Search Results (267)

Background/Objectives: Host factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease-serine 2 (TMPRSS2), furin paired basic amino acid cleaving enzyme (FURIN), and neuropilin-1 (NRP1) facilitate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry, but it is unclear whether their upper-airway expression after recovery is associated with the severity of the preceding acute illness. We examined the association between post-acute expression of these genes and previous coronavirus disease 2019 (COVID-19) severity. Methods: This multicenter cross-sectional study included 104 adults with polymerase chain reaction (PCR)-confirmed COVID-19 during the first two pandemic waves in Murcia, Spain. Nasal and/or oropharyngeal swabs were collected in the post-acute phase, at a median of 75 days after symptom onset, and transcript levels were quantified by quantitative real-time PCR. Severity was categorized using the World Health Organization (WHO) Clinical Progression Scale, and associations were evaluated using logistic regression adjusted for age, sex, and race/ethnicity. Results: In the primary analyses using continuous expression measures, none of the four genes was significantly associated with severity. In exploratory tertile-based analyses, the intermediate ACE2 tertile (odds ratio [OR] = 0.17, 95% confidence interval [CI] 0.05–0.61; p = 0.007) and intermediate NRP1 tertile (OR = 0.29, 95% CI 0.10–0.88; p = 0.030) were associated with lower odds of severe disease; no significant associations were observed for the high tertiles or for FURIN or TMPRSS2. Conclusions: Primary adjusted analyses did not reveal statistically significant associations between post-acute nasal expression of ACE2, TMPRSS2, FURIN, or NRP1 and COVID-19 severity. Because expression was measured after clinical recovery, these associations cannot be interpreted as predictors of acute severity and may instead reflect persistent molecular remodeling after more severe disease; reverse causation cannot be excluded. Longitudinal studies with acute-phase and serial post-acute sampling and healthy controls are needed. Full article

By combining next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE) PCR, we sequenced from adult bean flower thrips (Megalurothrips usitatus) the near-complete genome of a previously undescribed virus (related to Anopheline-associated C virus), which we named “Megalurothrips usitatus associated virus 1” (MUaV1). The viral genome comprises two linear, single-stranded, positive-sense RNA segments, designated RNA1 (3658 nt) and RNA2 (2041 nt), which shared 35.75% and 23.74% nucleotide identity with Anopheline-associated C virus (YP_009011225.1) and chronic bee paralysis virus (ASM62179.1), respectively. According to phylogenetic analysis, MUaV1 shares the closest evolutionary relationship with Anopheline-associated C virus, followed by chronic bee paralysis virus. A clear predominance of 22 nt vsiRNAs, characteristic of Dicer-mediated siRNA processing in insects, was observed, demonstrating that MUaV1 is capable of infecting the bean flower thrips and activating its antiviral RNAi response. Our study provides the first characterization of this virus in the bean flower thrips, enhancing our understanding of the bean flower thrips virome. Full article

Background/Objectives: Vitamin D deficiency is highly prevalent among children with obesity, but the mechanisms underlying the effectiveness of vitamin D supplementation remain poorly understood. This study examined the expression patterns of VDR-target genes across metabolically diverse tissues and compared these molecular measures with circulating serum 25-hydroxyvitamin D (25OHD), the current clinical marker of vitamin D status. We hypothesized that VDR-pathway gene expression would correlate within metabolically relevant tissues but would not be significantly associated with circulating serum 25OHD levels. Methods: A secondary analysis was performed on blood, intestinal, and visceral and subcutaneous adipose tissue (VAT and SAT, respectively) samples obtained from adolescents with obesity. Subject data included age, gender, race/ethnicity, and BMI. The tissues were analyzed via real-time qPCR to obtain quantitative levels of VDR-target gene expression, which included TLR4, THBD, and VDR in SAT and VAT and TRPV6, S100G, and VDR in intestinal tissue. Blood samples were analyzed for serum 25OHD. Results: Gene expression of THBD, VDR, and TLR4 in SAT and VAT significantly correlated with each other. In intestinal tissue, there was significant correlation between TRPV6, S100G, and VDR. No statistically significant associations were identified between the gene expression levels and serum 25OHD levels. Conclusions: VDR-target gene expression levels correlated with each other across diverse tissues but not with serum 25OHD levels. This discrepancy suggests that circulating 25OHD concentrations may not fully reflect vitamin D action. Full article

Next-generation sequencing (NGS) is currently regarded as a preferable method for detection of actionable alterations in non-small cell lung carcinomas (NSCLCs). RNA-based NGS is particularly efficient in detection of gene fusions and allows for mRNA expression analysis of relevant genes. This study describes a novel library preparation pipeline for targeted RNA sequencing, which is based on the 5′ rapid amplification of the cDNA ends (5′ RACE) and anchored multiplex PCR. The NGS panel was designed for the analysis of 15 genes relevant to NSCLC therapeutic decisions (ALK, BRAF, CD274 (PD-L1), EGFR, ERBB2 (HER2), KRAS, MET, NRAS, NRG1-2, NTRK1-3, RET and ROS1). The validation against PCR was performed for 168 NSCLC samples. The NGS assay successfully detected all 85 mutations previously identified by PCR. It also confirmed the absence of tested mutations in 82 out of 83 PCR-negative samples. The only discordant case was subsequently analyzed by digital droplet PCR and demonstrated low fraction of the mutated allele. The newly designed NGS panel was subsequently used for the analysis of 272 carcinomas from young (≤50 years old) patients with no driver mutations identified by PCR tests or with failed PCR analysis. NGS was successful in 240 (88.2%) cases, including 35 (64.8%) PCR-failed samples. Driver mutations were detected in 55 tumors, including three previously unreported fusions (PAPLN::ALK, CD55::ROS1 and FKBP15::RET). We conclude that 5′ RACE-based RNA sequencing is a viable approach for NSCLC molecular testing. Full article

Trichomonas vaginalis is the protozoan parasite responsible for trichomoniasis, a prevalent non-viral sexually transmitted disease where high Zn²⁺ concentrations in the male prostate microenvironment function as a natural infection barrier. This study evaluates the post-transcriptional mechanisms governing the gene expression and mRNA stability of the 50 kDa metalloproteinase (TvMP50), an immunogenic biomarker for male trichomoniasis, under zinc stress. Using T. vaginalis female (CNCD147) and male (HGMN01) isolates, mp50 expression was characterized through transcription inhibition assays with Actinomycin-D, 5′ RACE, 3′ RACE, poly (A) tail assays (PAT-PCR), and Mfold secondary structure modeling. Results demonstrated that while transcription consistently initiates at a 10-nucleotide sequence relative to the start codon, the presence of 1.6 mM Zn²⁺ significantly stabilizes the transcript, extending its experimental half-life from 30 to 47 min. Furthermore, PAT-PCR and sequencing confirmed that Zn²⁺ exposure alters poly (A) tract lengths, reaching up to ~700 nucleotides, and utilizes alternative polyadenylation and cleavage sites to form stable 3′ untranslated region stem-loop configurations. In conclusion, mp50 overexpression under zinc stress is driven by a post-transcriptional mechanism where the synergistic effect of alternative processing signals and longer poly (A) tails enhances mRNA stability, optimizing parasite virulence during male urogenital colonization. Full article

Wheat stripe rust (yellow rust), a devastating disease caused by the phytopathogen Puccinia striiformis f. sp. tritici (Pst), poses a major threat to global wheat (Triticum aestivum L.) production. Under ongoing climate warming, the highly virulent Pst race CYR34 has become predominant in China and exhibits increased tolerance to elevated temperatures. Although temperature is known to influence Pst urediniospore germination, the molecular mechanisms underlying temperature sensitivity during this process remain poorly understood. In this study, we combined histological examinations with transcriptome sequencing across multiple temperature regimes to identify temperature-responsive genes and characterize the predicted protein association networks in Pst. Urediniospore germination of three Pst races was tested; stronger heat tolerance was observed in CYR34-8 with 67.40–77.40% germination at 9–16 °C. RNA-Seq analysis identified 89 differentially expressed genes (DEGs) associated with temperature sensitivity, and their expression patterns were validated by qRT-PCR. The DEGs were significantly enriched in ubiquinone and other terpenoid-quinone biosynthesis, glycan degradation, and longevity-regulating pathways. Among these DEGs, genes annotated as encoding an ABC transporter, malate dehydrogenase, Hsp70, and a class 3 lipase were identified as putative hub genes in the predicted protein association network and may be associated with the coordination of temperature responses and metabolic processes. Full article

Skeletal muscle serves as the direct executor of movement, and the proliferation and migration capacities of its constituent cells represent the key physiological foundation determining equine endurance and speed. Previous whole-transcriptome analysis has shown that Insulin-like Growth Factor 1 (IGF1) is significantly upregulated in Yili horses following racing and is involved in the PI3K-Akt signaling pathway; however, its specific regulatory mechanism in equine skeletal muscle cells remains unclear. This study utilized primary equine skeletal muscle cells as a model. By constructing an IGF1 overexpression plasmid and screening efficient siRNA interference sequences, we employed RT-qPCR and Western Blot techniques to verify gene expression and the activation level of the PI3K/Akt pathway. Cell Counting Kit-8 (CCK-8) assays were used to detect cell proliferation viability, and cell scratch assays were conducted to evaluate migration capacity, aiming to clarify the regulatory effect of IGF1 on the phenotype and function of equine skeletal muscle cells. Functional experiments demonstrated that IGF1 overexpression significantly promoted the proliferation viability (p < 0.0001) and migration rate (p < 0.0001) of equine skeletal muscle cells, whereas knockdown of IGF1 significantly inhibited these cellular capabilities. Mechanistic studies revealed that IGF1 overexpression significantly increased Akt phosphorylation. Notably, IGF1 exerted its biological functions in conjunction with the activation of the PI3K/Akt signaling pathway. In conclusion, IGF1 enhances cell proliferation and wound-healing capacity of primary equine skeletal muscle cells, which correlates with its ability to activate the PI3K/Akt signaling pathway. This study is the first to reveal the critical role of IGF1 in equine skeletal muscle biology at the cellular level, providing experimental evidence for a deeper understanding of the molecular mechanisms underlying the formation of athletic performance in Yili horses. It also offers novel potential targets for the molecular breeding of sport horses and interventions for injury repair. Full article

Soybean (Glycine max) is a major economic and food crop whose yield is severely affected by frogeye leaf spot (FLS), caused by Cercospora sojina. Current knowledge of resistance genes remains insufficient for effective molecular breeding. In this study, a recombinant inbred line (RIL) population derived from a cross between the resistant parent, Henong 60 (H60), and the susceptible parent, Dongnong L13 (DN L13), was evaluated under field conditions in Acheng (AC) and Xiangyang (XY). Plants were artificially inoculated with physiological race 7 of C. sojina, and disease severity at the R3 growth stage was recorded. Genotyping using the SoySNP660K chip yielded 54,836 high-quality single-nucleotide polymorphism (SNP) markers. A genome-wide association study (GWAS) was performed using the 3VmrMLM model by integrating dual-environment phenotypic data, and four quantitative trait nucleotides (QTNs) significantly associated with resistance to FLS were identified on chromosomes 8 (1), 17 (1), and 20 (2). By the analysis of genomic annotation, functional enrichment, metabolic pathway analyses, haplotype–phenotype association and quantitative real-time PCR (qRT-PCR), Glyma.20G155700 and Glyma.17G070500 are intended to be candidate genes related to soybean resistance to race 7 of FLS. The findings of this study provide insights into the genetic mechanisms underlying resistance to FLS in soybean. The identified molecular markers and candidate genes may provide useful resources for marker-assisted breeding and the development of disease-resistant germplasm. Full article

Sea cucumber Apostichopus japonicus is one of the few animals capable of synthesizing saponins, which are critical components of its nutritional quality and health-beneficial properties. However, the specific mechanism underlying saponin biosynthesis in sea cucumbers remains unclear despite previous investigations. This study aimed to characterize the molecular pathway and regulatory mechanism of saponin biosynthesis in A. japonicus. Thirteen candidate genes involved in de novo saponin skeleton synthesis were identified from the A. japonicus genome, and their full-length cDNAs were obtained via PCR-RACE. Sequence analysis predicted the intracellular localization of these genes. Combined in situ hybridization and quantitative real-time PCR analyses revealed their high expression in coelomocytes, indicating coelomocytes as the primary saponin synthesis sites. Knockdown of mevalonate kinase (AjMVK) and two oxidosqualene cyclases (AjPS and AjLS) caused a more obvious decrease in saponin levels, identifying them as key biosynthetic enzymes. Yeast two-hybrid assays revealed that AjPS and AjLS interact with ficolins, complement component 3-2, O-linked β-N-acetylglucosamine transferase, and α-L-fucosidase, whose regulatory effects were further validated by RNA interference and saponin content measurements. These results suggest that saponin biosynthesis in A. japonicus is regulated by the complement lectin pathway and modulated by glycosylation enzymes, providing a molecular foundation for enhancing bioactive saponin production for pharmaceutical and nutraceutical applications. Full article

Seashore paspalum (Paspalum vaginatum), a salinity-tolerant turfgrass, lacks well-characterized viral profiles. This study reports the discovery of a novel virus, tentatively named Paspalum latent virus (PaLV), representing a new taxon within the Alphaflexiviridae. Using high-throughput sequencing and RACE PCR, the 6995 nt genome was determined, revealing five open reading frames. Notably, PaLV lacks the AlkB domain and exhibits unique features, including overlapping start-stop codons (ORF4/ORF5) and a second in-frame AUG in the coat protein (CP) region. Phylogenetic analysis of the replicase placed PaLV in a distinct clade, separate from Potexvirus and Lolavirus. Despite low sequence identity, AlphaFold2 revealed conserved CP structural domains. Genetic analysis of 11 isolates showed low diversity and strong purifying selection. Pathogenicity assays through mechanical transmission demonstrated a broad but latent host range, including Zea mays and Sorghum spp. These findings suggest PaLV represents a novel species within a putatively new genus, Paspalovirus. Given its 90% incidence rate and latent profile, the RT-PCR assay developed here is vital for routine molecular diagnostics in turfgrass management and germplasm conservation. Full article

Background/Objectives: Banana (Musa spp.) production is severely threatened by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), highlighting the need to identify genetic determinants of resistance. Methods: We performed a genome-wide analysis of NBS genes in Musa acuminata ssp. malaccensis, including phylogenetic, chromosomal, and microsynteny analyses. The genomic context and promoter regions of MamRGA2 were characterized, its response to defense-related phytohormones was evaluated by RT-qPCR, and its protein structure was predicted by homology modeling. Results: A total of 118 NBS genes were identified. Notably, we report for the first time in banana two NBS genes encoding proteins with integrated domains, corresponding to an ATP-binding cassette (ABC) transporter and a Nuclear Factor Y subunit A (NF-YA) transcription factor. Chromosomal mapping revealed a marked enrichment of NBS genes on chromosome 3, which harbors MamRGA2, an NBS gene associated with resistance to Foc TR4. RT-qPCR analyses showed that MamRGA2 is strongly induced by exogenous methyl jasmonate (MeJA) in the resistant wild genotype but not in a susceptible Cavendish cultivar, a pattern associated with divergence in promoter sequences between the two genotypes. Structural modeling suggested that the MamRGA2 protein possesses features consistent with a resistosome-like architecture. Conclusions: Overall, these findings expand current knowledge of NBS gene diversity in banana and provide a framework for future studies aimed at elucidating the molecular mechanisms underlying resistance to Foc TR4. Full article

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating threatening to global rice production. The narrow genetic background of modern rice cultivars exacerbates the shortage of durable resistance resources. In contrast, the wild rice species Oryza officinalis harbors abundant stress-resistance alleles and represents a valuable gene pool for identifying novel broad blast-resistance genes. The cloned resistance gene Pid2 is encoded in a receptor-like protein kinase conferring race-specific resistance against the M. oryzae isolate ZB15. In this study, three Pid2 homologs were isolated from O. officinalis. The special allele Pid2of-MD33 was transformed into “Yunjing 37(YG37), a blast-susceptible japonica rice cultivar” via Agrobacterium-mediated transformation. Quantitative real-time PCR analysis showed that Pid2of-MD33 was consistently expressed in various tissues of O. officinalis, with the highest transcript abundance detected in leaf mesophyll cells and plasma membranes. Inoculation with the M. oryzae isolate ZB15 revealed that transgenic YG37 lines expressing Pid2of-MD33 displayed significantly reduced lesion size and pathogen proliferation, suggesting recovered race-specific resistance. These results enrich the resistance gene resources for rice blast research and provide a promising candidate gene for rice blast resistance breeding. Full article

Allium spp. (alliums) are susceptible to rot-diseases caused by pathogenic Fusarium spp., including F. proliferatum (FP) and F. oxysporum (FO), which can cause severe crop losses. A series of pathogenicity tests of four FP isolates from garlic (Allium sativum), four FO isolates from garlic and three FO isolates from onion (Allium cepa var. cepa) were conducted on garlic seedlings and cloves, onion seedlings and bulbs, and shallot (Allium cepa var. aggregatum) bulbs to determine the virulence of the isolates. A combination of PCRs and whole-genome sequencing (WGS), using ONT long-read technology, was used to identify genes encoding putative effectors. The FP isolates caused moderate to severe symptoms in garlic and contained homologues of SIX2, CRX1 and CRX2, and either SIX9 or SIX13. The FOC ex onion isolates caused severe disease symptoms in all allium species tested, while FO from garlic caused moderate to severe disease in garlic but only mild symptoms in onion and shallot. Fusarium oxysporum f. sp. cepae ex onion potentially contained homologues of SIX3, SIX5, SIX7, SIX9, SIX10, SIX12, SIX14, C5, CRX1 and CRX2. The most pathogenic FO isolate to garlic was Fo_VPRI44630 ex garlic, which contained SIX9, SIX13, C5, CRX1 and CRX2. The difference in virulence and putative effector profiles suggests evidence of host-associated differentiation, and as such, the f. sp. or race designation between FO ex garlic and FO ex onion should be investigated further. This is an important finding for future research into best management practices and breeding for disease resistance to FO and FP in garlic. 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

Although the diterpenoid alkaloids of Aconitum carmichaelii Debx. have long been a research focus in phytochemistry and pharmacology, systematic studies on the growth and development of its daughter roots remain limited, yet this process critically determines the yield and quality of the medicinal material. This study utilized the Jiangyou-derived daughter root of A. carmichaelii as experimental material. Quantitative real-time polymerase chain reaction (qRT-PCR) and total lignin quantification demonstrated that both the expression level of AcPRX12 and total lignin relative content were consistently higher in the non-swollen (PB) parts than in the swollen (P) parts of the daughter roots. The complete cDNA sequence of the AcPRX12 (with a full length of 1357 bp and encoding 350 amino acids) was obtained by rapid amplification of cDNA ends (RACE). Bioinformatics analysis identified AcPRX12 as an extracellular class III peroxidase containing a secretory peroxidase domain, and further predicted its strong binding affinity for syringaldazine, an S-type lignin monomer analog. In addition, the heterologous expression of AcPRX12 in Arabidopsis thaliana resulted in a significant increase in lignin content, which inhibited plant growth, as evidenced by shorter roots, thinner stems, smaller leaves, and shorter siliques. Collectively, these results support a model in which AcPRX12 promotes lignin biosynthesis to modulate daughter root development, ultimately shaping its distinctive tapered morphology. In conclusion, our findings propose a lignin-mediated regulatory mechanism for daughter root development controlled by AcPRX12, offering a key gene resource and a theoretical basis for understanding its morphogenesis. Full article