Search Results (348)

Forensic DNA profiling commonly relies on polymerase chain reaction (PCR) amplification followed by capillary electrophoresis (CE) or massively parallel sequencing (MPS), which requires expensive, laboratory-based equipment that depends on a stable power supply and is unsuitable for field applications. Here, we present a proof-of-concept assay that uses recombinase polymerase amplification (RPA) combined with exo probe detection for rapid, isothermal genotyping of insertion–deletion (InDel) markers. To the best of our knowledge, this study represents the first demonstration of forensic DNA typing using RPA coupled with exo probes. The reaction proceeds at 39 °C and combines amplification and detection in a single 20 min step. Thirteen DNA samples were genotyped in triplicate across eight InDel loci using allele-specific fluorescent probes. Genotypes were derived from differential endpoint fluorescence between matched and mismatched probes. Compared with benchmark genotyping, 97.07% of genotypes (n = 307) were correct at 1 ng DNA input. Accurate profiles were reliably obtained for DNA inputs as low as 250 pg, and partial profiles were still detectable at 31 pg. The results demonstrate that RPA-based InDel genotyping is fast, sensitive, and reproducible. With further optimization, such as refined probe design and selection of robust loci, the assay has clear potential to achieve complete accuracy and to be integrated into portable lab-on-a-chip platforms for rapid, field-deployable forensic identification. Full article

Background/Objectives: Cell-free DNA (cfDNA) is a valuable biomarker for cancer diagnosis and therapy monitoring; however, its low abundance and fragmented nature present major challenges for reliable isolation, particularly from limited plasma volumes. Here, we report the development and evaluation of a novel magnetically assisted microfluidic chip with a three-inlet design for efficient cfDNA extraction from small-volume plasma samples. Methods: The platform enables controlled infusion of plasma, lysis buffer, and magnetic nanoparticle suspensions at defined flow rates. An external magnetic field selectively captures cfDNA-bound nanoparticles while efficiently removing background impurities. Results: Direct comparison with two in vitro diagnostic (IVD)-certified commercial cfDNA extraction kits showed that the microfluidic system achieved comparable cfDNA yields at standard plasma volumes and superior performance at reduced input volumes. High DNA purity and integrity were confirmed by quantitative PCR amplification of a housekeeping gene and clinically relevant targets. The complete workflow required approximately 9 min, used minimal equipment, reduced contamination risk, and enabled rapid processing with future potential for parallel multi-chip configurations. Conclusions: These findings establish the proposed microfluidic platform as a rapid, reproducible, and scalable alternative to conventional cfDNA extraction methods. By significantly improving recovery efficiency from small plasma volumes, the system enhances the clinical feasibility of liquid biopsy applications in cancer diagnostics and precision medicine. Full article

Improving wheat processing quality is a crucial objective in modern wheat breeding. Among various quality parameters, grain protein content (GPC) and wet gluten content (WGC) significantly influence the end-use quality of flour. These traits are controlled by multiple minor effect genes and highly influenced by environmental factors. Identifying stable and major-effect genetic loci and developing breeder-friendly molecular markers are of great significance for breeding high-quality wheat varieties. In this study, we evaluated the GPC and WGC of 310 diverse wheat varieties, mainly from China and Europe, across four environments. Genotyping was performed using the wheat 100K SNP chip, and genome-wide association analysis (GWAS) was employed to identify stable loci with substantial effects. In total, four loci for GPC were identified on chromosomes 1A, 3A, 3B, and 4B, with explained phenotypic variation (PVE) ranging from 6.0 to 8.4%. In addition, three loci for WGC were identified on chromosomes 4B, 5A, and 5D, which explained 7.0–10.0% of the PVE. Among these, three loci coincided with known genes or quantitative trait loci (QTL), whereas QGPC.zaas-3AL, QGPC.zaas-4BL, QWGC.zaas-4BL, and QWGC.zaas-5A were potentially novel. Seven candidate genes were involved in various biological pathways, including growth, development, and signal transduction. Furthermore, five kompetitive allele specific PCR (KASP) markers were developed and validated in a natural population. The newly identified loci and validated KASP markers can be utilized for quality improvement. This research provides valuable germplasm, novel loci, and validated markers for high-quality wheat breeding. Full article

Polymerase chain reaction (PCR) is vital in biological and medical research, but microfluidic PCR chips often suffer from limited reagent processing capacity and slow thermal response under high flow rates. To address this, we designed three serpentine microfluidic chips with double-sided heaters: a standard serpentine chip (case 1), one with unchamfered channel expansion areas (case 2), and one with chamfered expansions (case 3). Using numerical simulations, we analyzed temperature, velocity, and pressure distributions at flow rates of 75, 125, and 175 μL/min. At 175 μL/min, case 2 showed a 41% higher pressure drop than case 1, but also demonstrated significantly improved thermal performance: the constant-temperature zones were extended by 30 mm, 10 mm, and 30 mm at 95 °C, 72 °C, and 55 °C, respectively; the temperature gradient in expansion zones increased by 1.6 times; and the maximum temperature difference decreased by 80%. Case 2 achieved the best trade-off between thermal performance and flow resistance, making it suitable for high-flow-rate PCR applications. Full article

Background/Objectives: Antimicrobial resistance (AMR) represents a major global health challenge, driving the need for rapid and accurate diagnostic tools. Novel molecular assays, including multiplex PCR and DNA microarray-based systems, have emerged to detect antimicrobial resistance genes (ARGs) alongside bacterial identification. Methods: In this study, we evaluated the performance of the HybriSpot12 PCR AUTO (HS12a) system and the MDR Direct Flow Chip (MDR-FC) Kit—an automatic microarray assay based on reverse hybridization—for the detection of ARGs directly from positive blood culture (PBC) samples. A total of 111 Gram-negative bacterial isolates (92 Enterobacterales, 14 Acinetobacter baumannii, and 6 Pseudomonas spp.), previously characterized by whole-genome sequencing (WGS), were each used to generate a PBC, which was then analyzed with the HS12a/MDR-FC assay. Results: We demonstrated perfect agreement for the detection of macrolide resistance genes across all bacterial species and high agreement for genes conferring resistance to sulfonamides and β-lactams. In contrast, aminoglycoside resistance genes showed only moderate agreement, with minor discrepancies observed in Klebsiella pneumoniae and Escherichia coli, largely attributable to specific SNP variations. Conclusions: The HS12a/MDR-FC assay includes 51 ARGs, though not all were represented in our isolate set, and some false negatives were observed. Despite these limitations, its broad coverage and rapid turnaround remain advantageous compared to other rapid assays with fewer targets. Future refinements should aim at broader gene coverage, inclusion of key mutations, and detection of emerging variants, making this approach a promising tool for rapid AMR surveillance and antimicrobial stewardship. Full article

Growth and carcass traits are key economic traits in beef cattle production, and identifying their associated genetic markers is crucial for improving breeding efficiency. Charolais cattle, as a superior beef breed, exhibit excellent performance in growth rate and meat production. The aim of this study was to utilize the preferred high-coverage whole-genome resequencing (hcWGS) as a replacement for single nucleotide polymorphism (SNP) chips to identify significant SNPs and candidate genes associated with growth (body weight, body height, cross height, body length, and chest measurement across different growth stages) and carcass traits (live backfat thickness and eye muscle area at 18 months) in 240 Charolais cattle, thereby providing guidance for beef cattle breeding. Through hcWGS (approximately 13× coverage) and quality control, 4,088,633 SNPs were identified and subsequently used for genetic analyses. Through FarmCPU-based genome-wide association studies, 196 potentially significant SNPs associated with growth traits and 29 SNPs with carcass traits were identified. Annotation analyses revealed 353 candidate genes (such as RBM33, KCTD17, PTHLH, RAC2, CHD6, TRDN, WBP1L, TLL2, CH25H, and ST13) linked to growth traits and 26 candidate genes linked to carcass traits (such as CHST11, LRRK2, RIOK2, and INTS10). Additionally, three SNPs (g.8674692C>G, g.54418624G>T, and g.71085551G>A) were validated via polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), enabling efficient marker-assisted selection. Furthermore, eight SNPs in the Acyl-CoA oxidase 1 (ACOX1) gene were found to be associated with growth and backfat thickness traits. These findings provide valuable preliminary insights into the genetic mechanisms underlying growth and carcass traits in Charolais cattle, facilitating genome-assisted breeding. Full article

Background and Clinical Significance: Mastocytosis and mast cell activation syndrome (MCAS) include conditions in which patients manifest signs, symptoms, and laboratory findings consistent with mast cell activation and can only be diagnosed in the presence of specific criteria. Mutations of ZRSR2, a gene involved in RNA splicing, are not closely associated with mast cell disorders, but rather with myelodysplastic syndromes development. Case Presentation: We report a case of a 37-year-old man who was referred to our institution for anaphylaxis after a bee sting and elevated serum tryptase levels (17.8 ng/mL in the first sample and 19.2 ng/mL in the second sample). Complete blood count was unremarkable. Bone marrow biopsy showed signs of dysplasia and some CD25+ mast cells. ASO-qPCR and targeted myeloid NGS analysis did not detect the KIT p.D816V mutation, but rather showed the presence of a pathogenetic variant of the ZRSR2 gene (p.S447_R448del) with a variant allele frequency of 7.4%. Mastocytosis could not be diagnosed based on the established diagnostic criteria. The patient’s symptoms were not recurrent and tryptase release was not event-related; therefore, a diagnosis of MCAS could not be made either. Taken together, these findings led to the diagnosis of clonal hematopoiesis of indeterminate potential (CHIP). A watch and wait strategy consisting of clinical evaluations, blood tests, and cardiovascular risk assessment was initiated. Conclusions: This case report highlights the importance of combining clinical and laboratory findings, hematopathology, and molecular analyses to establish the most probable diagnosis in challenging cases. It also underscores the possible relevance of identifying predisposing conditions, such as CHIP, in order to guide counseling and follow-up strategy. Full article

The cultivated potato (Solanum tuberosum L.) is a globally important crop with a narrow genetic pool, making it vulnerable to biotic and abiotic stresses. The present study analyzed the relative content of the nuclear, mitochondrial, and plastid genomes and their contributions to agronomic traits in 30 diploid interspecific potato hybrids with diverse cytoplasmic types and pedigrees. The nuclear genome size (2C-value) was estimated using flow cytometry, while the organelle DNA content and cytoplasm types were determined by quantitative polymerase chain reaction (qPCR) and multiplex PCR, respectively. The genome size of individual diploid genotypes remained stable across cultivation conditions, such as in vitro or greenhouse environments. Significant variation was observed in genome size, organelle content, and cytoplasmic types, which were associated with differences in pollen fertility and starch content. Kendall’s correlation analysis revealed a strong positive correlation between the content of plastid and mitochondrial DNA, and between starch content and chip colour after cold storage. Principal component analysis (PCA) demonstrated that variation in plastid and mitochondrial DNA content explained differences among genotypes, with nuclear DNA content contributing independently. Notably, cytoplasmic male sterility was observed in some T-type cytoplasm genotypes, thus highlighting the role of nuclear–cytoplasmic interactions. The results obtained demonstrate that organelle genome composition exerts a significant influence on agronomic traits and offer valuable insights into the potential for the enhancement of potato breeding programmes through the analysis of cytoplasm and nuclear genomes. Full article

The master transcription factors that control cell adaptation under hypoxia are known as hypoxia-inducible factors or HIFs. HIF-2α is the second isoform, which has been studied less extensively, and its expression is limited to particular cell types and is associated with increased malignancy in tumors. Herein, we investigate the interaction of HIF-2α with Ataxin-10, an intracellular protein involved in cell survival and differentiation, as well as the mechanism and the effects of this interaction in cervical cancer (HeLa) and glioma (U-87MG) cells. The interaction was investigated by LC-MS/MS proteomic analysis, immunoprecipitation, and immunoblotting. HIF-2 transcriptional activity was measured by luciferase assays and quantitative RT-PCR of target genes specific to HIF-2. The mechanism of interaction was investigated using immunofluorescence microscopy analysis, subcellular fractionation, siRNA-mediated silencing, quantitative RT-PCR, in vitro binding assays, and chromatin immunoprecipitation (ChIP). Ataxin interacts specifically with HIF2α and binds to the HIF-2α carboxyterminal activation domain. The interaction of HIF-2α with Ataxin-10 increases HIF-2-transcriptional activity under hypoxia through the enhancement of HIF-2α binding to chromatin in Hypoxia Response Elements of HIF-2 specific target genes SERPINE1, CITED-2, and SOD-2. These new data highlight a novel HIF-2 fine-tuning mechanism and may offer new, effective therapeutic approaches for treating cancerous tumors. Full article

As synthetic biology advances toward precise design, the construction of high-quality mutant libraries has become essential for large-scale functional screening. Traditional approaches, such as random and saturation mutagenesis, often suffer from low accuracy, high bias, and limited coverage. An ideal method should offer controlled mutagenesis, comprehensive coverage, high throughput, operational simplicity, and controllable outcomes, enabling effective large-scale screening. Here, we developed a high-throughput, precisely controlled method for constructing a mutagenesis library based on chip-based oligonucleotide synthesis. Using PSMD10 as a model, we constructed a full-length amber codon scanning mutagenesis library with 93.75% mutation coverage. Among the five polymerases evaluated, KAPA HiFi HotStart, Platinum SuperFi II and Hot-Start Pfu DNA Polymerase demonstrated higher amplification efficiency and lower chimera formation rates, making them preferred enzymes for optimized library construction. Analysis of unmapped reads highlighted key technical factors, such as oligonucleotide synthesis errors and chimeric sequence formation caused by incomplete extension of DNA polymerase or synthesis across discontinuous templates during PCR. To improve efficiency and fidelity, we recommend refining PCR conditions and strengthening oligo synthesis quality control. We establish an efficient, scalable, precisely controlled mutagenesis library construction strategy tailored for high-throughput functional research and recommend using a high-fidelity, low-bias polymerase to ensure quality. Full article

Aflatoxins, which are potentially genotoxic and carcinogenic substances, are mainly produced by the Aspergillus section Flavi, including Aspergillus flavus and A. parasiticus. Current Aspergillus spp. detection is often based on molecular methods, such as real-time PCR and loop-mediated isothermal amplification (LAMP), targeting genes of the aflatoxin biosynthetic cluster. In this study, we developed a Lab-on-a-Chip (LoC) method based on real-time PCR and on LAMP for the specific detection of aflatoxigenic strains of A. flavus and A. parasiticus from infected hazelnuts. LoC-LAMP and LoC-real-time PCR assays were tested in terms of specificity, sensitivity, speed, and repeatability. The microfluidic chip allowed quick, specific, sensitive, simple, automatized, cheap, and user-friendly detection of aflatoxigenic strains of A. flavus and A. parasiticus. The LoC-LAMP showed a limit of detection (LOD) of 10 fg of DNA, while the LoC-real-time PCR showed a LOD of 10 pg of DNA. Achieving comparable sensitivity to that of LAMP and real-time PCR techniques, both LoC methods developed in this work offer the advantages of automation, minimal sample requirements, reagent requirements, and cost-effectiveness. Overall, the developed methods open the perspective for alternative monitoring of aflatoxigenic fungi in the agri-food industry. Full article

Background: Cervical cancer remains a major global health burden, and treatment failure due to radioresistance and secondary drug resistance severely limits clinical outcomes. Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator implicated in tumor progression. This study aimed to determine whether EZH2-mediated PTEN silencing drives afatinib resistance via AKT activation in radiation-resistant cervical cancer cells. Methods: A radioresistant cervical cancer cell line (HeLaR) was established following cumulative irradiation (70 Gy). Cell viability, clonogenic survival, methylation-specific PCR (MSP), chromatin immunoprecipitation (ChIP), and Western blot analyses were conducted. EZH2 (Dznep; tazemetostat), PI3K, and AKT inhibitors were tested in combination with afatinib. A xenograft mouse model was used for in vivo validation. Results: HeLaR cells exhibited upregulation of EZH2 and H3K27me3, downregulation of PTEN, and sustained AKT activation. EZH2 inhibition restored PTEN expression, attenuated AKT phosphorylation, and re-sensitized cells to afatinib. MSP and ChIP confirmed EZH2-mediated PTEN promoter silencing. PI3K inhibition reproduced these effects, whereas ERK inhibition had minimal impact. In xenograft models, combined treatment with Dznep and afatinib significantly suppressed tumor growth compared to single agents. Conclusions: EZH2-driven PTEN suppression promotes AKT-dependent afatinib resistance in radiation-resistant cervical cancer. Targeting the EZH2–PTEN–AKT axis may provide a potential therapeutic approach to mitigate combined radioresistance and chemoresistance in recurrent cervical cancer, although further preclinical and clinical validation is required. Full article

Leaf rust, a devastating fungal disease caused by Puccinia triticina (Pt), severely impacts wheat quality and yield. Identifying genetic loci for wheat leaf rust resistance, developing molecular markers, and breeding resistant varieties is the most environmentally friendly and economical strategy for disease control. This study utilized a recombinant inbred line (RIL) population of Doumai and Shi4185, combined with the wheat 90 K single nucleotide polymorphisms (SNPs) chip data and maximum disease severity (MDS) of leaf rust from four environments, to identify adult plant resistance (APR) loci through linkage mapping. Additionally, kompetitive allele-specific PCR (KASP) markers suitable for breeding were developed, and genetic effects were validated in a natural population. In this study, 5 quantitative trait loci (QTL) on chromosomes 1B (2), 2A and 7B (2) were identified through inclusive composite interval mapping, and named as QLr.lfnu-1BL1, QLr.lfnu-1BL2, QLr.lfnu-2AL, QLr.lfnu-7BL1 and QLr.lfnu-7BL2, respectively, explaining 4.54–8.91% of the phenotypic variances. The resistance alleles of QLr.lfnu-1BL1 and QLr.lfnu-1BL2 originated from Doumai, while the resistance alleles of QLr.lfnu-2AL, QLr.lfnu-7BL1 and QLr.lfnu-7BL2 came from Shi4185. Among these, QLr.lfnu-1BL2, QLr.lfnu-7BL1 and QLr.lfnu-7BL2 overlapped with previously reported loci, whereas QLr.lfnu-1BL1 and QLr.lfnu-2AL are likely to be novel. Two KASP markers, QLr.lfnu-2AL and QLr.lfnu-7BL, were significantly associated with leaf rust resistance in a diverse panel of 150 wheat varieties mainly from China. Totally, 34 potential candidate genes encoded the NLR proteins, receptor-like kinases, signaling kinases and transcription factors were selected as candidate genes for the resistance loci. These findings will provide stable QTL, available breeding KASP markers and candidate genes, and will accelerate the progresses of wheat leaf rust resistance improvement through marker-assisted selection breeding. Full article

Obesity is a risk factor associated with cardiovascular diseases that may lead to heart failure (HF). However, in HF, overweight and obese patients have longer survival than underweight patients, a phenomenon known as the obesity paradox. MiRNAs play a fundamental role in gene regulation involved in obesity and HF. The main objective of this study was to identify and validate differentially expressed circulating miRNAs in HF–obese and HF–lean patients. This case–control study was carried out in two phases: discovery and validation. In the discovery phase, plasma samples from 20 HF patients and from 10 healthy controls were analyzed using the miRNA 4.0 Affymetrix GeneChip array. Differentially expressed miRNAs were ranked and selected for validation. In this phase, plasma miRNAs -451a, -22-3p, and -548ac from 80 patients and controls were analyzed by qPCR. Target analysis and functional enrichment analysis were performed. When comparing HF–lean and HF–obese groups compared to controls, miRNAs -451a and -22-3p were up-regulated in both discovery and validation phases, while -548ac was down-regulated in the discovery phase and up-regulated in the validation phase, indicating that miRNA changes are independent of obesity. These miRNAs regulate genes and different biological processes associated with metabolic, morphological, and functional outcomes. Full article

Background/Objective: Aberrant acetylation and methylation of histone and non-histone proteins contribute to carcinogenesis. Among non-histone proteins, wild-type (wt) p53 is particularly notable for the critical role that acetylation and methylation play in regulating its stability and function. Although with opposite outcomes, these post-translational modifications (PTMs) can also affect mutant forms of p53 (mutp53), which are frequently detected in cancers. These proteins may acquire oncogenic properties, activating signaling pathways that promote carcinogenesis. Acetylation activates wtp53, while this PTM has been shown to destabilize mutp53, reducing cancer aggressiveness and improving the efficacy of anticancer therapies. In this study, we investigated the possibility of targeting mutp53 in pancreatic cancer cells by using a combination of EZH2 and HDAC inhibitors. Methods: Western blotting, qRT-PCR, and ChIP experiments were performed to address this question. Results: We found that the EZH2 inhibitor Valemetostat (DS) in combination with the histone deacetylase inhibitor SAHA displaced the SET/TAF-Iβ oncoprotein from mutp53 and increased its interaction with the acetyltransferase p300, which was responsible for p53 acetylation. Moreover, mutp53 was downregulated, p21 was upregulated, and CHK1 was reduced, increasing DNA damage and leading to a stronger impairment of pancreatic cancer cell survival compared with single-agent treatments. Conclusions: Our results reveal that combining epigenetic drugs such as Valemetostat and SAHA could be exploited to target mutp53 and improve the outcome of treatments for aggressive tumors harboring it, such as in pancreatic cancer. Full article