Search Results (127,453)

Lagenaria siceraria is an essential horticultural and medicinal crop that is used for its edible fruits and ornamental purposes. WRKY transcription factors have been extensively studied in plant responses to environmental stress; however, there is limited information on their specific functions in L. siceraria. In this study, 51 LsWRKY genes were identified in the L. siceraria genome. The 51 LsWRKYs were divided into classes I, II, and III based on evolutionary analysis. Members of each class have similar conserved motifs and exon-intron structures, and promoter analysis helped identify many cis-regulatory elements associated with growth, hormones, and stress responses. GO terms and KEGG analyses indicated the potential roles of LsWRKY in the regulation of bottle gourd development and acclimation to various environmental stressors. Significant differences in LsWRKY expression were observed between different tissues. The results of RNA-seq and qRT-PCR showed that LsWRKYs were expressed in a tissue- and development-specific manner under normal growth conditions. LsWRKY abundance showed a clear pattern of change related to stress when L. siceraria was exposed to unfavorable environmental conditions. This study provides new insights into the role of LsWRKYs in the growth and stress responses of cucurbits. Full article

Background/Objectives: Spheroid cultures in Matrigel are routinely used to study cell behaviour in complex 3D settings, thereby generating preclinical models of disease. Ideally, researchers would like to modulate gene expression ‘in situ’ for testing novel gene therapies while conserving the spheroid architecture. Here, we aim to provide an efficient method to transfect small RNAs (such as microRNAs and small interfering RNAs, i.e., siRNAs) into human induced pluripotent stem cell (iPSC)-derived 3D lung spheroids, specifically alveolar type II epithelial cells (iAT2) and basal cell (iBC) spheroids. Methods: Transfection of iAT2 spheroids within 3D Matrigel ‘in situ’, whole spheroids released from Matrigel or spheroids dissociated to single cells was explored via flow cytometry using a fluorescently labelled siRNA. Validation of the transfection method was performed in iAT2 and iBC spheroids using siRNA and miRNA mimics and measurement of specific target expression post-transfection. Results: Maximal delivery of siRNA was achieved in serum-free conditions in whole spheroids released from the Matrigel, followed by whole spheroids ‘in situ’. ‘In situ’ transfection of SFTPC-siRNA led to a 50% reduction in the SFTPC mRNA levels in iAT2 spheroids. Transfection of miR-29c mimic and miR-21 pre-miR into iAT2 and iBC spheroids, respectively, led to significant miRNA overexpression, together with a significant decrease in protein levels of the miR-29 target FOXO3a. Conclusions: This study demonstrates successful transfection of iPSC-derived lung spheroids without disruption of their 3D structure using a simple and feasible approach. Further development of these methods will facilitate functional studies in iPSC-derived spheroids utilizing small RNAs. Full article

The effects of soil acidification on the phoD-harboring microbial community and the fractions of soil phosphorus in tea plantation soils are still unclear. In this study, tea plantations with different soil pH were used as the research object to analyze changes in soil phosphorus fractions, phoD gene abundance, phoD-harboring microbial community composition, and their relationship with tea yield and quality. The results showed that the content of tea polyphenols, caffeine, free amino acids, theanine, and tea yield decreased significantly after acidification. Moreover, the content of total phosphorus in the acidified soil also decreased significantly. Further analysis of soil phosphorus fractions showed that the acidification of the tea plantation soil resulted in a significant decrease in the content of different types of labile and moderately labile phosphorus, whereas the content of non-labile phosphorus exhibited the opposite trend. As the content of soil NaHCO₃-Po, NaOH-Po, Resin-Pi, NaHCO₃-Pi, NaOH-Pi, and HCl-Pi decreased significantly after acidification, its organic and inorganic phosphorus content also decreased significantly. Its phosphorus activation capacity decreased by 4.75% after soil acidification. Soil acidification significantly reduced the diversity of phoD-harboring microbial communities by 61.89%. Analysis of the phoD-harboring microbial community composition suggested that the microbial abundance of Acidobacteria and Proteobacteria showed a decreasing trend in acidified soils, while for Nitrospirae, Verrucomicrobia, Actinobacteria, and Planctomycetes, it showed an increasing trend. Correlation analysis showed that microorganisms with significantly decreasing abundance in tea plantation soils were significantly and positively correlated with soil pH, labile phosphorus, moderately labile phosphorus, phosphorus activation coefficients, and tea yield and quality after soil acidification. It is evident that soil acidification inhibited soil phosphorus availability by shifting phoD-harboring microbial community composition in tea plantation soils, thus affecting the yield and quality of above-ground tea leaves. Full article

Precision medicine approaches rely on accurate somatic variant detection, where the DNA input into genomic workflows is a key variable. However, there are no gold standard methods for total DNA quantification. In this study, a pentaplex reference gene panel using digital PCR (dPCR) was developed as a candidate reference method. The multiplex approach was compared between two assay chemistries, applied to healthy donor genomic DNA and plasma cell-free DNA (cfDNA) to measure the ERBB2 (HER2) copy number variation in cancer cell line DNA. The multiplex approach demonstrated robust performance with the two assay chemistries, demonstrating comparable results and a wide dynamic range. Ratios of reference genes were close to the expected 1:1 in healthy samples; however, some small but significant differences (<1.2-fold) were observed in one of the five targets. Expanded relative measurement uncertainty was 12.1–19.8% for healthy gDNA and 9.2–25.2% for cfDNA. The multiplex approach afforded lower measurement uncertainty compared to the use of a single reference for total DNA quantification, which is an advantage for its potential use as a calibration method. It avoided potential biases in the application to CNV quantification of cancer samples, where cancer genome instability may be prominent. Full article

Finding the causative mutations for musculoskeletal system development and health status is of a higher priority for all sport horse breeders’ associations. Of the regulating proteins involved in animal ossification, 15 gene polymorphisms were chosen to be identified as connected with the nine fetlock and 14 hock bone structures measurements of 198 horses. All measurements were taken using X-rays of the limbs, which were available at the beginning and end of the horse training. The analysis of variance (GLM, SAS program) was performed taking into account identified training and horse-connected characteristics, and gene polymorphism. The larger size of the bone structure was achieved in the fetlock for the heterozygotes of COL9A2, AOAH1, BMPER, HYAL3, and ELMO1. The heterozygotes were superior to homozygotes in the hock for the following genes: COL9A2, HYAL3, ANLN, and HYAL1. The lower homozygote values were obtained for GG in CPVL in fetlock measurements, TT for HYAL3 in fetlock, TT for ANLN in fetlock, CC for FRZB in the hock, TT for MATN in the hock, and TT for COL5A2 in the hock than their opposite homozygote and heterozygote variants. COL9A2 and HYAL3 are expressed in the same way for most of the bone structures in both joints. Full article

Cucumber green mottle mosaic virus (CGMMV) represents a serious threat in the production of watermelon. Small RNAs facilitate a mechanism known as RNA interference (RNAi), which regulates gene expression. RNAi technology employs foreign double-stranded RNAs (dsRNAs) to target and reduce the expression levels of specific genes in plants by interfering with their mRNAs. In this study, watermelon plants were treated with dsRNAs of CGMMV MET, IR, and HEL fragments that had been generated in E. coli HT115. We investigated variations in several factors, including viral accumulation, virus-derived small interfering RNAs (vsiRNAs), and symptom severity. MET-dsRNA, IR-dsRNA and HEL-dsRNA dramatically decreased the symptoms of CGMMV in plants in the growth chamber test. Plants treated with viral-derived dsRNA showed a considerable decrease in both virus titers and vsiRNA levels. We also explored the mobility of spray-on dsRNA-derived long dsRNA and discovered that it could be identified in both inoculated leaves and the systemic leaves. IR-dsRNA outperformed MET-dsRNA and HEL-dsRNA in dsRNA therapy. Illumina sequencing of small RNAs from watermelon plants treated with IR-dsRNA and those that were not treated showed that the decreased accumulation of vsiRNAs was consistent with interference with CGMMV infection in systemic leaves. dsRNA-treated plants showed a higher level of 24-nt viral siRNA and lower level of 22-nt viral siRNA accumulation, while 22-nt viral siRNA predominated in untreated plants, indicating that dsRNA treatment improved DCL3 activity. In conclusion, our research provides deeper insights into the mechanism of antiviral RNA interference and confirms the effectiveness of applying dsRNA locally to enhance plant antiviral activity. Full article

Background/Objectives: Neuroblastoma (NB) is an aggressive pediatric malignancy that accounts for nearly 15% of all childhood cancer-related deaths, with high-risk cases showing a poor 20% prognosis and limited response to current therapies. Survivin, encoded by the BIRC5 gene, is an anti-apoptotic protein frequently overexpressed in NB and linked to treatment resistance and unfavorable clinical outcomes. Methods and Results: An analysis of 1235 NB patient datasets revealed a significant association between elevated BIRC5 expression and reduced overall and event-free survival, highlighting survivin as an important therapeutic target in NB. To explore this strategy, we evaluated the efficacy of YM-155, a small-molecule survivin inhibitor, across multiple NB cell lines. YM-155 displayed potent cytotoxic activity in six NB cell lines with IC₅₀ values ranging from 8 to 212 nM and significantly inhibited colony formation and 3D spheroid growth in a dose-dependent manner. Mechanistic analyses revealed that YM-155 downregulated survivin at both mRNA and protein levels, induced apoptosis by about 2–7-fold, and caused G0/G1 phase cell cycle arrest. Moreover, YM-155 treatment enhanced p53 expression, suggesting reactivation of tumor suppressor pathways. Notably, combining YM-155 and the chemotherapeutic agent etoposide resulted in synergistic inhibition of NB growth with ED75 values ranging from 0.17 to 1, compared to either agent alone. In the xenograft mouse model, YM-155 inhibited tumor burden in contrast to controls by about 3-fold, and without any notable toxic effects in vivo. Conclusion: Overall, our findings identify YM-155 as a promising therapeutic agent for high-risk NB by directly targeting survivin and enhancing chemosensitivity. These results support continued preclinical development of survivin inhibitors as part of rational combination strategies in pediatric cancer treatment. Full article

Tree peony is an important horticultural plant with both ornamental and oil value. The tree peony genome and databases were used to search for calmodulin family genes to explore their function in the pollination of tree peony. The CaM gene family was identified, and then the basic protein characteristics of the family members, such as gene structure, isoelectric point, molecular weight, subcellular localization, and conserved protein domain, were analyzed. The expression levels of these genes in the pistil tissue of Paeonia ostii ‘Fengdan’ at different developmental stages after pollination were also analyzed. Further, qRT-PCR was used to detect the expression levels of six PsCaMs during the development process of the pistil under bee pollination conditions. The results showed that there were six CaM family members located on three chromosomes and one non-chromosome. There were a large number of hormone response and stress response elements on the gene promoter of this family. During the development of pistil tissue after pollination, CaM family gene expression showed a trend of first increasing and then decreasing, which may be related to its function during pollination. The purpose of this study is to identify the gene characteristics and expression patterns of the CaM family during pollination, and to lay a foundation for the functional study of the CaM family in tree peony pollination. Full article

Breast cancer is a heterogeneous disease and a leading cause of cancer-related deaths worldwide, underscoring the urgent need for effective biomarkers to guide diagnosis, prognosis, and therapeutic decisions. Bioinformatics methodologies, including genomics, transcriptomics, proteomics, and metabolomics data analysis, are essential for deciphering the complex molecular landscape of breast cancer. Bioinformatics tools facilitate the identification of differentially expressed genes, non-coding RNAs, and proteins, unraveling crucial pathways involved in tumor initiation, progression, and metastasis. By constructing and analyzing protein–protein interaction networks and signaling pathways, bioinformatics approaches can identify potential diagnostic, prognostic, and predictive biomarkers. Herein, we explore the role of bioinformatics in breast cancer research and its potential application in identifying novel therapeutic targets and predicting drug response, ultimately enabling the development of tailored treatment strategies. We also address the challenges and future directions in utilizing bioinformatics for biomarker discovery and validation, emphasizing the need for robust statistical methods, standardized data analysis pipelines, and collaborative efforts to translate bioinformatics insights into improved clinical outcomes for breast cancer patients. Full article

Pulmonary fibrosis (PF) is a progressive and fatal lung disease characterized by irreversible alveolar destruction and pathological extracellular matrix (ECM) deposition. Currently approved agents (pirfenidone and nintedanib) slow functional decline but do not reverse established fibrosis or restore functional alveoli. Multifunctional bioscaffolds present a promising therapeutic strategy through targeted modulation of critical cellular processes, including proliferation, migration, and differentiation. This review synthesizes recent advances in scaffold-based interventions for PF, with a focus on their dual mechano-epigenetic regulatory functions. We delineate how scaffold properties (elastic modulus, stiffness gradients, dynamic mechanical cues) direct cell fate decisions via mechanotransduction pathways, exemplified by focal adhesion–cytoskeleton coupling. Critically, we highlight how pathological mechanical inputs establish and perpetuate self-reinforcing epigenetic barriers to regeneration through aberrant chromatin states. Furthermore, we examine scaffolds as platforms for precision epigenetic drug delivery, particularly controlled release of inhibitors targeting DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) to disrupt this mechano-reinforced barrier. Evidence from PF murine models and ex vivo lung slice cultures demonstrate scaffold-mediated remodeling of the fibrotic niche, with key studies reporting substantial reductions in collagen deposition and significant increases in alveolar epithelial cell markers following intervention. These quantitative outcomes highlight enhanced alveolar epithelial plasticity and upregulating antifibrotic gene networks. Emerging integration of stimuli-responsive biomaterials, CRISPR/dCas9-based epigenetic editors, and AI-driven design to enhance scaffold functionality is discussed. Collectively, multifunctional bioscaffolds hold significant potential for clinical translation by uniquely co-targeting mechanotransduction and epigenetic reprogramming. Future work will need to resolve persistent challenges, including the erasure of pathological mechanical memory and precise spatiotemporal control of epigenetic modifiers in vivo, to unlock their full therapeutic potential. Full article

The management of Meloidogyne incognita often depends on chemical nematicides, which pose environmental and health risks. This study investigated the potential of bacterial strains isolated from uncultivated native soil as biocontrol agents and plant growth-promoting rhizobacteria (PGPR) in tomato plants artificially infected with this nematode. Fifteen strains were screened in vitro for nematicidal and ovicidal activity, and four promising strains (307, GB16, GB24, and GB29) were selected for greenhouse trials. All strains reduced the nematode reproduction factor and the number of nematodes/g of root. Strains 307 and GB24 showed the highest reductions, 61.39 and 57.24%, respectively. Despite some positive physiological trends, Bacillus spp. did not promote a significant increase in plant growth. Metabolomic analysis revealed that the strains produced a wide range of primary metabolites with potential nematicidal activity. All strains also secreted proteases and chitinases, enzymes linked to nematode cuticle degradation. Preliminary identification based on the 16S rRNA gene and phylogenetic analysis grouped the four strains into the Bacillus subtilis group (strains GB16, GB29 and 307) or Bacillus cereus group (strain GB24); however, genome sequencing will be required in future studies. Overall, strains 307 and GB24 demonstrated strong biocontrol potential, supporting their use as sustainable and complementary alternatives to chemical nematicides. Full article

The methylation of CpG sites with 5mC mark is a dynamic epigenetic modification. However, the relationship between the methylation and the surrounding genomic sequence context remains poorly explored. Investigation of the allele methylation provides an opportunity to decipher the interplay between differences in the primary DNA sequence and epigenetic variation. Here, we performed high-coverage long-read whole-genome direct DNA sequencing of one individual using Oxford Nanopore technology. We also used Illumina whole-genome sequencing of the parental genomes in order to identify allele-specific methylation sites with a trio-binning approach. We have compared the results of the haplotype-specific methylation detection and revealed that trio binning outperformed other approaches that do not take into account parental information. Also, we analysed the cis-regulatory effects of the genomic variations for influence on CpG methylation. To this end, we have used available Deep Learning models trained on the primary DNA sequence to score the cis-regulatory potential of the genomic loci. We evaluated the functional role of the allele-specific epigenetic changes with respect to gene expression using long-read Nanopore RNA sequencing. Our analysis revealed that the frequency of SNVs near allele-specific methylation positions is approximately four times higher compared to the biallelic methylation positions. In addition, we identified that allele-specific methylation sites are more conserved and enriched at the chromatin states corresponding to bivalent promoters and enhancers. Together, these findings suggest that significant impact on methylation can be encoded in the DNA sequence context. In order to elucidate the effect of the SNVs around sites of allele-specific methylation, we applied the Deep Learning model for detection of the cis-regulatory modules and estimated the impact that a genomic variant brings with respect to changes to the regulatory activity of a DNA loci. We revealed higher cis-regulatory impact variants near differentially methylated sites that we further coupled with transcriptomic long-read sequencing results. Our investigation also highlights technical aspects of allele methylation analysis and the impact of sequencing coverage on the accuracy of genomic phasing. In particular, increasing coverage above 30X does not lead to a significant improvement in allele-specific methylation discovery, and only the addition of trio binning information significantly improves phasing. We investigated genomic variation in a single human individual and coupled computational discovery of cis-regulatory modules with allele-specific methylation (ASM) profiling. In this proof-of-concept analysis, we observed that SNPs located near methylated CpG sites on the same haplotype were enriched for sequence features suggestive of high-impact regulatory potential. This finding—derived from one deeply sequenced genome—illustrates how phased genetic and epigenetic data analyses can jointly put forward a hypotheses about the involvement of regulatory protein machinery in shaping allele-specific epigenetic states. Our investigation provides a methodological framework and candidate loci for future studies of genomic imprinting and cis-mediated epigenetic regulation in humans. Full article

Elevated cholesterol levels play important mitogenic roles. Pseurotin A (PsA) is a fermentation product that has recently been reported as a dual inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion and protein-protein interaction (PPI) with the LDLR. PsA showed a high acute safety profile and therapeutic potential against metastatic castration-resistant prostate cancer (mCRPC). The study aims to uncover the chronic safety, distribution, and anti-mCRPC genomic and molecular mechanistic insights of PsA. A 90-day chronic safety assessment of PsA up to 80 mg/kg in Swiss albino mice showed no signs of hematological, biochemical, or major organ toxicity. PsA demonstrated rapid intravenous distribution and elimination in Swiss albino mice. PsA is biodistributed to multiple key organs but was not detected in the brain, indicating its inability to cross the blood-brain barrier. PsA effectively suppressed the recurrence of nude mice xenografted mCRPC, which was subjected to a neoadjuvant docetaxel and enzalutamide regimen, followed by surgical excision. Collected PsA and vehicle control-treated recurrent tumors were subjected to RNA-sequencing and pathway enrichment analysis (PEA) of differentially expressed genes (DEGs). PsA-treated tumors revealed multiple significantly enriched pathways associated with promoting tumor apoptosis and inhibiting both invasion and migration. The PPI network analyses for the downregulated DEGs displayed prominent networks of genes associated with the ubiquitin-proteasome system. Results provide comprehensive mechanistic and preclinical validations for PsA’s potential as a novel PC recurrence suppressive lead entity. Full article

The microbial community in a recirculating aquaculture system (RAS) is pivotal in fish health, contributing significantly to the productive performance during the growing-out phase. Classical and molecular methods using PCR for species-specific amplifications have traditionally been used for bacterial community surveillance. Unfortunately, these approaches mask the real bacterial diversity and abundance, population dynamics, and prevalence of pathogenic bacteria. In this study, we explored the use of Oxford Nanopore Technology to characterize the microbiota and functional metagenomics in a commercial freshwater RAS. Intestine samples from Atlantic salmon (Salmo salar (85 ± 5.7 g)) and water samples from the inlet/outlet water, settling tank, and biofilters were collected. The full-length 16S rRNA gene was sequenced to reconstruct the microbial community, and bioinformatic tools were applied to estimate the functional potential in the RAS and fish microbiota. The analysis showed that bacteria involved in denitrification processes were found in water samples, as well as metabolic pathways related to hydrogen sulfide metabolism. Observations suggested that fish classified as sick exhibited decreased microbial diversity compared with fish without clinical symptomatology (p < 0.05). Proteobacteria were predominant in ill fish, and pathogens of the genera Aeromonas, Aliivibrio, and Vibrio were detected in all intestinal samples. Notably, Aliivibrio wodanis was detected in fish showing abnormal clinical conditions. Healthy salmon showed higher contributions of pathways related to amino acid metabolism and short-chain fatty acid fermentation (p < 0.05), which may indicate more favorable fish conditions. These findings suggest the utility of nanopore sequencing methods in assessing the microbial community in RASs for salmon aquaculture. Full article

Cancer has remained a major global health challenge, with around 20 million new cases and 9.7 million fatalities recorded each year. Even though there has been recent progress in therapies such as radiotherapy, chemotherapy, immunotherapy, and gene therapy, cancer remains a major treatment challenge due to late diagnosis and difficulties in therapeutic effectiveness. Flavonoids, a substantial category of naturally occurring polyphenols, have received considerable interest in recent years for their potential involvement in cancer management and prevention, especially concerning breast cancer. These bioactive compounds, abundant in vegetables, fruits, and herbs, exhibit various therapeutic actions, including antioxidant, anti-inflammatory, and antimutagenic effects. The advanced therapeutic potential of flavonoids, when combined with FDA-approved medicines, offers synergistic effects and enhanced clinical results. Additionally, flavonoid-loaded nano-formulations, involving co-delivery systems, are being explored to increase solubility, stability, and bioavailability, enabling targeted delivery to cancer cells while reducing off-target adverse effects. This review examines the role of flavonoids in the prevention and management of breast cancer, focusing on their dietary sources, metabolism, and pharmacokinetic properties. Furthermore, we explore novel strategies, such as combination therapies with FDA-approved drugs and the application of flavonoid-based nanoformulations, which have the potential to enhance therapeutic outcomes. The clinical application of these strategies has the potential to improve breast cancer treatment and create new opportunities for the advancement of flavonoid-based therapies. Full article