of SOD and CAT activities. qRT-PCR results demonstrated upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic B-cell lymphoma 2 (Bcl-2), nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-2, and MMP-9. Conclusions: TQ potentiates the anticancer activity of MTX in MCF-7 breast cancer cells by synergistically inducing apoptosis, oxidative stress, and cell cycle arrest while suppressing metastasis-related genes. This combination may represent a promising therapeutic strategy for breast cancer, warranting further validation in in vivo and clinical studies. Full article