Search Results (354)

Background/Objectives: Operational DNA databases traditionally rely on static locus-count thresholds to determine search eligibility and report matches. While computationally straightforward, these rigid criteria routinely discard high-value investigative leads from degraded forensic profiles while simultaneously permitting adventitious matches when common alleles are involved. To overcome the limitations of static rules, this study introduces an automated framework for dynamic likelihood ratio (LR) thresholding. Methods: Utilizing a Fast Fourier Transform (FFT) algorithm, the system calculates the Probability Mass Function (PMF) for any specific combination of shared loci in real-time, natively incorporating the Balding–Nichols model to account for population substructure. Instead of applying an arbitrary locus count or fixed LR cutoff, the framework defines admissibility based on a user-defined maximum upper bound of acceptable false positives at a specified confidence (probability) level (e.g., 95%). Results: This empowers database custodians to precisely predict and adapt their search criteria to match an acceptable administrative workload, dynamically adjusting the required LR threshold to the exact size of the searched database. This approach was validated through massive-scale empirical simulations across five reference population groups. Receiver Operating Characteristic (ROC) and Poisson distribution analyses reveal that static thresholds inevitably collapse under the multiplicity effect of large-scale comparisons; for instance, a static locus rule that maintains safety within a small DNA database yields an unmanageable false positive risk when scaled to larger DNA databases or international networks like the Prüm DNA Exchange. Conclusions: By explicitly coupling the decision threshold to the database size and the genetic rarity of the evidence, this dynamic framework provides a mathematically rigorous and scalable solution. Most notably, it identifies rare, low-locus matches that static rules typically discard, offering a method to maintain a predefined expected false positive rate. Full article

In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 10⁰ copies/μL for H. influenzae and L. monocytogenes, and 10¹ copies/μL for C. neoformans using recombinant plasmids, as well as 10⁻⁷ to 10⁻⁶ ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted. Full article

Foodborne pathogens such as Klebsiella aerogenes pose a threat to food safety, highlighting the need for rapid, reliable detection methods amid rising contamination risks in production chains. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated to detect the histidine decarboxylase (HDC) gene of K. aerogenes. The assay was optimized for specificity and sensitivity, tested on pure bacterial genomic DNA and artificially contaminated food matrices (vegetables and meats), and evaluated against real-time PCR (qPCR). To evaluate performance under different DNA quality conditions and simulate laboratory versus on-site workflows, two extraction approaches were compared: a standard laboratory protocol yielding high-purity DNA and a crude extraction method producing low-purity DNA, mimicking the presence of inhibitors commonly encountered in routine analysis and enabling practical on-site detection where commercial kits are not feasible. The developed LAMP assay achieved maximum specificity with no cross-reactivity to related species, limits of detection of 240 fg/reaction for pure bacterial DNA and 0.4 pg/µL in K. aerogenes artificially contaminated food samples, and a reaction time under 30 min—outperforming real-time PCR in speed and robustness. This cost-effective method provides a scalable tool for near-real-time monitoring of K. aerogenes in food production, enhancing safety and enabling early outbreak detection. Full article

In the study of eggplant (Solanum melongena L.), a cross between the green peel line 19143 and the white peel line 19147 produced E4957 F₁ hybrids with a purple–brown peel. Self-fertilization of the F₁ hybrids yielded E4957 F₂ offspring with a segregation ratio of 27:9:21:7 among individuals with purple–brown, purple–red, green, and white peel colors, respectively, which was consistent with a genetic model controlled by reciprocal recessive epistasis between D and P, and Gv1 likely acting as a modifying factor. The green peel line 19143 exhibited higher chlorophyll but lower anthocyanin levels than the white peel line 19147, which contained low levels of both pigments, while the E4957 F₁ hybrids had elevated levels of both pigments. Two epistatic genes, D and P, associated with anthocyanin synthesis, were mapped on chromosomes 10 and 8, respectively. The putative modifying locus Gf, involved in chlorophyll accumulation in the flesh, was mapped on chromosome 8, and the localization interval was close to the previously reported Gv1 locus associated with chlorophyll synthesis in the peel. DNA markers (InDel22522, InDel5531, InDel-APRR2) were developed to genotype 237 F₂ individuals and correlate genotypes with phenotypes. Sequence analysis revealed a 6 bp deletion in the SmMYB1 (D) gene and a large deletion in the SmAPRR2-Like (Gv1) gene in the white peel line 19147, as well as a T to A mutation in the SmANS (P) gene in the green line 19143. This study provided evidence for inheritance between loci involved in anthocyanin and chlorophyll pathways contributing to eggplant peel color variation and provides molecular markers that may facilitate the breeding of eggplant varieties with diverse peel colors. Full article

Ochratoxin A (OTA) is a highly toxic mycotoxin commonly detected in food and agricultural products, requiring sensitive analytical methods for reliable monitoring. Herein, we report an ultrasensitive turn-on electrochemical aptasensor for OTA detection based on a target-induced displacement of an aptamer–complementary DNA (cDNA) duplex assembled on an electrochemically reduced graphene oxide (ERGO)-modified glassy carbon electrode (GCE). In the absence of OTA, a methylene blue (MB)-labeled aptamer hybridized with cDNA is immobilized on the ERGO surface via π–π stacking interactions, forming a rigid duplex that suppresses electron transfer and yields a low electrochemical signal. Upon OTA binding, the aptamer undergoes a conformational transition into a G-quadruplex structure, leading to dissociation of the cDNA strand. This target-induced folding brings the MB redox tag into close proximity to the ERGO surface, markedly accelerating electron transfer and enhancing the cathodic reduction current of MB, thereby producing a pronounced signal-on response in square-wave voltammetry (SWV). The ERGO-modified electrode provides a conductive and stable interface without chemical linkers. Under optimized conditions, the aptasensor shows a linear response to OTA from 10 fM to 100 pM with an ultralow LOD of 0.67 fM, together with high selectivity, good reproducibility, and satisfactory stability. This work demonstrates a simple and effective turn-on aptasensing strategy for sensitive electrochemical detection of OTA. Full article

Background/Objectives: The combination of hearing loss and visual impairment in a single patient strongly suggests a genetic aetiology. However, after conventional testing, a considerable proportion of deafblindness cases remain without a genetic diagnosis. The aim of this study was to address this diagnostic gap. Methods: We developed an enhanced exome strategy that uses a whole-exome backbone complemented by spike-in capture probes for (i) low-coverage coding segments and clinically validated, non-coding regions (including deep intronic splice-altering sites and untranslated exonic sequences) across 659 genes associated with hearing loss and/or visual impairment, and (ii) mitochondrial DNA. Results: With 66.6 million paired-end reads per sample, this methodology achieved coverage of at least 20 reads per base at 99.3% of target coding and non-coding positions of genes associated with deafness and/or blindness, as well as 98.8% of the whole exome. The enhanced exome approach correctly identified the genetic variants causative of deafness and/or blindness in 10 out of 10 cases with a previously known genetic cause, in 3 out of 10 additional cases that remained undiagnosed after extensive panel sequencing, and in 4 out of 4 cases that had not been genetically studied before. Comparison of the performance of two commercial bioinformatics platforms for enhanced exome interpretation revealed that eVAI consistently prioritised causative variants higher than, or as high as, VarSome Clinical, resulting in a tendency toward shorter interpretation times using the former. Both platforms offered the same diagnostic yield and both failed to correctly call one of the causative variants. Conclusions: In an era where many centres operate exome analysis through virtual panels, enhanced exome sequencing leverages the advantages of whole-exome and custom panel sequencing: it provides panel-like sensitivity for clinically actionable loci, while offering the flexibility to periodically reanalyse data and discover candidate genes. Full article

Background: low-quantity or degraded samples are often studied in forensic genetics. Therefore, it is important to efficiently obtain all the available DNA from the biological sample analyzed to provide the most reliable results. This is particularly challenging in bone marrow processing due to its hydrophobic molecular structure, as for other lipid-rich tissues, especially if rancid. In fact, during adipose tissue decomposition, the putrefaction of fatty acids can in some instances give a compact cerous consistency to the lipidic tissue, hardly susceptible to the nucleic acid extraction mechanisms. According to environmental circumstances, this condition is notably observable in submerged bodies or in putrefied bone marrow. Thus, this study is focused on developing an optimized nucleic acids extraction protocol for putrefied bone marrow. Methods: genetic analyses were performed on putrefied yellow bone marrow collected from 20 human femora recovered from bodies in different decomposition stages. The optimized method was developed by integrating additional steps, reagents and time intervals on a silica-based column commercial kit. This strategy was compared in DNA yield to a standard extraction protocol, represented by the same commercial kit, but following the manufacturer’s directions. Both these strategies were tested in nucleic acid isolation efficiency by performing DNA typing, including real-time PCR quantification, Short Tandem Repeats (STR) amplification and fragments analysis steps. The analytical parameters evaluated were allele count, DNA concentration (ng/µL) and Degradation Index (DI). Results: for allele count and DNA concentration parameters, the optimized protocol showed clear and significant qualitative and quantitative improvements compared with the standard protocol, supporting its potential applicability in forensic casework and laying the foundation for future studies. Conclusions: prior to appropriate laboratory internal validation, the optimized protocol can be used for tough lipid-rich tissues processing without the need to purchase a dedicated system and using a same commercial kit routinely adopted for other forensic genetics matrices. Full article

Background/Objectives: Habitat degradation and fragmentation reduce population size, genetic diversity, and connectivity, increasing extinction risk in small and isolated populations. Coastal wetlands of northwestern Peru have undergone extensive anthropogenic modification, yet the genetic and ecological status of resident carnivore populations remains poorly documented. This study aimed to assess genetic diversity, relatedness, demographic signals, and diet composition of a Pampas cat (Leopardus garleppi) population inhabiting the Mangroves San Pedro de Vice (MSPV), a Ramsar-listed coastal wetland. Methods: We combined noninvasive fecal genotyping using eight nuclear microsatellite loci with vertebrate DNA metabarcoding. Scat samples were collected across three field seasons (2019–2021). Individual identification, genetic diversity metrics, genetic mark–recapture estimation of census size (Nc), effective population size (Ne), bottleneck tests, and relatedness analyses were performed to evaluate population status and kin structure. Dietary composition was characterized using metabarcoding and assessed for sex-specific differences. Results: Sixty-eight scats yielded multilocus genotypes for nine individuals (six males, three females). Genetic analyses revealed moderate diversity (mean allelic richness = 3.47; observed heterozygosity = 0.69; expected heterozygosity = 0.58) and evidence consistent with a recent genetic bottleneck. Genetic mark–recapture analyses estimated a small census size (Nc = 9; 95% CI: 7.0–9.0), while the effective population size was markedly low (Ne = 2.4; 95% CI: 1.5–7.4), yielding an Ne/Nc ratio of ~0.27. Multiple first-order kin dyads were detected, indicating strong local kin structure and limited external recruitment. Metabarcoding identified eight vertebrate prey species, with diet dominated by the native rodent Aegialomys xanthaeolus. No significant sex-specific differences in diet composition were detected. Conclusions: The MSPV Pampas cat population represents a small, kin-structured range-edge population showing signatures consistent with recent genetic erosion and restricted connectivity. These patterns align with isolation in a degraded coastal wetland landscape, highlighting the importance of habitat protection, prey resource conservation, and restoration of functional connectivity to support long-term population persistence. Full article

Pseudostellaria heterophylla, an important traditional medicinal plant in China, has suffered increasing yield and quality loss due to leaf spot disease in recent years. In this study, the causal agent was conclusively identified as Sclerotiophoma versabilis through detailed morphological characteristics and multi-locus phylogenetic analyses based on the internal transcribed spacer regions (ITS), the 28S large subunit of the nrDNA (LSU), RNA polymerase II (rpb2), and ß-tubulin (tub2) sequences. Pathogenicity tests fulfilled Koch’s postulates, thereby resolving previous taxonomic inconsistencies regarding this disease. The effects of environmental and nutritional factors on mycelial growth, conidial germination, and infection were systematically evaluated. Optimal mycelial growth occurred at 20–25 °C, pH 6–8, under continuous light. Optimal mycelial growth occurred at 20–25 °C, pH 6–8, under continuous light, while conidial germination was maximized at 20–25 °C and pH 6–7 under continuous light. Starch and glycine were identified as the most favorable carbon and nitrogen sources for the fungal mycelial growth, respectively. Infection assays indicated an incubation period of approximately 3 d and maximal disease development at moderate temperatures under low-light conditions, with 6 d-old cultures exhibiting the greatest infectivity. Microscopic observations revealed that S. versabilis penetrated host tissues directly or via stomata without forming specialized infection structures. These findings integrate taxonomic resolution with ecological and infection biology analyses, providing mechanistic insight into the environmental drivers of leaf spot epidemics and a scientific basis for disease-risk assessment and management in P. heterophylla production systems. Full article

The objective of this study was to investigate the relationships between ruminal microbial communities and carcass traits associated with adipose accumulation in two Tibetan sheep breeds—Gannan and Tianzhu. A total of twenty Tibetan sheep (ten from each breed) were slaughtered, and samples of ruminal contents along with carcass trait data were collected for analysis. Ruminal microbial DNA was analyzed by 16S rRNA gene sequencing, and correlations between microbial composition and carcass traits were examined using correlation analysis and one-way ANOVA. The results showed that marbling score (p = 0.001) and longissimus lipid content (p = 0.007) were positively correlated with the Chao1 richness index, indicating that individuals with higher intramuscular fat content had greater ruminal microbial species richness. At the phylum level, Rikenellaceae RC9 gut group, Ruminococcaceae NK4A214 group were negatively correlated (p ≤ 0.05) with the above fat traits, whereas the abundance of the bacterial family Ruminococcus 1 was positively correlated with marbling score (p = 0.002). Stratified analysis by marbling grade further revealed associations with microbial richness (p ≤ 0.063), diversity (p = 0.044), and Ruminococcus 1 abundance (p < 0.001). However, microbial metabolic pathway prediction showed no significant differences (p ≥ 0.05) between the high- and low-marbling groups. In addition, several microbial taxa were positively correlated (p ≤ 0.05) with rib fat thickness and yield grade. In summary, ruminal microbial composition was closely associated with variations in carcass fat traits. Notably, most of the bacterial taxa associated with intramuscular and subcutaneous fat deposition did not overlap, suggesting that microbial metabolites may regulate fat deposition by influencing distinct adipogenic pathways in the host. Full article

In people taking antiretroviral therapy (ART) for HIV infection, the methods to characterize latent and active HIV reservoirs remain costly and labor-intensive. Our objective was to develop a relatively low-cost technique to amplify and sequence the proviruses that persist during ART along with the site in the human genome where each provirus is integrated. We developed a novel HIV-specific Multiple Displacement Amplification (HIV-MDA) assay that specifically amplifies HIV-1 proviruses and their associated integration site. Upon comparison of our HIV-MDA to an established commercial kit designed to amplify cellular DNA, we found that the HIV-MDA (1) typically yielded a greater number of HIV integration site (HIV IS) sequences per 150,000 cells analyzed; (2) improved rates of proviral DNA amplification; and (3) amplified HIV IS at a fraction of the cost (13.6 times less expensive). Thus, the HIV-MDA method appears to be a more sensitive and cost-effective approach to sequencing HIV IS and the associated proviruses compared to a commercial kit. Full article

Early detection of bladder cancer poses a major challenge for liquid biopsy due to limited tumor burden and low abundance of tumor-derived DNA. In such low-signal settings, detection sensitivity critically depends on both biofluid selection and effective integration of weak, distributed molecular signals. We analyzed Enzymatic Methyl-seq (EM-seq) data on 41 matched urine–plasma pairs, which demonstrated that urine samples exhibited significantly higher tumor fractions and greater concordance with tissue methylation profiles than plasma. Based on this observation, we developed a urine-based bladder cancer detection framework using EM-seq. We profiled 143 urine samples (68 bladder cancer and 75 healthy controls) and 14 bladder cancer tissues. Methylation markers (113,052 regions) were identified by comparing cancer tissues (n = 14) with urine from healthy individuals (n = 14). Using XGBoost, possible features and their combinations were evaluated, with the combination of methylation and copy number variations (CNV) yielding the best performance as the final ensemble model. When evaluated on an independent test set, the model achieved 91.9% sensitivity at 80% specificity, with an area under the curve (AUC) of 0.932 for bladder cancer detection and 0.928 for non-muscle invasive bladder cancer (NMIBC) detection. Notably, the model successfully detected four of seven mutation-negative cases, demonstrating complementary value to mutation-based approaches. Full article

Colorectal cancer (CRC) is traditionally considered a “cold tumor” characterized by low immunogenicity and limited responsiveness to immune checkpoint inhibitors (ICIs). However, recent findings reveal that cytotoxic modalities can reprogram this immunologically inert landscape. This review integrates these evolving concepts to guide the optimization of future treatments. Radiotherapy induces extensive DNA double-strand breaks, which may generate de novo mutations through error-prone repair while simultaneously exposing cryptic antigens via increased transcriptional instability, alternative splicing, and enhanced proteasomal processing. Chemoradiation also amplifies epigenetic and epitranscriptomic sources of neoepitope diversity, including RNA editing and stress-induced splicing alterations, expanding the immunopeptidome beyond canonical mutation-driven neoantigens. These changes collectively enhance antigen presentation and facilitate T-cell priming. Chemotherapy further reduces immunosuppressive cell populations and promotes dendritic cell activation, creating a permissive milieu for subsequent immune engagement. Clinically, the VOLTAGE studies demonstrated that long-course chemoradiotherapy can sensitize even mismatch repair–proficient rectal cancers to PD-1 blockade, yielding clinically meaningful pathological responses. In contrast, mismatch repair–deficient rectal tumors may respond completely to ICIs alone. Short-course radiotherapy combined with chemotherapy and ICIs has also shown encouraging activity in the setting of total neoadjuvant therapy. Collectively, these findings support a paradigm in which radiotherapy, chemotherapy, and epigenetic/epitranscriptomic alterations—including RNA editing—act as potent modulators of tumor antigenicity. By expanding the neoantigen repertoire and reshaping the tumor microenvironment, these strategies can transform CRC from a cold tumor into one that is increasingly responsive to immunotherapy. Full article

This study investigated the genetic diversity and population structure of five Hainan indigenous pig breeds (147 individuals from 7 populations representing 5 breeds: 3 Duntou pig subpopulations (DT-DZ, DT-SJ, and DT-SG) and four additional breeds (Wuzhishan, Wenchang, Lingao, and Tunchang)) to address germplasm conservation needs driven by exotic crossbreeding, African swine fever, and inadequate genetic evaluation. After strict quality screening, we used 147 qualified samples for microsatellite genotyping and 104 samples for mtDNA D-loop sequencing. The analyses integrated 17 FAO-recommended microsatellite markers and mtDNA D-loop sequencing. In total, 15 out of 17 loci exhibited high polymorphism (PIC > 0.6), with Wuzhishan pigs exhibiting the highest genetic diversity (He = 0.666, I = 1.279). Pairwise Fst values indicated significant genetic differentiation among all populations (p < 0.05), and AMOVA attributed 87.32% of the genetic variation to within-population differences. Three complementary clustering methods (UPGMA, PCoA, and STRUCTURE with the optimal K value of 2 identified via the ΔK algorithm) divided the populations into two clades, clearly separating the Duntou subpopulations from other breeds. mtDNA D-loop sequencing of 104 individuals yielded a 1175 bp fragment, identifying 12 haplotypes and a high haplotype diversity (Hd = 0.688) low nucleotide diversity (π = 0.00193) pattern; Lingao pigs showed no genetic variation, while Duntou and Wuzhishan pigs had the highest Hd. NJ phylogenetic analysis indicated that Hainan pigs form an independent subclade within Chinese indigenous pigs, closely related to Luchuan pigs. These findings confirm the high overall genetic diversity and distinct population-level divergence in Hainan pigs, with Duntou pigs representing a unique lineage. This work provides a scientific basis for targeted conservation strategies, including prioritizing the conservation of Duntou and Wuzhishan pigs and restoring genetic variation in Lingao pigs. Full article

Proton therapy offers superior dose localization, yet the biological effects of low-energy protons relevant to superficial tissues remain underexplored. We report the design and validation of a proton irradiation setup developed at the Tandem Accelerator of NCSR “Demokritos” for controlled radiobiological experiments. Monte Carlo simulations using Geant4 and Monte Carlo Damage Simulation (MCDS—Monte Carlo Damage Simulation) were used to determine proton energy spectra, linear energy transfer (LET), and predicted DNA damage yields. A single layer (15–20 μm in thickness) of human keratinocytes (HaCaT) was irradiated at doses from 0.65 to 3.65 Gy, and γ-H2AX foci were quantified as markers of tracks including one or more DNA double-strand breaks. The system achieved a uniform dose rate of 0.37 Gy/min, as calculated with Geant4, with a mean proton energy of 4.1 MeV (LET ≈ 8 keV/μm). A strong correlation (R² = 0.93) was observed between proton dose and γH2AX foci per nucleus (~10 foci/Gy), reflecting damage-inducing proton tracks rather than individual DNA double-strand breaks. At higher doses, an increased fraction of cells exhibited pan-nuclear γH2AX staining, characterized by a diffuse γH2AX signal throughout the nucleus and commonly associated with extensive or clustered DNA damage and global chromatin phosphorylation. These responses are consistent with the well-established dense ionization patterns produced by low-energy protons, as indicated by the LET spectrum and supported by MCDS-predicted clustered damage yields. While the γH2AX assay does not directly resolve simple versus complex DNA lesions, the agreement between Monte Carlo modeling and the observed cellular stress responses indicates that the irradiation platform reliably reproduces the expected biological signatures of low-energy proton exposure. Consequently, the developed system provides a robust experimental tool for systematic investigations of cellular radiosensitivity and radiotoxicity, with potential applications in skin dosimetry and radioprotection. Full article