ND2 Gene Research Articles

Climbing on the La Canna Volcanic Sea Stack to Obtain First-Hand Data on the Tiniest Population of the Critically Endangered Aeolian Wall Lizard Podarcis raffonei.

Among the extant populations of the critically endangered Aeolian wall lizard, the most vulnerable is the one surviving on La Canna, a columnar volcanic stack off the Filicudi Island. Here, I report the results of the first climbing expedition by a biologist on La Canna, that contributed direct observations and updated information on the size, morphology, and genetic variability of this population. Lizard density at the sampling site (a small terrace at 50 m of elevation) was 1.7 m-2, twice of a previous estimate. Standard methods for estimating population size are unsuitable for La Canna. An educated guess of about a hundred individuals can be drawn, considering the extent of habitat available on the stack and the number of observed lizards. Lizards on La Canna were not fearless, despite what was reported by alpinists, possibly because of aggressive intraspecific interactions or high environmental temperatures during sampling. Biometric data significantly extend the body size of La Canna's lizards and indicate that it is not smaller than other P. raffonei populations. A complete lack of genetic diversity was found at the mitochondrial nd4 gene, in line with previous allozyme data and with estimates on other microinsular Podarcis populations. The small size of the La Canna population implies severe genetic drift and an extremely high level of inbreeding, as supported by low heterozygosity found across the genome. Detrimental effects of inbreeding depression are evident as cephalic malformations observed in all captured lizards of La Canna and might represent the more immediate threat to the persistence of this population.

Pleistocene Glacial Refugia in the Don River Basin: Witness from the Endangered Depressed River Mussel

The depressed river mussel Pseudanodonta complanata (Rossmässler, 1835) is an endangered freshwater bivalve with sparse distribution across Europe. Despite its high conservation priority, available data on the current population genetics and biogeography of P. complanata are very limited. In this paper, we studied the genetic diversity and differentiation of P. complanata based on newly sequenced COI and ND1 gene fragments, and on available sequences from the NCBI GenBank. We discovered a localized and highly divergent lineage in the Don River basin and a widespread but low diversified lineage distributed from the Volga River basin to the Oise River in France. A star-like haplotype network, results of mismatch analysis, and significant negative values of neutrality tests reveal that the pan-European lineage went through a bottleneck event. The discovery of the separate lineage in the Don River basin is and additional evidence of the existence of Pleistocene glacial refugia in the south of the Russian Plain. Moreover, this finding confirms the significance of the Azov Sea drainage as a freshwater biodiversity hotspot of great conservation importance.

Ecological and physiological analysis of immune reactions of <i>Pelophylax ridibundus</i> and <i>P. lessonae</i> (Amphibia: Ranidae) in anthropogeneously transformed territories

The purpose of the work is to assess the state of Anura populations living in an anthropo-genic territory according to a set of indicators of the body’s immune homeostasis. The objects of the study were Pelophylax ridibundus (Pallas, 1771) and P. lessonae (Camerano, 1882), living in the natural populations of reservoirs in Nizhny Novgorod. Priority chemical pollutants of the water bodies were determined by spectrophotometry. Species were identified using a multiplex PCR test system. The identification results were confirmed by sequencing of the mitochondrial ND2 gene and a fragment of the SAI protein. We counted the numbers of erythrocytes and leukocytes, determined the leukocyte profile and the level of immune complexes in all frogs. An excess of the water quality standard for the content of heavy metals was revealed in all the water bodies. Molecular genetic diagnostics showed the presence of both “pure” P. ridibundus and individuals with introgressive mtDNA of the Anatolian form of the lake frog (P. cf. bedriagae) in the sample of lake frogs. In the sample of pond frogs, all studied individuals had only species-specific mt- and nDNA markers of P. lessonae. P. ridibundus differed from P. lessonae by an increased content of erythrocytes, neutrophils, basophils, small immune complexes, and a reduced content of lymphocytes. The revealed changes in the immunohematological parameters of green frogs were caused by the complex henotoxic effect of pollutants in the water bodies. A decrease in the lymphocytes/eosinophils ratio index was shown with an increase in the concentration of nitrites, an increased activity of humoral immune responses in conditions of sulfate pollution of the aquatic environment, an increase in the proportion of myelocytes in the blood of frogs with an increased concentration of manganese and nitrates in water. Under conditions of environmental stress, the blood regulatory systems of frogs reflected a stress-induced reaction, which was more pronounced in the body of lake frogs compared to pond ones.

An expanded description of Cnemaspis gracilis (Beddome 1870) (Squamata: Gekkonidae) based on recent material.

Cnemaspis gracilis is a poorly known species that has been reported from across southern India, with 11 named species within the clade. The species is known only from the type series, as many other records from areas outside the type locality have turned out to represent distinct species. We provide an expanded morphological description of the species based on topotypic material and other samples that were assigned using genetic data. Cnemaspis gracilis is now known with certainty from three localities in the vicinity of Palakkad, Palakkad District, Kerala, and Valparai town, Coimbatore district, Tamil Nadu, besides from an introduced population in the Timber Market, Kolhapur city, Kolhapur district, Maharashtra, India. The species is 7.6-15.8 % divergent on the ND2 gene from described members of the gracilis clade and can be diagnosed by a combination of morphological characters including body size, number of paravertebral tubercles between limb insertions, number of dorsal tubercle rows, number of ventral scale rows across the belly, number of femoral and precloacal pores and poreless scales separating these series in males, and the presence of a single central black ocellus on the neck and a smaller one on the occiput. The assignment of extant populations to the species, a detailed morphological description and genetic data will allow the description of many more species from within this diverse clade of diurnal geckos.

Mitochondrial DNA methylation in metabolic associated fatty liver disease.

Hepatic lipid accumulation and mitochondrial dysfunction are hallmarks of metabolic associated fatty liver disease (MAFLD), yet molecular parameters underlying MAFLD progression are not well understood. Differential methylation within the mitochondrial DNA (mtDNA) has been suggested to be associated with dysfunctional mitochondria, also during progression to Metabolic Steatohepatitis (MeSH). This study further investigates whether mtDNA methylation is associated with hepatic lipid accumulation and MAFLD. HepG2 cells were constructed to stably express mitochondria-targeted viral and prokaryotic cytosine DNA methyltransferases (mtM.CviPI or mtM.SssI for GpC or CpG methylation, respectively). A catalytically inactive variant (mtM.CviPI-Mut) was constructed as a control. Mouse and human patients' samples were also investigated. mtDNA methylation was assessed by pyro- or nanopore sequencing. Differentially induced mtDNA hypermethylation impaired mitochondrial gene expression and metabolic activity in HepG2-mtM.CviPI and HepG2-mtM.SssI cells and was associated with increased lipid accumulation, when compared to the controls. To test whether lipid accumulation causes mtDNA methylation, HepG2 cells were subjected to 1 or 2 weeks of fatty acid treatment, but no clear differences in mtDNA methylation were detected. In contrast, hepatic Nd6 mitochondrial gene body cytosine methylation and Nd6 gene expression were increased in mice fed a high-fat high cholesterol diet (HFC for 6 or 20 weeks), when compared to controls, while mtDNA content was unchanged. For patients with simple steatosis, a higher ND6 methylation was confirmed using Methylation Specific PCR, but no additional distinctive cytosines could be identified using pyrosequencing. This study warrants further investigation into a role for mtDNA methylation in promoting mitochondrial dysfunction and impaired lipid metabolism in MAFLD.

A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics

BackgroundMitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed.ResultsWe found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology.ConclusionsMany human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

Complete mitochondrial genomes of Gritzenko's charr Salvelinus gritzenkoi and Miyabe charr Salvelinus malma miyabei and their interpretation regarding intraspecific taxonomy.

Salvelinus is one the most species-rich genera of salmonids (Salmoniformes: Salmonidae), and mitogenomic sequence data have served as a rich source of information for resolving fish phylogenies and identifying new charr species. However, current reference databases contain limited mitochondrial genome information on narrow-ranged, endemic charrs with disputable origin and systematic status. More comprehensive mitochondrial genome-based phylogenetics will help enhance understanding of the relationship and delineate charr species. In the present study, the complete mitochondrial genomes of three taxa (S. gritzenkoi, S malma miyabei, and S. curilus) were sequenced (PCR and Sanger dideoxy sequencing) and compared with those of other charr species whose mitochondrial genomes are already reported. Results indicate that the mitochondrial genomes of the three taxa were similar in length (16,652bp and 16,653bp for S. curilus and S. malma miyabei, to 16,658bp for S. gritzenkoi). Analyses of nucleotide composition showed that the newly sequenced five mitochondrial genomes skewed strongly towards high AT (54.4%) content, which is typical of Salvelinus. No large deletions and insertions were found in the mitochondrial genomes, including samples from isolated populations. Heteroplasmy associated with single-nucleotide substitution in the ND1 gene was found in one case (S. gritzenkoi). In maximum likelihood and Bayesian inference trees, S. gritzenkoi and S malma miyabei were clustered with S. curilus with strong branch support. Our results provide the basis for a possible reclassification of S. gritzenkoi to S. curilus. The results of this study may be useful for future genetic studies on charrs of the genus Salvelinus toward the in-depth phylogenetic study and proper assessment of the conservation status of the disputed taxa.

Aspirin alleviates cisplatin-induced acute kidney injury through the AMPK-PGC-1α signaling pathway

Cisplatin (CIS) is a widely used clinical chemotherapeutic agent for solid malignancies such as lung, testicular and ovarian cancers, but the development of nephrotoxicity has limited the use of this class of drugs. Some studies have shown that aspirin can reduce cisplatin-induced nephrotoxicity, but the mechanism of protection is not yet clear. By establishing a mouse model of cisplatin-induced acute kidney injury and a mouse model of aspirin combination, we observed a reduction in creatinine, blood urea nitrogen, and tissue damage, thus verifying that aspirin can alleviate cisplatin-induced acute kidney injury in mice. Aspirin was found to have a significant protective effect against cisplatin-induced acute kidney injury, as evidenced by the reduction in levels of ROS, NO, and MDA and the increase in T-AOC, CAT, SOD, and GSH. Furthermore, aspirin was observed to down-regulate the expression of pro-inflammatory factors TNF-α, NF-κB, IL-1β, and IL-6 mRNA and proteins, increase the expression of BAX and Caspase3 as indicators of apoptosis, decrease the expression of Bcl-2, and improve the reduced expression of mtDNA, ATP content, ATPase activity and mitochondrial respiratory chain complex enzyme-related genes ND1, Atp5b, and SDHD. These findings suggest that the protective effects of aspirin are associated with its anti-inflammatory, antioxidant, anti-apoptotic properties, and its ability to maintain mitochondrial function, as demonstrated by the detection of AMPK-PGC-1α pathway-related genes. The results showed that the reduced expression of p-AMPK and mitochondrial production-related mRNA PGC-1α, NRF1, and TFAM in the kidney tissue of mice in the cisplatin group was alleviated by the effect of aspirin, indicating that aspirin could activate the p-AMPK, regulate mitochondrial production and alleviate cisplatin acute kidney injury through the AMPK-PGC-1α pathway. In summary, certain doses of aspirin protect the body from acute kidney injury by alleviating the cisplatin-induced inflammatory response oxidative stress, mitochondrial dysfunction, and apoptosis. Further studies have shown that the protective effect of aspirin is associated with AMPK-PGC-1α pathway activation.

Stronger selective constraints on the mitochondrial genome in flying fishes

Flying fishes, which use their wing-like pectoral fins and hypocercal caudal fin to glide through the air to avoid underwater predators, have independently evolved flight behavior, making them ideal for the study of adaptive evolution. To investigate the adaptation of flight behavior in flying fishes and the origin of Beloniformes fishes, this study obtained the complete mitochondrial genomes of Cheilopogon nigricans and Oxyporhamphus micropterus and constructed the DNA sequences extracted from these newly sequenced mitochondrial genomes with the DNA sequences of 32 previously published mitochondrial genomes into a dataset for reconstructing the phylogenetic relationships of Beloniformes fishes. The phylogeny that emerged strongly supported the possibility that flying fishes developed from halfbeaks and the progressive transition of flying fishes from two-wing to four-wing gliding. The divergence time analysis showed that the split between the suborder Belonidei and the family Adrianichthyidae occurred roughly 77.08 Mya, which fell within the period of evolution of the Indian plate in the late Cretaceous. Selection analyses revealed that flying fishes have a lower dN/dS ratio than the other members of Beloniformes, indicating that flying fishes experienced stronger purifying selection to eliminate deleterious mutations to maintain efficient energy metabolism to adapt to flight behavior. Moreover, this work found the positively selected signal in the ND4 gene, suggesting that different mitogenomic genes might have undergone different selective patterns during adaptive evolution.

Novel mitochondrial genome rearrangements including duplications and extensive heteroplasmy could underlie temperature adaptations in Antarctic notothenioid fishes

Mitochondrial genomes are known for their compact size and conserved gene order, however, recent studies employing long-read sequencing technologies have revealed the presence of atypical mitogenomes in some species. In this study, we assembled and annotated the mitogenomes of five Antarctic notothenioids, including four icefishes (Champsocephalus gunnari, C. esox, Chaenocephalus aceratus, and Pseudochaenichthys georgianus) and the cold-specialized Trematomus borchgrevinki. Antarctic notothenioids are known to harbor some rearrangements in their mt genomes, however the extensive duplications in icefishes observed in our study have never been reported before. In the icefishes, we observed duplications of the protein coding gene ND6, two transfer RNAs, and the control region with different copy number variants present within the same individuals and with some ND6 duplications appearing to follow the canonical Duplication-Degeneration-Complementation (DDC) model in C. esox and C. gunnari. In addition, using long-read sequencing and k-mer analysis, we were able to detect extensive heteroplasmy in C. aceratus and C. esox. We also observed a large inversion in the mitogenome of T. borchgrevinki, along with the presence of tandem repeats in its control region. This study is the first in using long-read sequencing to assemble and identify structural variants and heteroplasmy in notothenioid mitogenomes and signifies the importance of long-reads in resolving complex mitochondrial architectures. Identification of such wide-ranging structural variants in the mitogenomes of these fishes could provide insight into the genetic basis of the atypical icefish mitochondrial physiology and more generally may provide insights about their potential role in cold adaptation.

Mito-nuclear discordance in the phenotypically variable Andean hummingbirdsCoeligena bonaparteiandCoeligena helianthea(Trochilidae)

AbstractThe interplay among evolutionary mechanisms like gene flow and selection may result in discordant signals between mitochondrial DNA, nuclear markers and phenotypes. The Andean hummingbirds Coeligena bonapartei and Coeligena helianthea showed differentiation in the gene ND2 which is discordant with plumage coloration but consistent with geography. We analysed complete mitochondrial genomes of individuals from Coeligena bonapartei bonapartei, Coeligena bonapartei consita, Coeligena helianthea helianthea, and Coeligena helianthea tamai to inform their evolutionary history. We found genetic structure despite low genetic differentiation among these populations. Phylogenetic and network analyses based on mitogenomes showed a northern vs. southern differentiation pattern which is discordant with the relationships based on nuclear markers and the coloration phenotypes (serving as a basis for taxonomy). Mitogenomes of the two nominate subspecies are indistinguishable, suggesting incomplete lineage sorting or introgression, while those of C. b. consita and C. h. tamai are more similar to each other than they are to their respective nominate subspecies. Our results indicate that various evolutionary mechanisms drove the divergence in phenotypes, and nuclear and mitochondrial genomes of Coeligena hummingbirds, playing out over a complex biogeographic scenario likely involving periods of isolation and secondary contact. We outline hypotheses to be tested with future analyses of genome-wide variation.

Adaptive evolution characteristics of mitochondrial genomes in genus Aparapotamon (Brachyura, Potamidae) of freshwater crabs

BackgroundAparapotamon, a freshwater crab genus endemic to China, includes 13 species. The distribution of Aparapotamon spans the first and second tiers of China's terrain ladder, showing great altitudinal differences. To study the molecular mechanisms of adaptive evolution in Aparapotamon, we performed evolutionary analyses, including morphological, geographical, and phylogenetic analyses and divergence time estimation. We sequenced the mitogenomes of Aparapotamon binchuanense and Aparapotamon huizeense for the first time and resequenced three other mitogenomes of Aparapotamon grahami and Aparapotamon gracilipedum. These sequences were combined with NCBI sequences to perform comparative mitogenome analysis of all 13 Aparapotamon species, revealing mitogenome arrangement and the characteristics of protein-coding and tRNA genes.ResultsA new species classification scheme of the genus Aparapotamon has been detected and verified by different aspects, including geographical, morphological, phylogenetics and comparative mitogenome analyses. Imprints from adaptive evolution were discovered in the mitochondrial genomes of group A, including the same codon loss at position 416 of the ND6 gene and the unique arrangement pattern of the tRNA-Ile gene. Multiple tRNA genes conserved or involved in adaptive evolution were detected. Two genes associated with altitudinal adaptation, ATP8 and ND6, which experienced positive selection, were identified for the first time in freshwater crabs.ConclusionsGeological movements of the Qinghai-Tibet Plateau and Hengduan Mountains likely strongly impacted the speciation and differentiation of the four Aparapotamon groups. After some group A species dispersed from the Hengduan Mountain Range, new evolutionary characteristics emerged in their mitochondrial genomes, facilitating adaptation to the low-altitude environment of China's second terrain tier. Ultimately, group A species spread to high latitudes along the upper reaches of the Yangtze River, showing faster evolutionary rates, higher species diversity and the widest distribution.

A Fisherman's Tale: An Unusual Observation of the Ozark Cavefish, Troglichthys (= Amblyopsis) Rosae (Eigenmann)

Troglichthys (= Amblyopsis) rosae (Ozark Cavefish) is currently known from 83 locations within the Ozark Highlands ecoregion. We found a cavefish at a new location in the Grand Lake O' the Cherokees on the western side of the Neosho River (Delaware County, OK), which is on the northwest periphery of the Ozark Cavefish range. Examination of the mitochondrial ND2 gene supports that the specimen is an Ozark Cavefish, but distinct (4.6–9.2% pairwise distance) from other specimens that have been genetically sampled, and could represent a unique population. Future research should focus on expanding sampling efforts and conducting a range-wide genetic analysis of the Ozark Cavefish.

Five new species of the pelodryadid genus Litoria Tschudi from the southern versant of Papua New Guinea’s Central Cordillera, with observations on the diversification of reproductive strategies in Melanesian treefrogs

New Guinea has the most diverse insular frog fauna in the world, and rates of species discovery and description have increased rapidly in the last two decades. Pelodryadid treefrogs are the second most diverse family of anurans on the island but their taxonomy, relationships, and especially ecology remain poorly documented. Based on differences in morphology, advertisement calls (where available) and phylogenetic analyses of a 787 base pair alignment from the mitochondrial ND4 gene and flanking tRNA, we describe five new species of small treefrogs from hill and lower montane forests in the high rainfall belt that straddles the southern versant of Papua New Guinea's Central Cordillera. Three of these species are known only from forest growing on karst substrates, adding to the growing number of herpetofauna species currently known only from the extensive karst habitats of Papua New Guinea's South-fold Mountains. We also describe the arboreal breeding strategies of two of the new species, and report obligate treehole (phytotelm) breeding in New Guinean frogs for the first time. The new phytotelm-breeding species has juveniles with colour and patterning that closely resemble bird droppings, suggesting defensive mimicry or masquerade. A preliminary phylogeny suggests that arboreal-breeding frogs do not form a monophyletic group and that arboreal breeding has evolved multiple times within the New Guinean pelodryadid radiation. A further striking feature of the phylogeny is poor support for most basal nodes in the most diverse radiation of Melanesian Pelodryadidae, suggesting rapid ecological diversification and speciation, potentially following colonisation from Australia and/or mountain uplift. These new taxa and observations highlight previously unrecognised ecological and reproductive diversity in the Melanesian Pelodryadidae.

Abstract 1965: Racially divergent mitochondrial ND5 gene mutation promotes prostate cancer progression and confers therapeutic resistance

Abstract Compared to European American (EA), African American (AA) men often develop lethal forms of prostate cancer (PCa) and experience high mortality despite having a primary diagnosis of low-risk indolent disease. Understanding the molecular basis of this disparity is thus a dire need for better prevention, accurate monitoring, and guided therapeutics. Aggressive cancer growth is intimately associated with the reprogramming of metabolism and apoptosis, where mitochondrial DNA (mtDNA) alterations play a critical role. In the mitochondrial energy generation cascade, respiratory complex I (RCI) serves as the entry gate to initiate electron transfer through the oxidative phosphorylation system (OXPHOS). Alteration in RCI function could alter metabolism and attenuate apoptosis, hallmarks of malignant progression. Through next-generation sequencing of the entire mitochondrial genome, we identified a prevalence of RCI gene mutations in the AAPCa compared to the EAPCa patients. Among the RCI mutations, a homoplasmic 13709C>G mutation in NADH dehydrogenase 5 (ND5) was identified as the most frequently occurring mtDNA mutation among AA men. Forced expression of the 13709C>G mutation in the mitochondria of the AA-PCa patient-derived MDA PCa 2b and EA-PCa patient-derived LNCaP cells, remarkably enhanced their growth and malignant potential. The increased tumorigenicity was associated with a significant increase in the production of mitochondrial reactive oxygen species (ROS), O2 consumption, overall respiration, and ATP production. The ND5-mutant PCa cells exhibited remarkable resistance to androgen deprivation and docetaxel treatment. Induction of CXCR4, HIF-1α, and NF-kB was also noted in these ND5-mutant PCa cells. In clinical tissue samples, an abundance of CXCR4 protein expression was evident in AA-PCa patients harboring ND5 gene mutation but not in the AA or EA-PCa patients with wild-type ND5. Blockade of CXCR4 expression in the ND5-mutant PCa cells diminished NF-kB activity, thereby suggesting a potential role of the CXCR4/HIF-1α/NF-kB signaling axis in ND5 mutation-driven PCa tumorigenesis. The RCI-ND5 could be an attractive target for therapeutic and biomarker development for the better management of PCa in racially disparate populations. Citation Format: Kunwar Somesh Vikramdeo, Christopher E. Keel, Martin J. Heslin, Seema Singh, Ajay Pratap Singh, Santanu Dasgupta. Racially divergent mitochondrial ND5 gene mutation promotes prostate cancer progression and confers therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1965.

Species-Level Recognition and Redescription of the Kentucky Arrow Darter, Etheostoma spilotum Gilbert (Percidae: litocara)

The Kentucky Arrow Darter, Etheostoma spilotum, endemic to the upper Kentucky River basin of eastern Kentucky, is redescribed and recognized as a distinct species closely related to E. sagitta in the upper Cumberland River basin and E. nianguae in the Osage River drainage (Missouri River basin). Originally described as a subspecies of E. nianguae, it was later considered a full species and then a subspecies of E. sagitta, based on close geographic proximity to Cumberland basin populations and overlapping meristic variation interpreted as character intergradation. We present meristic, morphometric, and genetic data that support species-level recognition of E. spilotum. It differs from E. sagitta by lower counts of total and pored lateral scales, lower counts of caudal peduncle scales, fewer second dorsal-fin rays, and fewer pectoral-fin rays. Interspecific divergence of E. spilotum and E. sagitta is further demonstrated through analyses of variation in the mitochondrial nd2 gene and species delimitation using genome-wide double digest restriction-site associated DNA sequencing. Although allopatrically distributed, both species inhabit upland headwater streams on the Cumberland Plateau and have similar life history characteristics. Endemism, fragmented distributions, and low densities and genetic diversity within populations make these species extremely vulnerable to anthropogenic activities. Etheostoma spilotum was federally listed as threatened in 2016 due to degradation of stream habitat and water quality in the upper Kentucky basin that has eliminated the species from a significant portion of its range.

Assessment of population genetic diversity and genetic structure of the North Chinese leopard (Panthera pardus japonensis) in fragmented habitats of the Loess Plateau, China

Leopard (Panthera pardus) populations on the Chinese Loess Plateau have experienced severe declines and were once on the verge of extinction. Currently, leopards in the Loess Plateau region are mainly scattered in human-dominated fragmented habitats in Shanxi, Shaanxi and Ningxia. The lack of information on the genetic diversity and genetic structure makes difficult to assess the resistance and persistence of this metapopulation to various factors. In this study, we used mitochondrial ND-5 gene sequences and eight microsatellite loci to genetically analyze 97 faecal samples collected from three leopard ranges on the Loess Plateau of China. Among these samples, 62 feces were successfully identified from 22 individuals (6 females, 16 males). Mitochondrial haplotypes showed four different haplotypes and the genetic diversity was moderate (Hd = 0.331, Pi = 0.0036), while SSR showed high diversity (PIC = 0.673). STRUCTURE and TESS analyses revealed that leopard populations on the Loess Plateau appeared to have a distinct population boundary (K = 2) associated with a spatial-geographic barrier (the Yellow River). We estimate that the time of population divergence occurred between 8700 and 8300 years BP. Based on this research, we suggest that the leopard population on both sides of the Yellow River should be divided into two conservation management units to improve the efficiency of conservation management and contribute to the better recovery of the North Chinese leopard population.

Evaluation of Mitochondrial Function in Blood Samples Shows Distinct Patterns in Subjects with Thyroid Carcinoma from Those with Hyperplasia

Metabolic adaptations are a hallmark of cancer and may be exploited to develop novel diagnostic and therapeutic tools. Only about 50% of the patients who undergo thyroidectomy due to suspicion of thyroid cancer actually have the disease, highlighting the diagnostic limitations of current tools. We explored the possibility of using non-invasive blood tests to accurately diagnose thyroid cancer. We analyzed blood and thyroid tissue samples from two independent cohorts of patients undergoing thyroidectomy at the Hospital Universitario 12 de Octubre (Madrid, Spain). As expected, histological comparisons of thyroid cancer and hyperplasia revealed higher proliferation and apoptotic rates and enhanced vascular alterations in the former. Notably, they also revealed increased levels of membrane-bound phosphorylated AKT, suggestive of enhanced glycolysis, and alterations in mitochondrial sub-cellular distribution. Both characteristics are common metabolic adaptations in primary tumors. These data together with reduced mtDNA copy number and elevated levels of the mitochondrial antioxidant PRX3 in cancer tissue samples suggest the presence of mitochondrial oxidative stress. In plasma, cancer patients showed higher levels of cfDNA and mtDNA. Of note, mtDNA plasma levels inversely correlated with those in the tissue, suggesting that higher death rates were linked to lower mtDNA copy number. In PBMCs, cancer patients showed higher levels of PGC-1α, a positive regulator of mitochondrial function, but this increase was not associated with a corresponding induction of its target genes, suggesting a reduced activity in cancer patients. We also observed a significant difference in the PRDX3/PFKFB3 correlation at the gene expression level, between carcinoma and hyperplasia patients, also indicative of increased systemic metabolic stress in cancer patients. The correlation of mtDNA levels in tissue and PBMCs further stressed the interconnection between systemic and tumor metabolism. Evaluation of the mitochondrial gene ND1 in plasma, PBMCs and tissue samples, suggested that it could be a good biomarker for systemic oxidative metabolism, with ND1/mtDNA ratio positively correlating in PBMCs and tissue samples. In contrast, ND4 evaluation would be informative of tumor development, with ND4/mtDNA ratio specifically altered in the tumor context. Taken together, our data suggest that metabolic dysregulation in thyroid cancer can be monitored accurately in blood samples and might be exploited for the accurate discrimination of cancer from hyperplasia.

Men's ND1 gene genetic makeup Toxoplasmosis and Oligospermia affecting couples' infertility

Introduction: Toxoplasma infection was higher in infertile couples than fertile couples, probably due to anti-sperm antibodies that were higher in couples with Toxoplasmosis. Investigations of T. gondii infections in men with infertility showed that among 100 cases of men's infertility, 36% were serologically positive for Toxoplasma-IgG and IgM. It has been concluded that T. gondii can affect men's fertility and result in infertility. Materials and Methods: Selective infertile males were asked about days of sexual abstinence. Seminal fluid samples were collected following a minimum of 2 days and a maximum of 7 days from abstinence. Every patient was given a clean, wide mouth, sterile, dry, graduated plastic and warm disposable container. The samples were obtained by masturbation in a private room near the semen analysis lab to reduce seminal exposure to temperature fluctuations and control the time from collection to analysis. Results: For the ND1 gene, samples of 8 different fertility groups have been sequenced. These sequences have been compared to reference sequences taken from the NCBI database. Several mutations in various nucleotide positions of the ND1 regions have been detected in samples from multiple groups. The base substitution has been positioned on the nucleotides (nts) 3480, 3567, 3591, 3693, and 4216. The T to C evolution was notorious at nt 3480 in ND1 genes. The SNP was detected in an asthenospermia human (Sample code: 010480). Keywords: Sequence, ND1 gen, Oligospermia Toxoplasmosis, Couples infertility

Leber Hereditary Optic Neuropathy in a Family of Carriers of MT-ND5 m.13042G>T (A236S) Novel Variant.

A Slovenian three-generation family with 3 individuals with bilateral optic neuropathy and 2 unaffected relatives with a novel homoplasmic missense variant m.13042G > T (A236S) in the ND5 gene is described. A detailed phenotype at initial diagnosis and a follow-up of bilateral optic neuropathy progression is presented for 2 affected individuals. A detailed phenotype analysis with clinical examination in the early and chronic phase with electrophysiology and OCT segmentation is presented. Genotype analysis with full mitochondrial genome sequencing was performed. Two affected male individuals (maternal cousins) had a profound visual loss at an early age (11 and 20 years) with no recovery. The maternal grandmother exhibited bilateral optic atrophy with a history of visual loss at the age 58 years. The visual loss of both affected male individuals was characterized by centrocecal scotoma, abnormal color vision, abnormal PERG N95, and VEP. Later with disease progression, retinal nerve fiber layer thinning was observed on OCT. We observed no other extraocular clinical features. Mitochondrial sequencing identified a homoplasmic novel variant m.13042G > T (A236S) in the MT-ND5 gene, belonging to a haplogroup K1a. Novel homoplasmic variant m.13042G > T (A236S) in the ND5 gene in our family was associated with Leber hereditary optic neuropathy-like phenotype. However, predicting the pathogenicity of a novel ultra-rare missense variant in the mitochondrial ND5 gene is challenging. Genetic counseling should consider genotypic and phenotypic heterogeneity, incomplete penetrance, haplogroup type, and tissue-specific thresholds.