Search Results (1,590)

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

Vincristine is a classical chemotherapeutic agent widely used for its ability to disrupt microtubule polymerization, yet additional molecular effects may contribute to its anticancer activity. G-quadruplexes (G4s), non-canonical nucleic acid structures enriched in regulatory regions of the genome and in mitochondrial DNA, have emerged as relevant modulators of cellular homeostasis. In this study, we investigated whether vincristine can influence G4 biology. Cancer cells treated with vincristine were analyzed by immunofluorescence, revealing a consistent increase in nuclear and mitochondrial G4 foci. In particular, mitochondrial G4s were significantly elevated by approximately 1.5–2.5 fold compared to untreated cells, an effect accompanied by a detectable reduction in membrane potential, indicative of impaired organelle function. In addition, biophysical analyses on representative G4-forming sequences were carried out. Proton nuclear magnetic resonance titrations showed localized chemical shift perturbations upon vincristine addition, circular dichroism confirmed preservation of G4 topology, and isothermal titration calorimetry indicated weak but enthalpically favorable interactions. Taken together, these results suggest that vincristine perturbs both the cellular G4 landscape and mitochondrial homeostasis, while also engaging G4 DNA in vitro. Although additional studies are required to establish the mechanistic details, this work provides proof-of-concept for a previously unrecognized dimension of vincristine’s anticancer action. Full article

This study evaluated the diffusion of Listeria monocytogenes (LM) in a beef steak tartare production chain, aiming to (1) evaluate Listeria spp. diffusion in finishing farms supplying beef cattle, (2) evaluate LM prevalence in carcasses, and (3) map LM diffusion in the production plant. A detection rate of 6/76 was observed in the farm, while carcasses after skinning and before refrigeration tested positive in 19/30 and 11/30, respectively. During tartare production, 57/154 meat and 35/191 environmental samples tested positive. A total of 114 LM isolates were characterized via a whole-genome sequencing approach. Five clonal complexes (CCs) and seven sequence types (STs) were identified, with CC9-ST580 being the most prevalent. Four clusters were identified from both the slaughtering and production phases. Genes related to resistance to fosfomycin, quinolones, sulfonamides, lincosamide, and tetracycline were detected. Two hypervirulent strains (CC6-ST6 and CC2-ST145), harboring a full-length inlA, several virulence genes, and stress islands, were detected. Stress Survival Islet 1 was found in almost all the isolates. The wide diffusion of LM in steak tartare requires the management of some critical phases of the production chain (mainly slaughtering); genomic methodologies could be useful in describing the circulation and virulence of LM strains. Full article

Potato (Solanum tuberosum L.) is a globally important food crop, but its tetrasomic inheritance and diploid self-incompatibility have limited the discovery of potato genes and progress in breeding. Here, we developed an F₂ segregating population consisting of 174 lines by crossing a self-compatible genome-homozygous diploid line (Y8, female parent) with a heterozygous diploid line (IVP101, male parent), followed by selfing. Using whole-genome resequencing, we constructed a high-density genetic map containing 4464 recombinant bin markers with an average physical distance of 165.51 Kb. Phenotypic evaluation of 8 traits related to yield, tuber shape, and tuber eye number across three environments revealed significant parental differences and wide phenotypic variation within the F₂ population. QTL (Quantitative trait loci) mapping using this genetic map and multi-environment phenotypic data identified 89 QTLs, including 7 previously reported QTLs/genes. In addition, 10 QTLs were stably detected across multiple seasons (stable QTLs). Further genetic effect analysis showed that favorable alleles of these stable QTLs significantly enhanced phenotypic values. Notably, two pleiotropic QTLs were identified on chromosomes 5 and 12; the major-effect QTL on chromosome 12 (qTY-12-6, qTS-12-3, and qTE-12-4) exhibited high phenotypic variance explained (PVE). Its favorable allele from Y8 significantly increased mean tuber weight, tuber number per plant, and promoted rounder tuber shape while reducing eye number, simultaneously improving yield and quality. Collectively, this study provides a reference for genetic mapping using homozygous and heterozygous diploid parents, and the identified QTLs offer valuable genetic resources for potato breeding and molecular mechanism research, enhancing our understanding of the genetic regulation of yield, tuber shape, and eye number in potato. Full article

Bahaba taipingensis (Chinese bahaba) is a critically endangered fish endemic to China’s coastal waters, valued for both ecological and economic reasons and known as the “panda of the sea”. Captive breeding and stock enhancement are key conservation strategies, yet the genetic composition of released individuals directly affects program outcomes. This study combined mitochondrial and whole-genome resequencing to compare F₁-generation fish with wild populations. At the mitochondrial level, 60 SNPs were detected in F₁ individuals and 72 in wild populations, with haplotype analyses revealing retention of most common maternal lineages but reduced diversity. Nuclear genome analysis showed comparable genetic diversity between groups. Nucleotide diversity (π) was 0.000423 in F₁ fish and 0.000401 in the wild population. However, the F₁ cohort exhibited a higher inbreeding coefficient (F_IS = −0.030) than the wild group (F_IS = −0.118), suggesting early allele frequency shifts, thereby suggesting early genotype frequency shifts. Runs of homozygosity (ROH) analysis showed that the total number and length of ROH regions in the F₁ cohort (686, 283,089.25 kb) were significantly greater than those in the wild population (171, 52,607.30 kb). Genome-wide F_ST between groups was 0.035, and PCA indicated genetic homogenization in F₁ fish. N_e analysis showed that the wild population declined rapidly over generations and stabilized at a low level, indicating genetic diversity loss under environmental stress and highlighting the role of artificial breeding. These findings highlight the need for improved broodstock management and long-term genetic monitoring. Full article

Taraxacum kok-saghyz Rodin, an important rubber-producing plant, has emerged as a potential alternative crop for the natural rubber industry. Geranyl diphosphate synthase (GDS) catalyzes the condensation of dimethylallyl pyrophosphate and isopentenyl pyrophosphate into geranyl pyrophosphate in the mevalonate pathway in plants. However, its specific functions in natural rubber biosynthesis in T. kok-saghyz remain unclear. Methods: We conducted genome-wide analyses of TkGDS genes, followed by transient transformation assay, expression profiling, natural rubber quantification, and analysis of T. kok-saghyz photosynthesis. Results: Seven TkGDS genes are located on chromosomes A6 and A7 with an uneven distribution. All encoded TkGDS proteins contain FARM and SARM motifs. TkGDS1, TkGDS2, and TkGDS7 possess lspA domains, while TkGDS3, TkGDS4, TkGDS5, and TkGDS6 contain PLN02890 domains; both subgroups share similar domain architecture. TkGDS1, TkGDS2, and TkGDS7 exhibit interspecies collinearity with Arabidopsis thaliana; no intraspecies collinearity was detected. The putative cis-acting elements in promoter region of TkGDS genes mainly comprised abscisic acid responsiveness, anaerobic induction, light responsiveness, and MeJA responsiveness. Transient expression assays confirmed chloroplast localization of all TkGDS proteins. A strong positive correlation was observed between TkGDS1/TkGDS7 expression and natural rubber content, as confirmed by both transcriptome and qPCR analyses in T. kok-saghyz lines. Furthermore, overexpression of TkGDS1 and TkGDS7 improved photosynthetic efficiency and significantly increased natural rubber content (OE-TkGDS1: 6.08 ± 0.16%; OE-TkGDS7: 5.62 ± 0.32%; WT: 4.76 ± 0.28%). Conclusions: Our study elucidates the role of GDS1 and GDS7 in promoting growth and latex content, offering a genetic strategy for enhancing rubber accumulation in T. kok-saghyz. Full article

Stalk lodging constitutes a primary constraint on achieving consistently high yields in maize. Genetic improvement of lodging resistance requires the identification of stable quantitative trait loci (QTL) to facilitate the application of genomics-assisted breeding for improving selection efficiency in breeding programs. In this study, we performed a meta-analysis to identify consensus loci and functionally characterized candidate genes associated with stalk lodging-related traits. Through meta-analysis integrating 889 reported lodging-related QTLs using the IBM2 2008 Neighbors high-density genetic map, we identified 67 meta-QTLs (MQTLs), of which 32 were determined as core MQTLs. Among them, 67% were validated by co-localized marker–trait associations from genome-wide association studies (GWAS). Comparative genomics further revealed 40 evolutionarily conserved orthologs via protein alignment with rice lodging genes, while screening of core MQTL regions detected 802 candidate genes with KEGG enrichment implicating galactose degradation II in cell wall reinforcement, supported by transcriptomic evidence of their roles in lignin biosynthesis pathways modulating mechanical strength. In conclusion, the MQTL identified and validated in our study have significant scope in marker-assisted selection (MAS) breeding and map-based cloning programs for improving maize stalk lodging. Full article

Insights into the state of individual cells within a living organism are essential for identifying diseases and abnormalities. The internal state of a cell is reflected in its morphological features and changes in the localization of intracellular molecules. Using this information, it is possible to infer the state of the cells with high precision. In recent years, technological advancements and improvements in instrument specifications have made large-scale analyses, such as single-cell analysis, more widely accessible. Among these technologies, imaging flow cytometry (IFC) is a high-throughput imaging platform that can simultaneously acquire information from flow cytometry (FCM) and cellular images. While conventional FCM can only obtain fluorescence intensity information corresponding to each detector, IFC can acquire multidimensional information, including cellular morphology and the spatial arrangement of proteins, nucleic acids, and organelles for each imaging channel. This enables the discrimination of cell types and states based on the localization of proteins and organelles, which is difficult to assess accurately using conventional FCM. Because IFC can acquire a large number of single-cell morphological images in a short time, it is well suited for automated classification using machine learning. Furthermore, commercial instruments that combine integrated imaging and cell sorting capabilities have recently become available, enabling the sorting of cells based on their image information. In this review, we specifically highlight practical applications of IFC in four representative areas: cell cycle analysis, protein localization analysis, immunological synapse formation, and the detection of leukemic cells. In addition, particular emphasis is placed on applications that directly contribute to elucidating molecular mechanisms, thereby distinguishing this review from previous general overviews of IFC. IFC enables the estimation of cell cycle phases from large numbers of acquired cellular images using machine learning, thereby allowing more precise cell cycle analysis. Moreover, IFC has been applied to investigate intracellular survival and differentiation signals triggered by external stimuli, to monitor DNA damage responses such as γH2AX foci formation, and more recently, to detect immune synapse formation among interacting cells within large populations and to analyze these interactions at the molecular level. In hematological malignancies, IFC combined with fluorescence in situ hybridization (FISH) enables high-throughput detection of chromosomal abnormalities, such as BCR-ABL1 translocations. These advances demonstrate that IFC provides not only morphological and functional insights but also clinically relevant genomic information at the single-cell level. By summarizing these unique applications, this review aims to complement existing publications and provide researchers with practical insights into how IFC can be implemented in both basic and translational research. Full article

The disruption of the epigenetic mechanisms of gene expression regulation due to the emergence of pathogenic variants in genes-encoding elements of epigenetic machinery leads to the development of chromatinopathies. This group of hereditary diseases includes 179 syndromes, some of which present with overlapping phenotypes. Despite the variety of approaches to molecular diagnostics of chromatinopathies, it is not always possible to establish the molecular diagnosis by traditional methods; thus, the issue of optimizing diagnostic algorithms remains relevant. One of the most rapidly expanding areas of post-genomic molecular diagnostics is episignature detection, which relies on genome-wide DNA methylation analysis. This article aims to represent an original approach to indirect diagnostics of chromatinopathies on the example of Rubinstein–Taybi syndrome 1, which is based on the analysis of the methylation level of a limited set of loci designed to reproduce its classic episignature. In the current study, we apply two methods of targeted quantitative analysis of DNA methylation, which are relatively accessible and can be integrated into diagnostic practice. We demonstrate that Rubinstein–Taybi syndrome 1 episignature may be successfully reduced to a single locus of human genome, and that quantitative bisulfite DNA methylation analysis at this locus allows accurate identification of the Rubinstein–Taybi syndrome 1 patients. Full article

Background/Objectives: Detecting high-order epistatic interactions in genome-wide association studies (GWAS) is essential for understanding complex diseases, yet most existing approaches are limited to pairwise interactions. We propose GPBSO (Gene Pool-Based Brain Storm Optimization for Epistasis Detection), a novel stochastic framework that integrates Brain Storm Optimization with a dynamic gene pool to efficiently explore high-order SNP combinations. Methods: Epistasis is evaluated using the k2 Bayesian network scoring criterion and the G-test, with iterative updates to the gene matrix enhancing search diversity. Results: Comparative experiments on simulated datasets generated from five epistatic models demonstrated that GPBSO consistently outperformed a set of well-established methods—DECMDR, SNPHarvester, AntEpiSeeker, HS-MMGKG, and SEE—in terms of F-measure and statistical power, particularly for third-order interactions. Conclusions: GPBSO provides an effective and scalable approach for detecting high-order epistatic interactions, offering methodological advancements for genetic epidemiology and complex disease analysis. Full article

The Xinglong buffalo is a local swamp buffalo breed adapted to tropical regions in China. To facilitate the protection and utilization of valuable genetic resources, we first developed the breed-specific single nucleotide polymorphism (SNP) liquid-phase chip based on genotyping-by-target-sequencing (GBTS) technology. Whole-genome resequencing data from 143 buffaloes, resulting in 34,757,694 SNPs, were used to identify 1208 breed-specific and 2889 background sites. This chip also incorporates 965 functional SNP sites derived from literature, including SNPs significantly associated with immunity, reproduction, growth, and production. A total of 5062 SNP sites were successfully identified for the development of a 5K liquid-phase genome-wide breeding chip for the Xinglong buffalo. The validation of the chip using 93 samples showed a high detection rate with good repeatability and consistency. In addition, the chip exhibits strong capabilities in clustering and kinship analysis. Results of kinship analysis underscored the importance of a breed-specific chip for the Xinglong buffalo. These results highlight the advantages of a low-density, cost-effective, and breed-specific SNP chip for accurate genotyping. This chip will support future endeavors in molecular breeding, conservation, and genetic evaluation of Xinglong buffalo, thereby facilitating the sustainable utilization of this valuable indigenous germplasm resource. Full article

Chemosensory systems are fundamental for insects to regulate behaviors such as prey detection, oviposition, and pollination. Despite their importance, the molecular mechanisms underlying chemosensation remain poorly understood in many insect groups. Hoverflies (Syrphidae), whose larvae are efficient aphid predators and adults act as pollinators, represent a functionally important but understudied lineage. Building on the genome of Eupeodes corollae that we recently published, we selected this dominant and widespread species as a representative model and performed a genome-wide identification and analysis of odorant-binding proteins (OBPs) to provide a molecular foundation for understanding chemosensory recognition mechanisms. Accordingly, a total of 47 OBPs were identified and classified into Classic, Minus-C, and Plus-C subfamilies, with conserved motifs and structural features observed within each group. Next, phylogenetic analysis revealed that several EcorOBPs are homologous to functionally characterized OBPs in other Diptera, suggesting conserved evolutionary roles. Moreover, chromosomal mapping showed that Minus-C EcorOBPs cluster on chromosome 2, and Ka/Ks analysis indicated strong purifying selection, reflecting evolutionary stability. In addition, synteny analysis demonstrated that E. corollae shares more collinear OBP gene pairs with predatory hoverflies (Episyrphus balteatus and Scaeva pyrastri) than with the saprophagous species Eristalis tenax, consistent with ecological divergence. Finally, transcriptomic profiling revealed tissue-specific expression patterns, including antennal-biased EcorOBP1 linked to olfaction and reproductive tissue-biased EcorOBP11 linked to reproduction, highlighting candidate genes for functional studies. Together, these findings provide a comprehensive characterization of OBPs in E. corollae and offer molecular insights into chemosensory mechanisms that support both pest control and pollination services. Full article

Copaifera langsdorffii is a neotropical tree widely distributed in the Brazilian Atlantic Forest and Brazilian Savanna. Population genetic analyses can identify the scale at which tree species are impacted by human activities and provide useful demographic information for management and conservation. Using a Restriction site Associated DNA Sequencing approach, we assessed the genomic variability of six C. langsdorffii population relicts in a transition zone between the Seasonal Atlantic Forest and Savanna biomes in Southeastern Brazil. We identified 2797 high-confidence SNP markers from six remnant populations, with 10 to 29 individuals perpopulation, in a transition zone between the Seasonal Atlantic Forest and Savanna biomes in Southeastern Brazil. Observed heterozygosity values (0.197) were lower than expected heterozygosity (0.264) in all populations, indicating an excess of homozygotes. Differentiation among populations (F_ST) was low (0.023), but significant (0.007–0.044, c.i. 95%). A clear correlation was observed between geographic versus genetic distances, suggesting a pattern of isolation by distance. Bayesian inferences of population structure detected partial structuring due to the transition between the Atlantic Forest and the Brazilian Savanna, also suggested by spatial interpolation of ancestry coefficients. Through the analysis of F_ST outliers, 28 candidates for selection have been identified and may be associated with adaptation to these different phytophysiognomies. We conclude that the genetic variation found in these populations can be exploited in programs for the genetic conservation of the species. Full article

Mitochondria are essential organelles for cellular energy production, playing a central role in driving metabolic processes and supporting critical intracellular functions. Neurometabolic disorders encompass a wide variety of conditions characterized by mitochondrial dysfunction. Owing to their bacterial ancestry, mitochondria possess an independent genome consisting of a circular DNA molecule (mtDNA), which has been reported to be subject to methylation. However, the technical challenges in the detection of mtDNA methylation have led to debates on its existence. One of the concerns is that the compactness of mtDNA can lead to suboptimal bisulfite conversion, thereby causing mtDNA methylation overestimation. To address this, liquid chromatography tandem mass spectrometry (LC-MS/MS) offers a bisulfite-independent readout; however, this method requires mtDNA samples devoid of nuclear DNA (nDNA) contamination. To diminish nDNA contamination, we isolated mtDNA from the TRIzol RNA phase. Importantly, pyrosequencing showed no significant difference in the methylation levels of mtDNA isolated from the TRIzol RNA phase compared to those from the TRIzol DNA phase, or isolated via total genomic DNA (gDNA). Across different human cell lines, LC-MS/MS detected significantly lower global methylation levels for DNA isolated from the TRIzol RNA phase than those from the TRIzol DNA or gDNA isolation. Moreover, using mtDNA isolated from the TRIzol RNA phase, LC-MS/MS validated the enhanced mtDNA methylation in HepG2 transgenic cell lines expressing mitochondrial-targeted DNA methyltransferases (means of 2.89% and 2.03% for MCviPI and MSssI transgenic cell lines, respectively), compared to two negative control cell lines (1.36 and 1.39%). When applying it to clinically relevant material, LC-MS/MS demonstrated a significantly lower global methylation level for platelet DNA isolated from the TRIzol RNA phase (mean of 1.98%) compared to gDNA isolations (mean of 4.32%). Similar findings were confirmed in mouse brain tissue, in which a significantly lower methylation level was detected in DNA isolated from the TRIzol RNA phase (1.79%) compared to that from gDNA isolation (5.12%). In conclusion, isolating mtDNA from the TRIzol RNA phase holds significant potential in future studies, particularly for the quantification of mtDNA global methylation by LC-MS/MS, a technique that is independent of bisulfite conversion and bioinformatic analysis. Full article

Improving reproductive efficiency in beef cattle remains a key objective for sustainable genetic progress, particularly in extensively managed autochthonous breeds such as the Spanish Retinta. In this study, we applied a weighted single-step genome-wide association approach (wssGWAS) to identify genomic regions associated with four fertility-related traits: age at first calving (AFC), interval between first and second calving (IC12), average calving interval (ACI), and reproductive efficiency (RE). A total of 215,125 calving records from 44,032 cows and the genomic information of 1030 animals (Axiom™ Bovine Genotyping v3 Array 65k) were analyzed. Heritability was estimated using a single-step genomic best linear unbiased prediction (ssGBLUP) that incorporated both pedigree and genomic data, and estimates ranged from 0.15 (0.008) for AFC to 0.27 (0.012) for ACI. The wssGWAS identified 96 1 Mb-windows explaining over 1% of additive genetic variance (40 of them are common for more than one trait and 46 windows are unique), notably on chromosomes 2 and 5. Candidate genes related to folliculogenesis, steroidogenesis, immune modulation, and cell cycle control were identified, including ACVR1B, AMHR2, CYP27B1, CDK2, and IFNG. Additionally, a significant proportion of lncRNAs were detected, suggesting regulatory roles in reproductive processes through the modulation of gene expression at different levels. These findings enhance our understanding of the genetic architecture underlying female fertility in beef cattle and provide valuable markers for incorporation into genomic selection programs aimed at improving reproductive performance and long-term sustainability in the Retinta breed. Full article