Search Results (185)

Small, isolated, and fragmented populations often exhibit low levels of genetic diversity as a result of genetic drift, limited gene flow, and inbreeding. Huemul (Hippocamelus bisulcus) is a medium-sized South American deer categorized as endangered by the International Union for Conservation of Nature (IUCN). Huemul in its northernmost range was previously distributed in Central Chile between 36° S and 37° S, but its current distribution and conservation status in the region are poorly documented. We used non-invasive genetic approaches to assess the genetic diversity, population connectivity, and demographic history of the huemul’s northernmost population using nuclear microsatellite markers from fecal samples of wild individuals. Observed nuclear DNA genetic variation (Ho = 0.2958 ± 0.0318) was moderate, and allelic richness was low (Ar = 3.43–4.01), consistent with the theoretical expectation that isolated populations may retain heterozygosity while losing allelic richness more rapidly. However, the estimated nuclear DNA effective population size was low (Ne = 47; 95% CI: 19.2–∞). Demographic simulations project continued loss of genetic diversity under all scenarios modeled. Our results provide a foundation for further study of this population and provide the genetic data necessary to design detailed management plans to ensure the persistence of healthy populations of this rare and elusive deer. Full article

The Fusobacterium nucleatum complex, which comprises oral lineage 1 (L1) strains, is strongly associated with colorectal cancer (CRC). NusG (N-utilisation substance G) is a transcription elongation factor that is universally conserved. This study aimed to develop and validate a novel nusG-directed polymerase chain reaction (PCR) assay to specifically and sensitively detect ectopic Fusobacterium L1 colonisation in clinical CRC patient samples. Four L1-specific primer pairs targeting the nusG gene were designed using MEGA11 software (Molecular Evolutionary Genetics Analysis, version 11.0.13) and successfully employed in 40 stool samples from CRC patients and healthy controls (HC). Additionally, five species-specific primer pairs were designed for the L1 species F. animalis clades 1 and 2, F. nucleatum, F. polymorphum, and F. vincentii, and were successfully applied to stool and saliva samples. Their specificity was verified via Sanger sequencing. Two L1-specific primer pairs (NusG5-F/NusG6-R and NusG2a-F/NusG5-R) demonstrated robust performance in our cohort, showing statistical significance (padj < 0.05) and a large effect size (|r| ≥ 0.5) in the difference in Ct values and absolute cell counts between CRC patients and the HC group. These primer pairs also exhibited promising preliminary diagnostic potential, with respective area under the curve (AUC) values of 0.909 and 0.883. However, Fusobacterium L1 abundance in saliva samples did not differ significantly between groups, indicating that definitive conclusions cannot be drawn due to the limited power of the salivary sub-cohort. The data indicates that nusG-based PCR primers could be used as reliable, non-invasive biomarkers as a complementary tool for early CRC diagnostics. While potentially applicable in the context of other Fusobacterium-implicated diseases, further validation in larger, ethnically diverse cohorts remains essential. Full article

Molecular genetic approaches have become indispensable in veterinary medicine for early tumour detection, confirming histomorphological diagnoses, and non-invasive diagnosis using urine samples. In canine urothelial carcinoma (UC), molecular testing includes BRAF^V595E mutation analysis and the identification of specific copy number alterations (CNAs). CNAs affect multiple genes simultaneously, leading to their overexpression or underexpression. Recurrent gene gains on canine chromosomes (CFA)13 and CFA36 and losses on CFA19 are highly prevalent in canine UCs. This study evaluates CNA test performance and the influence of tumour purity on CNA test results using 76 histologically confirmed UC tissue samples. The tumour region of each histopathological section was measured, and its proportion relative to the total tissue area was calculated. In total, 58/76 cases (76.3%) were CNA-positive, defined by a ratio greater than 1.23 for both CFA13/19 and CFA36/19. A total of 14/18 negative cases had a ratio > 1.23 for CFA13/19 or CFA36/19. The sensitivity of this CNA assay depends on the applied thresholds, with a tumour-to-total tissue ratio of ≥20% yielding positive CNA results in 84% of cases, rising to 89% at ≥40%. These findings indicate that <20% tumour content and a moderate to high proportion of copy number-neutral cells may be associated with reduced CNA detectability. Full article

Background: Urinary cellular biomarkers detected by flow cytometry have emerged as promising non-invasive tools for the diagnosis, prognosis, and monitoring of renal and urological diseases. However, a comprehensive synthesis of their clinical applicability is lacking. Objectives: This review aimed to systematically evaluate and summarize the evidence of urinary cellular biomarkers identified through flow cytometry in human populations with renal or urological diseases. Methods: A systematic search of PubMed, Scopus and Web of Science was conducted from inception to January 2025. Observational studies analyzing urinary samples by flow cytometry were included, whereas animal studies, genetic-only analyses and studies with incomplete data were excluded. Data extraction and risk of bias assessment were performed independently by two reviewers using a standardized form and the Newcastle-Ottawa Scale. Due to heterogeneity in study populations, designs, and cytometry methods, results were synthesized narratively. Results: Of 3938 records screened, 23 studies met the inclusion criteria. All studies applied flow cytometry to characterize urinary cellular biomarkers across renal diseases. Several studies reported promising diagnostic and monitoring applications, but substantial heterogeneity in study design, cytometry protocols, and marker panels limited comparability. Longitudinal analyses and robust prognostic validation were infrequently reported. Conclusions: Urinary cellular biomarkers assessed by flow cytometry represent a promising non-invasive approach for evaluating renal and urological diseases. However, clinical implementation remains constrained by heterogeneity and insufficient validation. Future research should focus on standardized methodologies and large prospective studies to establish their diagnostic and prognostic utility. Full article

Background/Objectives: Child malnutrition is a global health challenge linked to poor growth, impaired development, weakened immunity, and adverse outcomes. Early risk detection is essential, but current screening tools differ in accuracy and feasibility. This study aimed to develop and validate new bedside pediatric malnutrition screening models based on machine learning and evolutionary computation methods that can capture complex patterns in non-invasive clinical indicators while remaining practical for routine ward use. Methods: We conducted a cross-sectional study including 180 hospitalized children (1 month–18 years) recruited consecutively from six pediatric wards. The required sample size (minimum 138 participants) was calculated a priori using national prevalence estimates of pediatric undernutrition (4–9.5%) to ensure adequate precision at a 95% confidence level. Data collection included a questionnaire, anthropometry, subjective malnutrition risk assessment, and the Subjective Global Nutritional Assessment (SGNA) tool. Screening models were developed using decision trees, random forests, XGBoost, lasso regression, artificial neural networks, ANFIS, and genetic programming. Their performance was evaluated against the SGNA tool and physician-based subjective malnutrition risk assessment using sensitivity, specificity, AUC, and Cohen’s κ. Results: Machine learning and intelligent evolutionary models (GP, ANN, and ANFIS) showed the best performance in this sample, with substantial to high agreement (κ = 0.81–1.00) and high diagnostic accuracy (AUC = 0.92–1.00) with the subjective malnutrition risk assessment. The GP model demonstrated the highest apparent accuracy in this dataset, but also higher complexity, whereas simpler models such as decision trees showed lower accuracy but greater interpretability and feasibility for routine clinical use. However, validation was performed on a relatively small independent sample, and no external validation was conducted, which may limit the generalizability of the findings. Conclusions: While complex models may serve as digital assessment instruments, simpler models are rapid and more suitable for bedside screening. All developed models are non-invasive and cost-effective and show potential for supportive approaches for early detection of malnutrition risk at hospital admission. However, given the limited validation sample and the absence of external validation, these findings should be interpreted with caution, and further large-scale, multicenter studies are required to confirm generalizability and clinical applicability. Full article

This scientometric review examines the evolution and scope of invasive (blood and tissue) and non-invasive (faeces, hair, and saliva) sampling in terrestrial mammal population genetics, with particular emphasis on the comparability of observed heterozygosity (Ho), expected heterozygosity (He), and the inbreeding coefficient (Fis) between studies published from 1985 to 2026. Searches in Web of Science and Scopus, filtered under PRISMA/PRISMA-S criteria, yielded a compendium of articles analysed with Bibliometrix and VOSviewer 1.6.20 to quantify temporal production, keyword evolution, collaborative networks, and publication outlets. Searches in Web of Science and Scopus, filtered under PRISMA/PRISMA-S criteria, yielded a broad corpus of 145 articles for general scientometric analyses, of which 85 met the eligibility criteria for the focused analysis of Ho, He, and Fis. The field shows steady growth (annual rate ≈ 6.1%), substantial authorship and international collaboration, and increasing thematic diversity. Adoption of non-invasive sampling has accelerated, broadening spatial and taxonomic coverage, but also increasing exposure to DNA degradation and genotyping error when laboratory quality control is insufficient. Across the literature, reporting of quality control practices (e.g., extraction blanks, negative PCR controls, multi-tube replication, and error-rate estimation) has improved over time but remains inconsistent. Comparisons indicate that differences in Ho, He, and Fis between invasive and non-invasive sampling are generally modest once marker system and species are taken into account. These findings indicate that quality control and transparency in reporting, rather than invasiveness per se, are the main factors determining comparability among studies. The scientometric patterns also reveal a methodological transition from microsatellites to SNP-based and reduced representation approaches, with implications for synthesis across marker types. Overall, this review identifies geographic and taxonomic biases in research effort and highlights the need for standardised reporting of DNA quality indicators, inclusion thresholds, and validation protocols to strengthen genetic monitoring in mammalian conservation. Full article

Misiones, Argentina, holds one of the largest remnants of the Atlantic Forest, with almost 1.4 million hectares of native forest, representing a critical landscape for sustainable biodiversity conservation. However, connectivity across this ecoregion is increasingly threatened by habitat conversion, landscape fragmentation, and poaching pressures that extend beyond protected area boundaries, undermining long-term sustainability of wildlife populations. Using conservation detection dogs, we located, collected, and genetically confirmed 198 scats belonging to four game species: 20 lowland tapir (Tapirus terrestris), 72 white-lipped peccary (Tayassu pecari), 55 collared peccary (Pecari tajacu), and 51 Azara’s agouti (Dasyprocta azarae). Analyses examining species-specific habitat associations emphasized the importance of extending inference beyond point locations to encompass species’ home ranges, with native forest consistently identified as a key component of habitat use. The high prevalence of scats in mosaics of human-modified habitats outside protected areas, especially along their borders, underscores the importance of managing these areas as part of a broader sustainable landscape matrix. While native forest fragments outside of protected areas may serve as important refugia supporting species persistence, their contribution to sustainable management depends on reducing poaching pressure across these landscapes. There is an urgent need to expand antipoaching efforts beyond protected areas and across the Atlantic Forest in the Green Corridor of Misiones while preventing ongoing deforestation and the expansion of monoculture plantations. Achieving sustainable wildlife management in this region will require integrated strategies that promote sustainable land use, conservation planning, and rural development. Full article

Nitrate (NO₃⁻) serves as a pivotal molecule with dual functions in nutrient supply and signaling during plant growth and development. Precise monitoring of its spatiotemporal dynamics in planta is therefore essential for dissecting the regulatory mechanisms underlying plant nitrogen metabolism. However, conventional nitrate detection methods suffer from inherent limitations, including destructive sampling, insufficient spatiotemporal resolution, and an inability to achieve real-time whole-plant monitoring. Here, we report a genetically encoded nitrate biosensor, designated NitNRCL1, constructed using a split firefly luciferase complementation system. Functional validation in both prokaryotic and eukaryotic systems demonstrates that NitNRCL1 responds to changes in nitrate availability and generates stable chemiluminescent signals in bacteria and diverse plant species. Importantly, NitNRCL1 enables non-invasive, real-time, and whole-plant monitoring of nitrate levels in living plants. Using NitNRCL1, we successfully imaged the spatiotemporal dynamics of nitrate signaling in Arabidopsis thaliana. Collectively, our findings establish NitNRCL1 as a robust and novel tool for investigating nitrate transport, signaling, and metabolic pathways in plants. This biosensor advances our mechanistic understanding of plant nitrate biology and provides a technical foundation for breeding nitrogen-use-efficient crops and developing precision fertilization strategies. Full article

Background: Pangolins are critically endangered mammals, and a comprehensive understanding of their genetic diversity is crucial for effective conservation. The mitochondrial genome serves as a vital molecular marker for phylogenetic and population genetic studies. Obtaining genetic material from these elusive animals non-invasively remains a challenge. This study aimed to sequence and characterize the complete mitochondrial genome of Manis javanica and explore the phylogenetic relationships among pangolin species. Methods: The complete mitochondrial genome was sequenced from a saliva-derived sample. Standard procedures for DNA extraction, amplification, and sequencing were employed. The genome was assembled and annotated using bioinformatic tools. Phylogenetic analysis was conducted based on the cytochrome c oxidase subunit I (COXI) gene sequences from nine pangolin species, with the resulting tree constructed using the maximum-likelihood method. Results: The complete mitochondrial genome of M. javanica (GenBank accession: PP110760) is a circular molecule of 16,573 bp, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. The overall base composition showed a lower GC content (43.83%) than AT content (56.17%). Phylogenetic analysis based on COXI sequences delineated the nine species into three distinct genera: Manis, Phataginus, and Smutsia. Within the genus Manis, Manis pentadactyla was identified as the closest relative to M. javanica. The newly described species Manis mysteria was found to be closer to Manis culionensis and Manis crassicaudata than to other congeners. Furthermore, the analysis indicated that African pangolins diverged earlier than Asian pangolins. Conclusions: This study successfully demonstrates the feasibility of extracting and sequencing the complete mitochondrial genome from saliva samples, providing a valuable non-invasive method for future genetic studies on pangolins. The genomic data and phylogenetic results offer significant molecular insights that will benefit the genetic management and conservation of critically endangered pangolin resources. 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

Background/Objectives: Following centuries of systematic eradication, grey wolf (Canis lupus) populations across Europe have experienced a significant recovery over recent decades, which leads to concerns regarding, among others, anthropogenic hybridization. In Greece, the genetic status of the wolf population is largely unknown to date. Here, we genetically monitor and test for wolf–dog hybridization events in a recently established wolf population in the Parnitha Protected Area, in close vicinity to the capital city of Greece. Methods: One hundred and twenty-four wolf scat samples were genotyped at 20 canine-specific autosomal microsatellite loci and compared to available reference tissue samples from wolves and free-ranging dogs. Results: A minimum of 31 unique wolf individuals were identified, structured into at least three packs. No wolf–dog hybrids were detected in the study area. To validate the accuracy of the microsatellite analysis, an ancestry informative 93-SNP panel was applied to non-invasive wolf DNA samples from the study area, confirming the absence of hybrids among them. However, a possible wolf–dog hybrid was detected among reference wolf samples collected in Northern Greece, where individuals with atypical morphological traits are observed. The estimated census population size was in accordance with concurrently obtained camera trapping data, while heterozygosity values were low. Conclusions: This research represents the first systematic effort in Greece to genetically monitor wolves recently established in a protected area. It highlights the need for targeted management strategies based on genetic data to ensure balanced long-term conservation of wolves in peri-urban areas. Full article

Background: Increased nuchal translucency (NT) is associated with an elevated risk of genetic abnormalities and structural malformations. The clinical utility of invasive testing and the optimal diagnostic approach in mildly increased NT (3.0–3.4 mm) is debated. This study aimed to evaluate genetic and ultrasound findings in this subgroup and to assess the diagnostic yield of advanced genetic testing. Methods: We retrospectively included a total of 107 fetuses with NT between 3.0 and 3.4 mm from a single fetal medicine unit. Complete outcome data were available for 97 pregnancies. Invasive prenatal testing with standard karyotype, chromosomal microarray analysis (CMA) and RASopathy panel testing were offered. All patients underwent detailed ultrasound examination to detect structural abnormalities at 16 and 20 weeks, regardless of whether invasive testing was performed. Results: Invasive prenatal testing, amniocentesis or chorionic villus sampling, (CVS), was performed in 77/97 cases (79.4%). Genetic abnormalities were detected in 28/97 (28.9%). Overall, five rare genetic anomalies were identified; none would have been detected by quantitative fluorescent polymerase chain reaction (QF-PCR) or non-invasive prenatal testing (NIPT). Two anomalies were detectable by standard karyotype, two exclusively by CMA and one exclusively by RASopathy panel. When considering all cases undergoing advanced genetic testing (CMA or RASopathy panel, n = 35) the overall diagnostic yield was 8.5% (3/35). When calculated across the entire cohort with complete follow-up, the additional diagnostic yield was 3.1% (3/97). Major structural malformations were identified in 17/97 cases (17.5%), of which 10 (58.8%) were associated with genetic abnormalities. Conclusions: Fetuses with NT measurements between 3.0 and 3.4 mm show a substantially increased risk of genetic abnormalities and structural malformations. These findings support a comprehensive prenatal evaluation, including invasive testing with advanced genetic analysis and detailed ultrasound assessment, to optimize diagnosis and counseling. Full article

Background/Objectives: Non-invasive samples offer an attractive alternative to logistically challenging invasive approaches in wildlife genetic studies but often contain low-quality host DNA that limits downstream analyses. Here, we assessed the applicability of moulted Eurasian goshawk feathers as a DNA source for whole-genome re-sequencing. Methods: We analysed 75 moulted feathers collected opportunistically from breeding territories. Each feather was measured from tip to tip, and its condition was visually assessed. Whole-genome re-sequencing was performed with a target coverage of 13× using 150 bp paired-end reads. Results: Feathers yielded an average of 7.19 ± 10.93 ng/μL DNA. DNA yield was positively correlated with feather size and the presence of blood traces in the calamus. On average, feather samples performed well, producing 208.7 ± 59.82 million reads, of which 82.69 ± 27.15% aligned to the reference genome, resulting in 83.58 ± 19.02% of the genome being covered at least once. After quality filtering, 10.34 ± 3.11 million biallelic single-nucleotide variants remained, of which 457,745 were common variants (MAF > 0.05). Larger feathers in good condition, with higher DNA yields and blood traces in the calamus, tended to perform better throughout the re-sequencing workflow. Nevertheless, approximately 22.7% of samples failed due to high missing data or poor genotype quality. Conclusions: Performance varied substantially even among samples with similar characteristics, indicating that improved sample selection incorporating direct measures of host DNA quality may be beneficial. Despite these challenges, moulted feathers represent a readily available DNA source for genome-wide re-sequencing of medium- to large-sized raptor species. Full article

The recent recognition of the Neotropical otter (Lontra annectens) as a distinct species highlights the need to evaluate its genetic status and connectivity across fragmented tropical habitats. We analyzed genetic diversity, population structure, and recent demographic patterns of L. annectens from two contrasting regions in northern Costa Rica—Tortuguero National Park (TNP) and the Sarapiquí River Basin (SRB). Non-invasive fecal and anal-gland secretion samples collected during 2021–2022 were genotyped at ten nuclear DNA microsatellite loci. Genetic diversity was moderate across regions (mean allelic richness [AR] = 3.98–4.03, observed heterozygosity [Ho] = 0.52–0.58), expected heterozygosity [He] = 0.62–0.65) with no significant inter-regional differences. Bayesian clustering, principal component analysis, and pairwise F_ST (0.002) supported a near-panmictic population. Kinship analyses detected localized clusters of related individuals, suggesting weak but non-random structuring, while contemporary migration estimates indicated low-frequency, asymmetric gene flow from SRB to TNP. Bottleneck tests revealed signatures of recent demographic contraction in both regions, particularly in TNP. These findings demonstrate limited yet ongoing connectivity among riverine subpopulations and emphasize that increasing habitat fragmentation could erode this exchange. Maintaining hydrological corridors and monitoring genetically vulnerable subpopulations should be conservation priorities to preserve gene flow and long-term viability of L. annectens in northern Costa Rica. Full article

Objectives: This study aimed to characterize the types and frequencies of sex chromosome aneuploidies (SCAs) detected through invasive prenatal testing, evaluate the concordance between non-invasive prenatal testing (NIPT) and confirmatory diagnostic methods, and assess the challenges faced during genetic counseling following SCA diagnosis. Study Design: A retrospective review was conducted on 842 prenatal samples collected between 2020 and 2024 in a tertiary private medical center. Samples included amniotic fluid, chorionic villi, and products of conception. Testing involved rapid QF-PCR for aneuploidy detection, followed by SNP-based chromosomal microarray analysis (CMA). NIPT results with high risk for sex chromosomes aneuploidies were correlated with invasive testing outcomes in 19 cases. Results: Sex chromosome aneuploidies were identified in 67 cases (7.96%), with Turner syndrome (45, X) being the most frequent (23 cases, including six mosaics), followed by Klinefelter syndrome (18 cases), 47, XYY (14 cases), and trisomy X (12 cases). Among 19 NIPT-tested cases, 10 were true positives, 5 false positives, and 4 false negatives, including two mosaic Turner syndrome cases undetected by NIPT. Discordances were attributed to factors such as mosaicism and placental anomalies. Conclusions: Prenatal diagnosis of SCAs via invasive testing remains crucial due to NIPT’s limited sensitivity for mosaicism and false positives. Comprehensive genetic counseling is essential to navigate diagnostic uncertainties and optimize prenatal management and postnatal outcomes. Full article