Functional Validation Research Articles

Genome-wide identification and analysis of ERF transcription factors related to abiotic stress responses in Nelumbo nucifera

BackgroundEthylene-responsive factor (ERF) transcription factors belong to the APETALA2/ERF (AP2/ERF) superfamily, and play crucial roles in plant development process and stress responses. However, the function of ERF proteins (especially for their role in response to abiotic stresses) remains scarce in Nelumbo nucifera, which is an important aquatic plant with high ornamental, economic, and ecological values.ResultsA total of 107 ERF genes were identified from the N. nucifera genome, and phylogenetic analysis classified these genes into 11 groups. The NnERF genes in the same group exhibited similar gene structure and conserved motifs, and they were unevenly distributed across the 8 chromosomes, with three pairs of tandem duplications and 21 pairs of segmental duplications. Synteny analysis revealed 44 and 39 of NnERF genes were orthologous to those in Arabidopsis thaliana and Oryza sativa, respectively. Tissue-specific expression patterns analysis of NnERF showed that 26 NnERF genes were expressed in all tested tissues, in which five genes exhibited high expression levels. Furthermore, 16 NnERF genes were selected for exploring their responses to different abiotic stresses, including cold, salt, drought, and Cd stresses. qRT-PCR analysis revealed that all these 16 investigated genes were regulated by at least one stress treatment, and 12 genes responded to all the stress treatments with different expression patterns or levels, suggesting their potential roles in diverse abiotic stress tolerance of N. nucifera. Additionally, two representative stress-related NnERFs (Nn3g19628 and Nn1g06033) were confirmed to be nuclear-localized proteins and displayed transcriptional activation.ConclusionsIn this study, we conducted a genome-wide identification and analysis of NnERF gene family related to abiotic stress responses in N. nucifera, which provides valuable information for further functional validation of these genes in stress responses, and forms a foundation for stress tolerance breeding in N. nucifera and other aquatic ornamental plants.

Dual Transcriptome Analysis Reveals the Changes in Gene Expression in Both Cotton and Verticillium dahliae During the Infection Process

Cotton is often threatened by Verticillium wilt caused by V. dahliae. Understanding the molecular mechanism of V. dahlia–cotton interaction is important for the prevention of this disease. To analyze the transcriptome profiles in V. dahliae and cotton simultaneously, the strongly pathogenic strain Vd592 was inoculated into cotton, and the infected cotton roots at 36 h and 3 d post infection were subjected to dual RNA-seq analysis. For the V. dahliae, transcriptomic analysis identified 317 differentially expressed genes (DEGs) encoding classical secreted proteins, which were up-regulated at least at one time point during infection. The 317 DEGs included 126 carbohydrate-active enzyme (CAZyme) and 108 small cysteine-rich protein genes. A pectinesterase gene (VDAG_01782) belonging to CAZyme, designated as VdPE1, was selected for functional validation. VdPE1 silencing by HIGS (host-induced gene silencing) resulted in reduced disease symptoms and the increased resistance of cotton to V. dahliae. For the cotton, transcriptomic analysis found that many DEGs involved in well-known disease resistance pathways (flavonoid biosynthesis, plant hormone signaling, and plant–pathogen interaction) as well as PTI (pattern-triggered immunity) and ETI (effector-triggered immunity) processes were significantly down-regulated in infected cotton roots. The dual RNA-seq data thus potentially connected the genes encoding secreted proteins to the pathogenicity of V. dahliae, and the genes were involved in some disease resistance pathways and PTI and ETI processes for the susceptibility of cotton to V. dahliae. These findings are helpful in the further characterization of candidate genes and breeding resistant cotton varieties via genetic engineering.

Identification of Novel Genetic Loci Affecting Age at Onset of Parkinson's Disease: A Genome-wide Association Study.

The age at onset (AAO) of Parkinson's disease (PD) varies widely among individuals and significantly influences disease progression and prognosis. However, few genome-wide association studies (GWASs) have investigated genetic variants determining AAO, particularly in East Asian populations. To identify single-nucleotide polymorphisms (SNPs) affecting AAO of PD in Korean patients. We conducted a GWAS on AAO of PD in 1048 Korean patients using sex-adjusted linear regression models. Additionally, we conducted downstream analyses of our primary GWAS results. rs2134545 demonstrated genome-wide significance (β = -2.459; standard error [SE] = 0.851; P = 1.898 × 10-8) and is an intergenic SNP near the ALCAM gene associated with an average AAO reduction of 3.47 years. Additionally, rs4366309 (LYST; MIR1537) demonstrated suggestive significance (β = 2.949; SE = 1.072; P = 8.68 × 10-8) and was associated with an average delay of 3.05 years. The polygenic risk score based on known PD risk loci also affected the AAO for European and Korean PD risk loci, respectively (β = -0.149; P < 0.001 and β = -0.096; P = 0.002). However, the proportion of variance was small (r2 = 0.022 and 0.009, respectively). We identified a novel SNP associated with the AAO of PD near the ALCAM gene, distinct from previously reported PD risk loci. These findings need further functional validation; however, they suggest unique genetic pathways influencing the AAO of PD and highlight the need for further research in diverse populations. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

TORC1 Regulates Thermotolerance via Modulating Metabolic Rate and Antioxidant Capacity in Scallop Argopecten irradians irradians

Target of rapamycin complex 1 (TORC1) is a key regulator of metabolism in eukaryotes across multiple pathways. Although TORC1 has been extensively studied in vertebrates and some invertebrates, research on this complex in scallops is limited. In this study, we identified the genes encoding TORC1 complex subunits in the scallop Argopecten irradians irradians through genome-wide in silico scanning. Five genes, including TOR, RAPTOR, LST8, DEPTOR, and PRAS40, that encode the subunits of TORC1 complex were identified in the bay scallop. We then conducted structural characterization and phylogenetic analysis of the A. i. irradians TORC1 (AiTORC1) subunits to determine their structural features and evolutionary relationships. Next, we analyzed the spatiotemporal expressions of AiTORC1-coding genes during various embryo/larvae developmental stages and across different tissues in healthy adult scallops. The results revealed stage- and tissue-specific expression patterns, suggesting diverse roles in development and growth. Furthermore, the regulation of AiTORC1-coding genes was examined in temperature-sensitive tissues (the mantle, gill, hemocyte, and heart) of bay scallops exposed to high-temperature (32 °C) stress over different durations (0 h, 6 h, 12 h, 24 h, 3 d, 6 d, and 10 d). The expression of AiTORC1-coding genes was predominantly suppressed in the hemocyte but was generally activated in the mantle, gill, and heart, indicating a tissue-specific response to heat stress. Finally, functional validation was performed using the TOR inhibitor rapamycin to suppress AiTORC1, leading to an enhanced catabolism, a decreased antioxidant capacity, and a significant reduction in thermotolerance in bay scallops. Collectively, this study elucidates the presence, structural features, evolutional relationships, expression profiles, and roles in antioxidant capacity and metabolism regulation of AiTORC1 in the bay scallop, providing a preliminary understanding of its versatile functions in response to high-temperature challenges in marine mollusks.

Stem cell transplantation extends the reproductive life span of naturally aging cynomolgus monkeys

The ovary is crucial for female reproduction and health, as it generates oocytes and secretes sex hormones. Transplantation of mesenchymal stem cells (MSCs) has been shown to alleviate pathological ovarian aging. However, it is unclear whether MSCs could benefit the naturally aging ovary. In this study, we first examined the dynamics of ovarian reserve of Chinese women during perimenopause. Using a naturally aging cynomolgus monkey (Macaca fascicularis) model, we found that transplanting human embryonic stem cells-derived MSC-like cells, which we called M cells, into the aging ovaries significantly decreased ovarian fibrosis and DNA damage, enhanced secretion of sex hormones and improved fertility. Encouragingly, a healthy baby monkey was born after M-cell transplantation. Moreover, single-cell RNA sequencing analysis and in vitro functional validation suggested that apoptosis, oxidative damage, inflammation, and fibrosis were mitigated in granulosa cells and stromal cells following M-cell transplantation. Altogether, these findings demonstrate the beneficial effects of M-cell transplantation on aging ovaries and expand our understanding of the molecular mechanisms underlying ovarian aging and stem cell-based alleviation of this process.

Genetic Screening Reveals Cone Cell-Specific Factors as Common Genetic Targets Modulating Rival-Induced Prolonged Mating in male Drosophila melanogaster.

Male-male social interactions exert a substantial impact on the transcriptional regulation of genes associated with aggression and mating behavior in male Drosophila melanogaster. Throughout our comprehensive genetic screening of aggression-related genes, we identified that the majority of mutants for these genes are associated with rival-induced and visually-oriented mating behavior, longer-mating-duration (LMD). The majority of mutants with upregulated genes in single-housed males significantly altered LMD behavior but not copulation latency, suggesting a primary regulation of mating duration. Single-cell RNA sequencing revealed that LMD-related genes are predominantly co-expressed with male-specific genes like dsx and Cyp6a20 in specific cell populations, especially in cone cells. Functional validation confirmed the roles of these genes in mediating LMD. Expression of LMD genes like Cyp6a20, Cyp4d21, and CrzR was enriched in cone cells, with disruptions in cone cell-specific expression of CrzR and Cyp4d21 leading to disrupted LMD. We also identified a novel gene, CG10026/Macewindu, that reversed LMD when overexpressed in cone cells. These findings underscore the critical role of cone cells as a pivotal site for the expression of genes involved in the regulation of LMD behavior. This study provides valuable insights into the intricate mechanisms underlying complex sexual behaviors in Drosophila.

Genome-Wide Analysis of the Serine Carboxypeptidase-like (SCPL) Protein Family of Bitter Gourd and Functional Validation of McSCPL22 in Fusarium oxysporum f. sp. Momordicae (FOM) Resistance

Bitter gourd is increasingly being recognized for its value as a vegetable and medicinal use, but the molecular mechanisms of pathogen resistance remain relatively poorly understood. The serine carboxypeptidase-like (SCPL) protein family plays a key role in plant growth, pathogen defense, and so on. However, a comprehensive identification and functional characterization of the SCPL gene family has yet to be conducted in bitter melon. In this study, 32 SCPL genes were identified in bitter gourd and divided into three classes. The number of SCPL genes contained in the three clusters was 7, 7, and 18, respectively. Most SCPL gene promoters contain cis-acting elements with light, hormone, and stress responses. The RNA sequencing data showed that the expression of several SCPL genes changed significantly after pathogen infection. In particular, expression of the McSCPL4, 10, 17, 22, and 25 genes increased substantially in the resistant varieties after infection, and their expression levels were higher than those in the susceptible varieties. These results suggested that genes such as McSCPL4, 10, 17, 22, and 25 may play a significant role in conferring resistance to fungal infections. Moreover, the expression levels of the McSCPL10, 17, 22, 23, and 25 genes were likewise significantly changed after being induced by salicylic acid (SA) and jasmonic acid (JA). In situ hybridization showed that McSCPL22 was expressed in the vascular tissues of infected plants, which largely overlapped with the location of Fusarium oxysporum f. sp. Momordicae (FOM) infection and the site of hydrogen peroxide production. Our results showed that McSCPL22 may be involved in the regulation of the SA and JA pathways and enhance resistance to FOM in bitter gourd plants. This is the first study to perform SCPL gene family analysis in bitter gourd. McSCPL22 may have the potential to enhance FOM resistance in bitter gourd, and further investigation into its function is warranted. The results of this study may enhance the yield and molecular breeding of bitter gourd.

Selection on synonymous codon usage in soybean (Glycine max) WRKY genes

The WRKY transcription factor gene family in soybean [Glycine max (L.) Merr.] (GmWRKY) is critical for the plant’s development and stress responses. This study examines the evolutionary dynamics of the GmWRKY gene family, focusing on its synonymous codon usage bias (CUB) in a comprehensive set of 179 coding sequences. CUB was analyzed using various indices, revealing a preference for A/T-ending codons and relatively low codon bias. Codon adaptation index (CAI) analysis suggested that these genes are optimized for efficient translation despite relatively low bias, reflecting a balance between codon diversity and translation efficiency. Neutrality and NC plots indicated that selective forces dominate over mutational forces in shaping codon usage, while selection signature analysis showed purifying selection being prevalent across the gene family. However, episodic positive selection was also detected in certain clades, highlighting potential adaptive diversification in response to environmental stress. Additionally, promoter binding site analysis uncovered correlations between codon usage and transcriptional regulation, indicating a context-dependent relationship between CUB and gene expression. Phylogenetic analysis identified 11 well-supported clades in the modern GmWRKY gene family and ancestral sequence reconstruction revealed more relaxed codon preferences and reduced selection constraints in modern GmWRKY genes, potentially linked to neofunctionalization and adaptation to environmental changes. These findings provide a framework for optimizing gene expression in transgenic soybean crops with resilience. Further functional validation of positively selected genes is recommended to elucidate their role in stress responses.

Decoding the genomic terrain: functional insights into 14 chemosensory proteins in whitefly Bemisia tabaci Asia II-1

Genome-wide analysis of Bemisia tabaci Asia II-1 unravelled for the first-time full-length sequences of 14 chemosensory proteins (CSPs), their exon-intron boundaries, insertion sites of retrotransposons, and clustering patterns on chromosomes. All the CSPs sans CSP6 have an N-terminal signal peptide. The presence of OS-D superfamily and PhBP domains in different CSPs suggests their roles in chemosensory signal transduction and pheromone binding. Motif analysis reveals the conservation and cohesiveness of CSPs in hemiptera. The phylogenetic analysis uncovers the evolutionary lineages of Hemipteran CSPs. RT-qPCR analysis showed spatial expression of CSPs in different body tissues of B. tabaci adults. In-silico docking analysis showed high-affinity binding of CSP 1 and 5 with two insecticides, imidacloprid and fipronil, with energy values ranging from − 5.8 to -9.3 kcal/mol, along with the details of interacting aminoacidic residues in the hydrophobic binding pockets of these two CSPs. Further functional validation was done through insecticide bioassays and RNAi. This study provides novel insights into the genomic architecture of CSPs in B. tabaci Asia II-1, and functional characterisation suggests that CSP1 and 5 genes may have indirect roles in insecticide resistance. It lays the foundation for further research on developing new control strategies for B. tabaci.

A one-step integrated forming and curing process for smart thin-walled fiber metal laminate structures with self-sensing functions

This study proposes and analyzes a novel one-step integrated forming and curing (IFC) process for thin-walled fiber metal laminates (FMLs) structures embedded with fiber Bragg grating (FBG) sensors, and have achieved both high-performance properties and self-sensing functions in the formed structures. A prototype machine and testing setup have been developed to validate the process's feasibility by manufacturing high-performance FMLs flat and curvature parts with effective self-sensing capabilities for real-time manufacturing and in-service monitoring. Numerical models considering curing-induced deformation and heat transfer during manufacturing have also been developed to support the analysis and validation of the self-monitoring capabilities of the intelligent FMLs parts. The results reveal that with proper control of pressure (e.g., 0.6 MPa) and time during forming and curing, high tensile and impact performance of FMLs can be maintained with embedded FBG, with less than a 3% loss. Additionally, the IFC process can effectively lead to an apparent reduction of springback deformation in the formed FMLs (more than 80%). The validation of the self-sensing function during the manufacturing process has been achieved by comparing the strain monitoring results with finite element (FE) simulation results during curing, with a minimum discrepancy of 2.0%. For the in-service self-sensing function, comparison between FE analysis and surface-fixed strain gauges during the compression instability test confirmed the efficacy of FBG sensors, with a minimum discrepancy of 4.3%. The results show that the proposed novel IFC process enables the successful manufacture of smart thin-walled FMLs parts with high shape accuracy and mechanical properties in a single step and holds significant promise for manufacturing self-sensing smart structures in the aerospace and aviation industries.

DNA Damage Response Alterations Predict for Neoadjuvant Chemotherapy Sensitivity in Muscle-Invasive Bladder Cancer: A Correlative Analysis of the SWOG S1314 Trial.

Alterations in DNA damage response (DDR) genes, including ERCC2, have been correlated with response to neoadjuvant cisplatin-based chemotherapy (NAC) in patients with muscle-invasive bladder cancer (MIBC). The SWOG 1314 (S1314) trial enrolled patients with MIBC who received one of two NAC regimens followed by radical cystectomy. We examined the prevalence of DDR alterations in NAC responders versus nonresponders and correlated DDR alteration status with response. Pretreatment tumor specimens from 179 evaluable patients underwent next-generation sequencing (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets assay). Associations were determined between any or only deleterious alterations within nine predefined DDR genes, or any alterations in ERCC2, and progression-free survival (PFS) and overall survival using Cox regression, and, in a subset of evaluable patients, pathologic response (complete response, pT0, or downstaging to <pT2) using logistic regression, adjusting for clinical stage and performance status. Deleterious DDR alterations were detected in 41 (23%) of 179 patients. Of the 151 patients evaluable for pathologic response, patients with deleterious DDR alterations (n = 39) demonstrated a higher pathologic response rate than those without (odds ratio [OR], 3.24 [95% CI, 1.51 to 6.94]; P = .003). In 24 ERCC2-mutant patients, the OR for pT0 was 3.33 (95% CI, 1.35 to 8.22; P = .009) and for <pT2 was 2.33 (95% CI, 0.92 to 5.89; P = .073). The association between deleterious DDR alterations and PFS provided an estimate of hazard ratio, 0.54 (95% CI, 0.29 to 1.01; P = .053). Deleterious DDR alterations were associated with pathologic response following NAC in S1314. Functional validation of ERCC2 and other DDR alterations is underway to help refine such alterations as biomarkers of NAC in patients with bladder cancer.

Structure–function analysis of tRNA t6A-catalysis, assembly and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

The universal N6-threonylcarbamoyladenosine (t6A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t6A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t6A biosynthesis using recombinant TsaC, TsaD–TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD2B2 tetramer. Our TsaD2B2–TsaE–tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1–α2 of TsaD. A. aeolicus TsaD2B2 or TsaDB possesses a basal divalent ion-dependent t6A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t6A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t6A-catalytic activity of TsaD2B2–tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

VviMYB24 positively regulates flavonol biosynthesis in response to moderate drought stress in ‘Cabernet Sauvignon’ grape seedlings

Drought is a kind of adversity that has always severely restricted the growth of grapes. Flavonols, as flavonoid compounds, are abundant in grapes and have an indispensable function in resisting drought. Hereafter, the objectives of this investigation were to identify key drought-induced transcription factors in grape and explore their roles and regulatory networks in the flavonol-mediated drought response mechanism. In this study, one-year-old cutting seedlings of ‘Cabernet Sauvignon’ grape were used as experimental materials, cultivated in a solar greenhouse, and subjected to moderate drought treatment. The physiological and biochemical parameters related to stress and flavonol content in plant tissues were determined, and gene expression, functional validation, and transcriptional regulation relationships were explored. The results showed that the growth of grape plants was affected under drought conditions, the flavonol accumulation and the expression level of VviMYB24 were significantly induced by drought stress. Overexpression of VviMYB24 reduced the degree of cytoplasmic membrane damage and induced flavonol biosynthesis to confer drought tolerance in Arabidopsis plants and grape callus. Further study revealed that VviMYB24 can interact with the bHLH transcription factor VviMYC2b, which was positively responsive to drought, and they could activate VviFLS5 expression individually and/or in combination in the form of a complex. Interestingly, VviMYB24 could positively regulate VviMYC2b expression to form a transcriptional cascade, enhancing the function of the complex. Transient overexpression of VviMYB24, VviMYC2b, and VviFLS5 in grape leaves promoted the drought-responsive flavonol accumulation. Overall, the above data elucidated that the VviMYB24-VviMYC2b-VviFLS5 module positively regulates drought-induced flavonol biosynthesis in response to drought stress in grape, which contributes to further grape drought resistant breeding and variety improvement through biotechnology.

GWAS for identification of genomic regions and candidate genes in vegetable crops.

Genome-wide association Studies (GWAS), initially developed for human genetics, have been highly effective in plant research, particularly for vegetable crops. GWAS is a robust tool for identifying genes associated with key traits such as yield, nutritional value, disease resistance, adaptability, and bioactive compound biosynthesis. Unlike traditional methods, GWAS does not require prior biological knowledge and can accurately pinpoint loci, minimizing false positives. The process involves developing a diverse panel, rigorous phenotyping and genotyping, and sophisticated statistical analysis using various models and software tools. By scanning the entire genome, GWAS identifies specific loci or single nucleotide polymorphisms (SNPs) linked to target traits. When a causal SNP variant is not directly genotyped, GWAS identifies SNPs in linkage disequilibrium (LD) with the causal variant, mapping the genetic interval. The method begins with careful panel selection, phenotyping, and genotyping, controlling for environmental effects and utilizing Best Linear Unbiased Prediction (BLUP). High-correlation, high-heritability traits are prioritized. Various genotyping methods address confounders like population structure and kinship. Bonferroni correction (BC) prevents false positives, and significant associations are shown in Manhattan plots. Candidate genes are identified through LD analysis and fine mapping, followed by functional validation. GWAS offers critical insights for enhancing vegetable crop breeding efficiency and precision, driving breakthroughs through advanced methods.

Cardiovascular Disease Pathogenicity Predictor (CVD-PP): A Tissue-Specific In Silico Tool for Discriminating Pathogenicity of Variants of Unknown Significance in Cardiovascular Disease Genes.

Interpretation of variants of uncertain significance (VUSs) remains a challenge in the care of patients with inherited cardiovascular diseases (CVDs); 56% of variants within CVD risk genes are VUS, and machine learning algorithms trained upon large data resources can stratify VUS into higher versus lower probability of contributing to a CVD phenotype. We used ClinVar pathogenic/likely pathogenic and benign/likely benign variants from 47 CVD genes to build a predictive model of variant pathogenicity utilizing measures of evolutionary constraint, deleteriousness, splicogenicity, local pathogenicity, cardiac-specific expression, and population allele frequency. Performance was validated using variants for which the ClinVar pathogenicity assignment changed. Functional validation was assessed using prior studies in >900 identified VUS. The model utility was demonstrated using the Catheterization Genetics cohort. We identified a top-ranked model that accurately prioritized variants for which ClinVar clinical significance had changed (n=663; precision-recall area under the curve, 0.97) and performed well compared with conventional in silico methods. This model (CVD pathogenicity predictor) also had high accuracy in prioritizing VUS with functional effects in vivo (precision-recall area under the curve, 0.58). In Catheterization Genetics, there was a greater burden of higher CVD pathogenicity predictor scored VUS in individuals with dilated cardiomyopathy compared with controls (P=8.2×10-15). Of individuals in Catheterization Genetics who harbored highly ranked CVD pathogenicity predictor VUS meeting clinical pathogenicity criteria, 27.6% had clinical evidence of disease. Variant prioritization using this model increased genetic diagnosis in Catheterization Genetics participants with a known clinical diagnosis of hypertrophic cardiomyopathy (7.8%-27.2%). We present a cardiac-specific model for prioritizing variants underlying CVD syndromes with high performance in discriminating the pathogenicity of VUS in CVD genes. Variant review and phenotyping of individuals carrying VUS of pathogenic interest support the clinical utility of this model. This model could also have utility in filtering variants as part of large-scale genomic sequencing studies.

Cuproptosis-related lncRNAs emerge as a novel signature for predicting prognosis in prostate carcinoma and functional experimental validation

BackgroundProstate cancer (PCa) is one of the most common malignancies of the urinary system. Cuproptosis, a newly discovered form of cell death. The relationship between cuproptosis-related long non-coding RNAs (ClncRNAs) related to PCa and prognosis remains unclear. This study aimed to explore the clinical significance of novel ClncRNAs in the prognostic assessment of PCa.MethodsClncRNAs and differentially expressed mRNAs linked to these ClncRNAs were identified using Pearson’s correlation and differential expression analyses. A prognostic signature (risk score) comprising three ClncRNAs was established based on multivariable Cox regression analysis. The predictive performance of this ClncRNAs signature was validated using receiver operating characteristic curves and nomograms. Finally, further in vitro cell experiments were conducted for validation, including quantitative polymerase chain reaction (qPCR), western blot (WB), cell proliferation assays, cell migration assays, cell invasion assays, apoptosis, and cell cycle analysis.ResultsWe constructed a prognostic signature of ClncRNAs for PCa comprising three key differentially expressed ClncRNAs(AC010896-1, AC016394-2, and SNHG9). Multivariable Cox regression analysis indicated that clinical staging and risk scores of the ClncRNAs signature were independent prognostic factors for PCa. Compared to other clinical features, the ClncRNAs signature exhibited higher diagnostic efficiency and performed well in predicting the 1-, 3-, and 5-year progression-free intervals (PFIs) for PCa. Notably, in terms of immune activity, PCa patients with high-risk scores exhibited higher tumor mutational burden (TMB) levels, while their Tumor Immune Dysfunction and Exclusion (TIDE) scores were lower than those of PCa patients with low-risk scores. Additionally, in vitro cellular functional experiments, we knocked down SNHG9 that is the most significantly differentially expressed ClncRNA among the three key ClncRNAs. SNHG9 knockdown resulted in a significant increase in G1 phase cells and a decrease in S and G2 phases, indicating inhibition of DNA synthesis and cell cycle progression. Colony formation assays showed reduced clonogenic ability, with fewer and smaller colonies. Western blot analysis revealed the upregulation of the key cuproptosis-related mRNAs FDX1 and DLST. These findings suggested that SNHG9 promotes PCa cell proliferation, migration, and invasion.ConclusionBuilding on the three ClncRNAs, we identified a novel prognostic signature of PCa. The ClncRNA SNHG9 can promote PCa cell proliferation, migration, and invasion.

GWAS and post-GWAS functional study reveal regulatory mechanism of piglet diarrhea.

Piglet diarrhea poses a serious threat to piglet health and the livestock economy, and is one of the most pressing problems in animal husbandry. This study aims to investigate the genetic factors involved in piglet diarrhea and to identify key genes that regulate this condition. We screened 600 diarrheal piglets based on unique diarrhea scores for resequencing and conducted a genome-wide association study (GWAS). Through this process, we identified 308 single nucleotide polymorphisms (SNPs) and annotated 151 candidate genes. Extensive functional validation and systematic analysis were performed on key candidate genes KSR1, SKAP1, SLC35F6, and OR12. The study found that the four key genes were involved in the regulation of piglet diarrhea through various mechanisms. OR12 affects the levels of ZO-1 and claudin-1. Changes in the expression levels of KSR1 could alter the expression of IL1-β, IL6, and TNF-α, as well as cell migration and proliferation. SKAP1 could affect the expression of CD3 and CD4, and influence the migration and proliferation ability of cells. SLC35F6 is involved in cell apoptosis through the Bcl2/BAX/caspase3 pathway and can also affect mitochondrial membrane potential. The results of this study provide strong support for breeding programs aimed at disease resistance and offer potential solutions to the problem of piglet diarrhea.

Integrative 3D genomics with multi-omics analysis and functional validation of genetic regulatory mechanisms of abdominal fat deposition in chickens

Chickens are the most abundant agricultural animals globally, with controlling abdominal fat deposition being a key objective in poultry breeding. While GWAS can identify genetic variants associated with abdominal fat deposition, the precise roles and mechanisms of these variants remain largely unclear. Here, we use male chickens from two lines divergently selected for abdominal fat deposition as experimental models. Through the integration of genomic, epigenomic, 3D genomic, and transcriptomic data, we build a comprehensive chromatin 3D regulatory network map to identify the genetic regulatory mechanisms that influence abdominal fat deposition in chickens. Notably, we find that the rs734209466 variant functions as an allele-specific enhancer, remotely enhancing the transcription of IGFBP2 and IGFBP5 by the binding transcription factor IRF4. This interaction influences the differentiation and proliferation of preadipocytes, which ultimately affects phenotype. This work presents a detailed genetic regulatory map for chicken abdominal fat deposition, offering molecular targets for selective breeding.

ApCarE4 and ApPOD3 participate in the adaptation of pea aphids to different alfalfa varieties

The adaptability of insects to hosts has long been a focal point in the study of insect-plant interactions. The pea aphid (Acythosiphon pisum), a significant pest of numerous leguminous crops, not only inflicts direct economic losses but also disseminates various plant viruses. To understand how pea aphids adapt to diverse alfalfa varieties. We analyzed the differentially expressed genes (DEGs) of pea aphids in distinct alfalfa varieties using transcriptome sequencing, and subsequently conducted functional validation of these genes. Comparative analysis between pea aphids feeding on susceptible and resistant strains revealed that DEGs in aphids feeding on resistant strains were primarily associated with transcriptional enrichment in the sugar, amino acid, protein, and lipid metabolism pathways. Fourteen DEGs related to adaptation of the pea aphid to alfalfa were chosen, including five carboxylesterases (CarE), four cytochrome P450s, three glutathione S-transferases, and two peroxidases (POD). RT-qPCR results indicated significant up-regulation of two carboxylesterase genes and two peroxidase genes after 24 h of feeding resistant alfalfa (Gannong 5, GN5) compared to the susceptible varieties (Hunter River, LRH), particularly highlighting the high expression levels of ApCarE4 and ApPOD3. Simultaneously, RNAi-induced knockdown of ApCarE4 and ApPOD3 led to a higher mortality of pea aphids in the alfalfa Hunter River. These results indicate that ApPOD3 and ApCarE4 are involved in the detoxification of metabolic functions in the adaptation of pea aphids to host switching. These findings contribute to the understanding of pea aphid adaptation to host plants and lay a foundation for further exploration of the physiological roles of carboxylesterase and peroxidase genes in pea aphids.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.