Search Results (2,242)

To dissect the molecular mechanisms underlying chicken cecal development, this study used Liangshan Yanying chickens (a local slow-growing breed) and Arbor Acres (AA) chickens (a fast-growing breed) as experimental models. Cecal tissues were collected from healthy chickens at 1, 14, and 28 days of age (n = 10 per breed per day of age) to measure cecal length and perform transcriptome sequencing. Through the screening of differentially expressed genes (DEGs), functional enrichment analysis, construction of protein–protein interaction (PPI) networks, and qRT-PCR validation, temporal changes in cecal development between the two breeds were systematically compared. Results showed that cecal length of both breeds increased significantly with age (p < 0.05), with significant differences between breeds. A total of 18 high-quality samples were obtained from transcriptome analysis (Q30 ≥ 93%), with a mapping efficiency of 86.2–90.5%. The number of DEGs was highest between 1 and 28 days of age (1844 DEGs in Liangshan Yanying chickens and 1747 DEGs in AA chickens), and the number of inter-breed DEGs reached 2133 at 28 days of age. A total of 70 DEGs with consistent expression trends were identified (22 upregulated and 48 downregulated), which were enriched in Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as “B cell activation”, “peptide transport”, and “bile acid metabolism”. qRT-PCR validation indicated that the expression trends of genes (e.g., CD79B, IRF4) were highly consistent with sequencing results (R² = 0.91). PPI network analysis suggested that SLC15A1, ACE, and ENPEP were key hub genes, forming a “transport–metabolism” synergistic module. This study reveals the temporal dynamics of chicken cecal development and the molecular basis of inter-breed differences, providing a theoretical foundation for broiler genetic improvement. 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

Background: Fungal infections are considered a global health concern, resulting in high morbidity and mortality rates, especially in immunocompromised individuals. Traditional diagnostic techniques, such as microscopy, culture, and polymerase chain reaction (PCR), suffer from low sensitivity, long processing time, and accessibility challenges, especially in resource-limited settings. Artificial intelligence (AI) and machine learning (ML) tools have demonstrated potential to enhance diagnostic accuracy and efficiency. This systematic study assesses the progress, precision, and efficacy of AI-driven diagnostic tools for fungal infections within various clinical contexts in comparison to traditional procedures. Methods: A systematic review was conducted according to PRISMA principles. Literature searches were conducted in PubMed, ScienceDirect, Web of Science, and Ovid, focusing on research employing AI models to diagnose fungal infections. The inclusion criteria were research that compared AI-based tools with conventional diagnostic methods in terms of sensitivity, specificity, and accuracy. Data extraction and quality evaluation were performed utilizing validated instruments, such as the Methodological Index for Non-Randomized Studies (MINORS). Results: Eleven research studies met the inclusion criteria: six retrospective and five prospective investigations. AI models, such as convolutional neural networks (CNNs), Faster R-CNN, VGG19, and MobileNet, have improved diagnostic accuracy, sensitivity, and specificity compared to traditional methods. However, differences in dataset quality, model validation, and real-world applicability remain as limitations. Conclusions: AI-driven diagnostic technologies provide significant benefits in identifying fungal infections, improving the speed and accuracy of diagnoses. However, additional extensive investigations and clinical validation are required to improve model generalizability and facilitate smooth incorporation into healthcare systems. Full article

CRISPR-Cas9 technology has opened new perspectives in genome editing of clonally, asexually propagated and polyploid plants by enabling multiple allelic gene edits. Traditional Agrobacterium- and particle bombardment-mediated transformations, which rely on integration of gene-editing transgene cassettes, have been efficiently applied to several plants; however, concerns about the acceptability of resultant edited transgenic genotypes make these methods less attractive for vegetatively propagated crops. We leveraged and optimized the CRISPR-Cas9/sgRNA-RNPs system for delivery into protoplasts of the hexaploid sweetpotato cultivar PI-318846, targeting eukaryotic translation initiation factor isoform 4E genes to enhance resistance to SPFMV potyviruses. To evaluate the efficiency of pre-assembled Cas9/sgRNA-RNP in sweetpotato transfection, single guide RNAs were designed to target putative host susceptibility genes: IbeIF4E, IbeIF(iso)4E, and IbCBP. Freshly isolated leaf protoplasts were subjected to CRISPR-CAS9-RNP PEG-mediated transfection under different parameters. Sweetpotato regenerants screened using PCR-RE-T7 assay, sequencing, and Inference CRISPR Edit analyses of target-site amplicons revealed the most efficient editing conditions utilizing 25% PEG with a 3:1 (15 µg:45 µg) ratio of Cas9/sgRNA-RNP for 25 min and 48 h incubation period. Different allelic InDels were obtained with editing efficiencies of 10–20% in regenerated plantlets, demonstrating that PEG-mediated CRISPR-RNP transfection system is key for advancing DNA-free editing tools in polyploid and vegetatively propagated crops. Full article

The polymorphism of the bovine β-casein gene (CSN2) is of increasing interest due to its relevance for A2 milk production. This study genotyped 2773 Holstein-Friesian cows for five CSN2 alleles (A1, A2, A3, B, I) using both conventional DNA-based PCR and a newly evaluated direct PCR protocol. Eleven genotypes were detected, with A2/A2 (33.9%) and A1/A2 (30.3%) being the most common, resulting in an A2 allele frequency of 59.0%. Genetic diversity indices indicated moderate polymorphism and a significant deviation from Hardy–Weinberg equilibrium, consistent with ongoing selection for the A2 allele. Associations between CSN2 genotype and milk traits (305-day milk, fat, and protein yield; fat% and protein%) were evaluated using linear mixed-effects models including lactation number, age at calving, and calving year as covariates, and cow ID as a random intercept. Several genotype effects reached statistical significance (p < 0.05); however, all effect sizes were very small (partial η² < 0.01), indicating that any influence of CSN2 on production traits is negligible within this population and management context. These findings suggest that A2-oriented selection is unlikely to compromise productivity. The direct PCR genotyping method achieved 96–100% success and enabled substantially faster and more cost-efficient processing (approximately 80–90% reduction in reagent costs), providing a rapid and scalable approach for large herds. Full article

Accurate genotyping of small insertions and deletions (InDels; <5 bp) remains technically challenging in routine molecular breeding, largely due to the limited resolution of agarose gel electrophoresis and the labor-intensive nature of polyacrylamide-based assays. Here, we present the Tri-Primer Amplification Refractory Mutation System (TP-ARMS), a simple and cost-effective PCR-based strategy that enables high-resolution genotyping of small InDels using standard agarose gels. The TP-ARMS employs a universal reverse primer in combination with two allele-specific forward primers targeting insertion and deletion alleles, respectively. This design allows clear discrimination of homozygous and heterozygous genotypes using a two-tube PCR workflow. The method showed complete concordance with Sanger sequencing in detecting 1–5 bp InDels across multiple crop species, including rice (Oryza sativa) and quinoa (Chenopodium quinoa). In addition, using a TP-ARMS reduced experimental time by approximately 90% compared with PAGE-based approaches and avoided the high equipment and DNA quality requirements of fluorescence-based assays. The practical applicability of the TP-ARMS was demonstrated in breeding populations, including efficient genotyping of a 3-bp InDel in OsNRAMP5 associated with cadmium accumulation and a 6-bp promoter InDel in OsSPL10 underlying natural variation in rice trichome density across 370 accessions. Collectively, the TP-ARMS provides a robust, scalable, and low-cost solution for precise small InDel genotyping, with broad applicability in marker-assisted breeding and functional genetic studies. Full article

Sugarcane (Saccharum spp. L.) is a globally vital sugar and energy crop whose genetic improvement has been constrained by its complex polyploid–allopolyploid genome. To address this limitation, we developed a practical, high-throughput single-nucleotide polymorphism (SNP) genotyping system. Using specific-locus amplified fragment sequencing (SLAF-seq) on 107 diverse accessions, we identified 2,420,550 high-quality SNPs anchored to the Saccharum officinarum LA-Purple genome. Stringent filtering yielded 55,750 SNPs for population analysis, which revealed three distinct genetic groups consistent with breeding history and adaptation. From these resources, 329 SNPs were converted into PCR-based ligase detection reaction (PCR-LDR) markers, resulting in a validated panel of 177 highly reliable SNPs (151 core and 26 extended) organized into an efficient multiplex typing system. The panel exhibited exceptional discriminatory power, successfully distinguishing all 303 tested sugarcane varieties and clearly resolving 186 individuals from three segregated hybrid populations. Compared to existing SSR and SNaPshot platforms, this SNP system offers superior experimental reproducibility, enhanced varietal clustering, and broader genome coverage. This work provides a robust, efficient genotyping tool to advance sugarcane variety identification, germplasm management, pedigree analysis, and marker-assisted breeding, with potential applicability to other complex polyploid crops. Full article

Background: Sheep (Ovis aries) tail fat serves as a crucial energy reserve for adapting to harsh environments. However, excessive deposition can reduce farming efficiency and product quality. Elucidating the regulatory mechanisms of tail fat deposition is of great significance for genetic improvement in sheep. Methods: In this study, transcriptome sequencing was conducted on tail fat tissues from fat-tailed Kazakh sheep (KAZ), thin-tailed Suffolk sheep (SFK), and their F2 hybrid sheep (CSH) (3 individuals per group). Subsequently, qRT-PCR validation, Enrichr, and KEGG database analyses were performed to investigate the molecular pathways involved in tail fat deposition. Results: High-quality clean reads were obtained from sequencing, with a genome alignment rate ranging from 76.15% to 79.43% and good data reproducibility. Differential expression analysis revealed multiple differentially expressed genes (DEGs) between KAZ and CSH groups, KAZ and SFK groups, as well as SFK and CSH groups. Five core candidate genes (BDH1, EPHX1, BCAT2, FASN, ACACA) were identified, all enriched in the fatty acid synthesis pathway and highly expressed in fat-tailed sheep, which was confirmed by qRT-PCR. Additionally, 189 lncRNAs were identified to collectively regulate target genes (e.g., FABP family, AGPAT2), along with three common differentially expressed miRNAs (novel_120, novel_171, novel_440) targeting genes enriched in lipid transport and lipid droplet formation pathways. Conclusions: This study confirms that the lncRNA-mRNA-miRNA regulatory axis is a key pathway in tail fat formation, providing important theoretical support and molecular targets for genetic improvement of ovine tail fat deposition traits. Full article

Bacillus cereus is a common Gram-positive bacterium that poses a significant threat to food safety due to its environmental adaptability, spore-forming ability, and production of harmful toxins. Traditional detection methods for B. cereus are time-consuming and inaccurate. This study aimed to develop a rapid enrichment medium for B. cereus to improve detection efficiency. Five B. cereus strains and five non-B. cereus strains were used. The TSB medium was selected as the basic medium as it supported the best growth and spore germination of B. cereus among the tested media. Magnesium sulfate and inosine were identified as the most effective promoters for the growth of vegetative cells and spore germination respectively, while glycine and sodium nitrite were chosen as suitable inhibitors against non-B. cereus bacteria. Through orthogonal experiments, the optimal formulation of the rapid enrichment medium (BC-TSB) was determined. BC-TSB effectively inhibited the growth of non-target bacteria and significantly promoted the growth and spore germination of B. cereus compared to the TSB basic medium. It also efficiently facilitated the recovery of thermally injured B. cereus cells, with a 6 h recovery rate of 87%—shortening the incubation time required by traditional method from 48 h to 6 h. In the detection of artificially contaminated dairy samples, BC-TSB could effectively pre-enrich B. cereus, achieving a 100% detection rate in UHT milk, modified milk, and pasteurized milk using both traditional and PMAxx-qPCR methods. Overall, the developed BC-TSB medium has great potential for the rapid and accurate detection of B. cereus in food, which can help enhance food safety monitoring. Full article

Dilated cardiomyopathy (DCM) is a major contributor to heart failure and cardiac transplantation. This study investigated the metabolic potential of human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and subsequently cardiac sphere-derived cells (SDCs) obtained from healthy (non-dilated) and pathological (dilated) myocardial tissues. hmMSCs were isolated using the explant outgrowth method and expanded in monolayer culture. Small round cells loosely attached on hmMSCs were harvested and cultivated as cardiac spheroids for 1–3 days, subsequently obtaining SDCs. The cell morphology, proliferation rate, mitochondrial activity, and intracellular calcium levels were analyzed using flow cytometry, Seahorse metabolic assays, and spectrophotometry, while expression of cell progenitor and cardiac commitment genes were analyzed by quantitative PCR. Both healthy and pathological SDCs demonstrated significantly enhanced mitochondrial function—reflected by increased maximal respiration, ATP production, and coupling efficiency—along with reduced steady-state intracellular calcium levels compared with hmMSCs. SDCs from both healthy and dilated myocardium showed marked upregulation of several cardiac progenitor and lineage-commitment genes relative to hmMSCs. SDCs derived from both healthy and dilated myocardiums possess a more favorable metabolic, progenitor and cardiac commitment profile than conventional hmMSCs. hmMSCs and SDCs from dilated myocardium retain residual metabolic potential, which may be further enhanced under 3D culture conditions. Full article

To address the technical bottlenecks commonly encountered with real-time quantitative PCR instruments, such as insufficient ramp rates and uneven chamber temperature distribution, this study proposes an innovative design scheme for a temperature control system that incorporates key hot air circulation and temperature field regulation technologies. By combining the PCR instruments’ working principles and structural characteristics, the failure mechanisms associated with the temperature control system are systematically analyzed, and a reliability-oriented thermodynamic analysis model is constructed to clarify the functional positioning of core components and to systematically test the airflow uniformity, temperature dynamics, and nucleic acid amplification efficiency. An integrated fixture for airflow rectifier and cruciform frames is designed, which enables precise quantitative characterization of the system temperature uniformity, ramp rates, and amplification efficiency on a multi-condition comparison platform. Through modeling analysis combined with experimental validation, the thermal performance differences among various heating chamber structures are compared, leading to a multidimensional optimization of the temperature control system. The test results demonstrate outstanding core performance metrics for the optimized system: the up ramp reaches 7.5 ± 0.1 °C/s, the down ramp reaches 13.5 ± 0.1 °C/s, and the steady-state temperature deviation is only ±0.1 °C. The total duration for 35 PCR cycles is recorded at 16.3 ± 0.6 min, with a nucleic acid amplification efficiency of 98.9 ± 0.2%. The core performance metrics comprehensively surpass those of mainstream global counterparts. The developed temperature control system is well-suited for practical applications such as rapid detection, providing critical technological support for the iterative upgrade of nucleic acid amplification techniques while laying a solid foundation for the engineering development of high-performance PCR instruments. Full article

Molecular sex determination in Syrian hamsters (Mesocricetus auratus) has been limited by the incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene PRSSLY, which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved PRSSLY coding region produced a male-specific amplicon showing 100% concordance with results from the established KDM5C/KDM5D PCR assay in E15.5 tail biopsies. SYBR Green–based qPCR enables the accurate detection of PRSSLY, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Application of the PRSSLY assay to 417 placental samples from 39 dams demonstrated its suitability for large-scale sex genotyping, enabling sex assignment in the majority of samples despite the intrinsic complexity of placental tissue containing both maternal and embryonic genetic material. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters. Full article

In this work, we evaluate the efficiency of a DNA purification protocol from gynecological samples using locally synthesized Fe₃O₄@SiO₂ magnetic microparticles and a low-cost, guanidinium thiocyanate (GITC)-free lysis buffer. The microparticles were characterized by SEM, EDS, FTIR, and magnetic measurements, confirming the formation of compact silica-coated aggregates with suitable magnetic responsiveness for rapid and complete capture. Using this material in combination with a simple, GITC-free lysis buffer, we achieved DNA extraction yields comparable to those obtained with standard methods based on chaotropic salts. The purified DNA showed high compatibility with molecular assays for the detection of Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, and human papilloma virus. Clinical validation demonstrated excellent diagnostic performance, with only a few discrepancies observed in samples near the detection threshold of qPCR, a limitation shared with commercial kits. Overall, the method represents a low-cost, safe, and sustainable alternative for routine clinical and epidemiological applications, compared to methods based on chaotropic salt buffers. Furthermore, it reduces reliance on imported commercial consumables and minimizes the handling of hazardous reagents. Full article

Duck plague virus (DPV), a highly contagious α-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg/μL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings. Full article

Porcine reproductive and respiratory syndrome (PRRS) is one of the costliest diseases in swine production, causing >$1.2 billion USD in annual losses in the United States. Oral fluids are widely used for PRRS virus (PRRSV) surveillance, accounting for 42% of nearly 480,000 PRRSV RT-qPCR cases submitted to six Midwestern U.S. laboratories between 2020 and 2025. Despite this reliance, few studies have applied appropriate methodological approaches to compare the performance of commercial extraction and PRRSV RT-qPCR protocols for oral fluid specimens. In this study, we evaluated nine extraction-amplification protocols for PRRSV RNA detection, based on three commercial extraction kits and three commercial RT-qPCR assays. For each protocol, performance was evaluated using 314 oral fluid samples of known status (215 positive, 99 negative), collected under controlled conditions from 72 pigs assigned to five groups inoculated with contemporary PRRSV isolates and from one negative control group. The Cq values were normalized as efficiency-standardized Cqs (ECqs) and then analyzed by receiver operating characteristic (ROC) analysis. The mean amplification efficiencies ranged from 67 to 92%, repeatability from 0.98 to 0.99, and overall reproducibility was 0.91. The ROC AUCs ranged from 0.916 to 0.986, with significant pairwise differences (p < 0.05). At optimal ECq cutoffs, sensitivities ranged from 83 to 98.1% with 100% specificity. Normalization enabled objective protocol comparisons and statistically valid diagnostic cutoffs and supports future improvements in PRRSV diagnostics. Full article