Search Results (106)

Background/Objectives: The gene pool of the Apennine wolf is affected by admixture with domestic variants due to anthropogenic hybridisation with dogs. Genetic monitoring at the population level involves assessing the extent of admixture in single individuals, ranging from pure wolves to recent hybrids or wolf backcrosses, through the analysis of nuclear and mitochondrial DNA (mtDNA) markers. Although individually non-diagnostic, mtDNA is nevertheless essential for completing the final diagnosis of genetic admixture. Typically, the identification of wolf mtDNA haplotypes is carried out via sequencing of coding genes and non-coding DNA stretches. Our objective was to develop a fast real-time PCR assay to detect the mtDNA haplotypes that occur exclusively in the Apennine wolf population, as a valuable alternative to the demanding sequence-based typing. Methods: We validated a qualitative duplex real-time PCR that exploits the combined presence of diagnostic point mutations in two mtDNA segments, the NDH-4 gene and the control region, and is performed in a single-tube step through TaqMan-MGB chemistry. The aim was to detect mtDNA multi-fragment haplotypes that are exclusive to the Apennine wolf, bypassing sequencing. Results: Basic validation of 149 field samples, consisting of pure Apennine wolves, dogs, wolf × dog hybrids, and Dinaric wolves, showed that the assay is highly specific and sensitive, with genomic DNA amounts as low as 10⁻⁵ ng still producing positive results. It also proved high repeatability and reproducibility, thereby enabling reliable high-throughput testing. Conclusions: The results indicate that the assay presented here provides a valuable alternative method to the time- and cost-consuming sequencing procedure to reliably diagnose the maternal lineage of the still-threatened Apennine wolf, and it covers a wide range of applications, from scientific research to conservation, diagnostics, and forensics. Full article

A series of incidents involving damage to vehicle speed sensor cables occurred in an urban area in Japan. At the request of the police, DNA analysis was conducted to identify the animal species responsible. Swab samples collected from the damaged sections of the cables were subjected to PCR testing using mtDNA fragments. Sequencing analysis with universal primers (SCPH02500, SCPL02981) detected DNA from the Japanese marten (Martes melampus). A comprehensive examination that included morphological analysis of the cable damage and consideration of the ecological characteristics of the Japanese martens suggested that the damage was likely caused by this species. DNA analysis using mtDNA markers is a valuable tool for species identification in wildlife forensic veterinary investigations and serves as important scientific evidence in criminal cases involving animals. The findings from this case may contribute to future investigations in forensic veterinary science and ecological research and may also inform measures to prevent human–wildlife conflicts involving animals. Full article

The Eurasian lynx (Lynx lynx) is listed in CITES Appendix II and is protected under the Bern Convention and the EU Habitats Directive, yet it remains a frequent target of wildlife crime, highlighting the urgent need for reliable identification methods. This study focuses on determination and DNA quantification of the Lynx spp. using quantitative real-time PCR (qPCR). The Llynx Qplex quantification multiplex system effectively distinguishes Lynx spp. from other Feliformia species by targeting mitochondrial and nuclear markers. Additionally, we present the results of the developmental validation of the Llyn STRplex system for individual identification and databasing using six STR loci. This study followed ISFG recommendations for non-human DNA testing and developmental validation guidelines. Both systems demonstrate high sensitivity (5 pg genomic DNA for Llynx Qplex and 30 pg of mtDNA for Llyn STRplex) and high specificity to Lynx spp., confirmed by testing against 16 related Feliformia species. Robustness was evaluated, showing sensitivity to temperature variation, and both repeatability and reproducibility were successfully tested across replicates and conditions. Given that forensic casework often involves degraded and limited biological material, molecular tools must be both sensitive and specific to ensure accurate results. Developing precise and efficient tools is essential for supporting investigations of wildlife crime involving the Eurasian lynx, as well as efforts aimed at conserving the species. Full article

Background: Smilax glabra (S. glabra) and Smilax zeylanica (S. zeylanica), two medicinally important species within the genus Smilax, have been widely used in Traditional Chinese Medicine (TCM) for the treatment of rheumatism, traumatic injuries, and related ailments. Despite their medicinal significance, research on the mitochondrial DNA (mtDNA) of Smilax species remains limited. Methods: We utilized NovaSeq 6000 and PromethION sequencing platforms to assemble the complete mitochondrial genomes of Smilax glabra and Smilax zeylanica, and conducted in-depth comparative genomic and evolutionary analyses. Results: The complete mitochondrial genomes of S. glabra and S. zeylanica were assembled and annotated, with total lengths of 535,215 bp and 471,049 bp, respectively. Both genomes encode 40 unique protein-coding genes (PCGs), composed of 24 core and 16 non-core genes, alongside multiple tRNA and rRNA genes. Repetitive element analysis identified 158 and 403 dispersed repeats in S. glabra and S. zeylanica, respectively, as well as 123 and 139 simple sequence repeats (SSRs). RNA editing site predictions revealed C-to-U conversions in both species. Additionally, chloroplast-to-mitochondrial DNA migration analysis detected 34 homologous fragments in S. glabra and 28 homologous fragments in S. zeylanica. Phylogenetically, S. glabra and S. zeylanica cluster within the Liliales order and Smilacaceae family, closely related to Lilium species. Collinearity analysis indicated numerous syntenic blocks between Smilax and three other Liliopsida species, though gene order was not conserved. Conclusions: This study presents high-quality mitochondrial genome assemblies for S. glabra and S. zeylanica, providing valuable insights into molecular identification and conservation efforts of these traditional medicinal plants. Full article

This Systematic Literature Review (SLR) consolidates current research on mitochondrial DNA (mtDNA) analysis in horses, focusing on genetic variation, maternal lineage tracing, and haplogroup identification. The article selection process screened 1380 articles, with 76 fulfilling the inclusion criteria. Data extraction covered sampling techniques, studied mtDNA regions, sequencing methods, and haplogroup identification. Following the methodology of the PRISMA guidelines, this review encompasses studies published since 2012, obtained from Scopus, PubMed, Research4Life, Web of Science, and ScienceDirect. The major findings emphasise the use of mtDNA for tracing ancestry, validating maternal lineages, and identifying haplogroups along with their geographic distributions. This review identifies challenges, including the need to update the haplogroup classification system and potential information loss due to sequence trimming. Additionally, it examines promising avenues for future research, such as the implementation of next-generation sequencing and the merging of haplogroup data with performance traits, which could influence conservation initiatives and breeding programs. This review emphasises the necessity for standardised classification systems and further research on underrepresented breeds and regions to improve our understanding of equine genetic diversity. Full article

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers. Full article

Gliomas are the most common primary brain tumors, representing approximately 28% of all central nervous system tumors. These tumors are characterized by rapid progression and show a median survival of approximately 18 months. The therapeutic options consist of surgical resection followed by radiotherapy and chemotherapy. Despite the multidisciplinary approach and the biomolecular role of targeted therapies, the median progression-free survival is approximately 6–8 months. The incomplete tumor compliance with treatment is due to several factors such as the presence of the blood–brain barrier, the numerous pathways involved in tumor transformation, and the presence of intra-tumoral mutations. Among these, the interaction between the mutations of genes involved in tumor bio-energetic metabolism and the functional response of the tumor has become the protagonist of numerous studies. In this scenario, the main role is played by mitochondria, cellular organelles delimited by a double membrane and containing their own DNA (mtDNA), which participates in numerous cellular processes such as the regulation of cellular metabolism, cellular proliferation, and apoptosis and is also the main source of cellular energy production. Therefore, it is understood that the mitochondrion, specifically its functional alteration, is a leading figure in tumor transformation, including brain tumors. The acquisition of mutations in the mitochondrial DNA of tumor cells and the subsequent identification of the so-called mitochondria-related genes (MRGs), both functional (mutation of Complex I) and structural (mutations of Complex III/IV), have been seen to play an important role in metabolic reprogramming with increased proliferation, resistance to apoptosis, and the progression of tumorigenesis. This demonstrates that these mitochondrial alterations could have a role not only in the intrinsic tumor biology but also in the extrinsic one associated with the therapeutic response. We aim to summarize the main mitochondrial dysfunction interactions present in gliomas and how they might impact prognosis. Full article

Analysis of mitochondrial DNA, specifically the cytochrome b gene (cyt b), has become an essential tool for species identification. In the case of degraded samples, in which DNA is fractionated, universal primers, which are highly effective at amplifying the target region, are necessary. The material analysed in this study was a keychain made of bone, which was secured at a border crossing due to the suspicion that it was made of ivory. Due to processing of the bone and the likelihood of DNA degradation, five pairs of universal primers with different product lengths (from 148 to 990 base pairs) were used for species identification. Fragments of mtDNA from the cyt b and the 12S rRNA and 16S rRNA subunits were analysed. The analysis showed that only one pair of primers (L15601/H15748) enabled identification of the species, which is very common in samples with highly degraded DNA. The material was bone tissue belonging to the species Bos taurus (cattle). Species identification by molecular methods is extremely important in analysis of material when the species cannot be identified on the basis of morphological characteristics. Full article

Medical care for domestic dogs is now respected worldwide as being at a similar level to that of humans. We previously established a test method to determine whole mitochondrial DNA (mtDNA) using oral mucosal DNA that may be useful for medical care and welfare. However, the sample types tested in dogs are not limited to those obtained from the oral mucosa. Therefore, in the present study, we attempted to establish a test method to determine whole mtDNA sequences using feces, which represents the least invasive specimen. Two Japanese domestic dogs were used in the present study. DNA was extracted from approximately 100 mg of fresh feces from each dog, and PCRs were performed using four primer pairs that can amplify whole mtDNA. Following PCR, amplicons were pooled to create a DNA library using an experimental robot with an original program. Data were then acquired via NGS and data analysis was performed. The results showed that the whole mtDNA sequence of the two dogs was determined with high accuracy. Our results suggest that feces can be adapted for mitochondrial disease and individual identification testing and could serve as a useful testing method for the future medical care and welfare of domestic dogs. Full article

The introduction of new sequencing approaches into clinical practice has radically changed the diagnostic approach to mitochondrial diseases, significantly improving the molecular definition rate in this group of neurogenetic disorders. At the same time, there have been no equal successes in the area of in-depth understanding of disease mechanisms and few innovative therapeutic approaches have been proposed recently. In this regard, the identification of the molecular basis of phenotypic variability in primary mitochondrial disorders represents a key aspect for deciphering disease mechanisms with important therapeutic implications. In this study, we present data from proteomic investigations in two subjects affected by mitochondrial disease characterized by a different clinical severity and associated with the same variant in the TWNK gene, encoding the mitochondrial DNA and RNA helicase with a specific role in the mtDNA replisome. Heterozygous pathogenic variants in this gene are associated with progressive external ophthalmoplegia and ptosis, usually with adult onset. The overall results suggest an imbalance in glucose metabolism and ROS production/regulation, with possible consequences on the phenotypic manifestations of the enrolled subjects. Although the data will need to be validated in a large cohort, proteomic investigations have proven to be a valid approach for a deep understanding of these neurometabolic disorders. Full article

NSCLC is marked by low survival and resistance to platinum-based chemotherapy. The XPG endonuclease has emerged as a promising biomarker for predicting the prognosis of cisplatin-treated patients and its downregulation having been reported to increase cisplatin efficacy. This study presents an integrated strategy for identifying small molecule inhibitors of XPG to improve cisplatin therapy in NSCLC. A structure-based virtual screening approach was adopted, including a structural and physicochemical analysis of the protein, and a library of small molecules with reported inhibitory activities was retrieved. This analysis identified Lys84 as a crucial residue for XPG activity by targeting its interaction with DNA. After molecular docking and virtual screening calculations, 61 small molecules were selected as potential XPG inhibitors, acquired from the ChemBridge database and then validated in H1299 cells, a NSCLC cell line exhibiting the highest ERCC5 expression. The MTS assay was performed as a first screening approach to determine whether these potential inhibitors could enhance cisplatin-induced cytotoxicity. Overall, among the eight compounds identified as the most promising, three of them revealed to significantly increase the impact of cisplatin. The inherent cytotoxicity of these compounds was further investigated in a non-tumoral lung cell line (BEAS-2B cells), which resulted in the identification of two non-cytotoxic candidates to be used in combination with cisplatin in order to improve its efficacy in NSCLC therapy. Full article

Exposure to ionizing radiation can result in the development of a number of diseases, including cancer, cataracts and neurodegenerative pathologies. Certain occupational groups are exposed to both natural and artificial sources of radiation as a consequence of their professional activities. The development of non-invasive biomarkers to assess the risk of exposure to ionizing radiation for these groups is of great importance. In this context, our objective was to identify epigenetic and molecular biomarkers that could be used to monitor exposure to ionizing radiation. The impact of X-ray exposure on the miRNAs profile and the level of cf mtDNA were evaluated using the RT-PCR method. The levels of pro-inflammatory cytokines in their blood were quantified using the ELISA method. A significant decrease in miR-19a-3p, miR-125b-5p and significant increase in miR-29a-3p was observed in the blood plasma of individuals exposed to X-ray. High levels of pro-inflammatory cytokines and cf mtDNA were also detected. In silico identification of potential targets of these miRNAs was conducted using MIENTURNET. VDAC1 and ALOX5 were identified as possible targets. Our study identified promising biomarkers such as miRNAs and cf mtDNA that showed a dose-dependent effect of X-ray exposure. Full article

Two stranded Lepidochelys-like sea turtles were rescued from the Thai Andaman Sea coastline by veterinarians of the Phuket Marine Biological Center (PMBC), one in May of 2019 and another in July of 2021. They were first identified as olive ridley turtles (Lepidochelys olivacea), as the external appearance of both turtles was closer to that species than the other four species found in the Thai Andaman Sea. In fact, when carefully examined, an unusual pattern of the lateral scutes on each turtle was observed, specifically symmetric 5/5 and asymmetric 5/6, both of which are considered rare for L. olivacea and had never been reported in the Thai Andaman Sea. In contrast, this characteristic was more common for the closely related species, Kemp’s ridley (L. kempii), although this species is not distributed in the Indo-Pacific Ocean. Thus, we further investigated their genetic information to confirm species identification using two molecular markers, namely the mtDNA control region and nDNA RAG2. The results from the mtDNA control region sequences using the Basic Local Alignment Search Tool (BLAST) indicated that both individuals exhibited a higher percent identity with L. olivacea (99.81–100.00%) rather than L. kempii (94.29–95.41%) or any other species. A phylogenetic tree confirmed that these two turtles belonged to the L. olivacea clade. Moreover, the results of RAG2 also supported the mtDNA result, as both individuals shared the same RAG2 haplotype with L. olivacea. Thus, we have concluded that the two turtles with unusual lateral scute patterns exhibited genetic consistency with their original species, L. olivacea, which has brought attention to the importance of exploring rare phenotypes in sea turtle populations residing in Thai Seas. Full article

Colorectal cancer (CRC) currently ranks as the third most common cancer and the second leading cause of cancer-related deaths worldwide, posing a significant global health burden to the population. Recent studies have reported the emergence of a new clinical picture of the disease, with a notable increase in CRC rates in younger populations of <50 years of age. The American Cancer Society (ACS) now recommends CRC screening starting at age 45 for average-risk individuals. Dr. Aasma Shaukat’s Keynote Conference highlights the critical need for updated screening strategies, with an emphasis on addressing the suboptimal adherence rates and the effective management of the growing burden of CRC. Lowering the adenoma detection screening age can facilitate early identification of adenomas in younger asymptomatic patients, altering the epidemiologic landscape. However, its implications may not be as profound unless a drastic shift in the age distribution of CRC is observed. Currently, various screening options are available in practice, including stool-based tests like multitarget stool DNA (mtDNA) tests, fecal immunochemical testing (FIT), and imaging-based tests. In addition to existing screening methods, blood-based tests are now emerging as promising tools for early CRC detection. These tests leverage innovative techniques along with AI and machine learning algorithms, aiding in tumor detection at a significantly earlier stage, which was not possible before. Medicare mandates specific criteria for national coverage of blood-based tests, including sensitivity ≥ 74%, specificity ≥ 90%, FDA approval, and inclusion in professional society guidelines. Ongoing clinical trials, such as Freenome, Guardant, and CancerSEEK, offer hope for further advancements in blood-based CRC screening. The development of multicancer early detection tests like GRAIL demonstrates a tremendous potential for detecting various solid tumors and hematologic malignancies. Despite these breakthroughs, the question of accessibility and affordability still stands. The ever-evolving landscape of CRC screening reflects the strength of the scientific field in light of an altered disease epidemiology. Lowering screening age along with the integration of blood-based tests with existing screening methods holds great potential in reducing the CRC-related burden. At the same time, it is increasingly important to address the challenges of adaptation of the healthcare system to this change in the epidemiologic paradigm. Full article

Introduction: Cell-free nucleic acids (cf-NAs) represent a promising biomarker of various pathological and physiological conditions. Since its discovery in 1948, cf-NAs gained prognostic value in oncology, immunology, and other relevant fields. In peritoneal dialysis (PD), blood purification is performed by exposing the peritoneal membrane. Relevant sections: Complications of PD such as acute peritonitis and peritoneal membrane aging are often critical in PD patient management. In this review, we focused on bacterial DNA, cell-free DNA, mitochondrial DNA (mtDNA), microRNA (miRNA), and their potential uses as biomarkers for monitoring PD and its complications. For instance, the isolation of bacterial DNA in early acute peritonitis allows bacterial identification and subsequent therapy implementation. Cell-free DNA in peritoneal dialysis effluent (PDE) represents a marker of stress of the peritoneal membrane in both acute and chronic PD complications. Moreover, miRNA are promising hallmarks of peritoneal membrane remodeling and aging, even before its manifestation. In this scenario, with multiple cytokines involved, mtDNA could be considered equally meaningful to determine tissue inflammation. Conclusions: This review explores the relevance of cf-NAs in PD, demonstrating its promising role for both diagnosis and treatment. Further studies are necessary to implement the use of cf-NAs in PD clinical practice. Full article