Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n = 40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.     

Analysis of functional consequences of haplogroup J polymorphisms m.4216T > C and m.3866T > C in human MT-ND1: Mutagenesis of homologous positions in Escherichia coli

MtDNA sequence variation is presumed to be neutral in effect, but associations with diseases and mtDNA haplogroups have been reported. The aim here was to evaluate the functional consequences of m.4216T > C present in haplogroup J. Furthermore, we evaluated m.3866T > C in MT-ND1, a variant detected in a child belonging to haplogroup J and with an isolated complex I deficiency. Homologous substitutions were introduced into Escherichia coli. NADH dehydrogenase domain activity of NDH-1 with either one or both mutations was markedly decreased suggesting that m.4216T > C and m.3866T > C may have an effect on the structural integrity of complex I.     

Next generation molecular diagnosis of mitochondrial disorders

Mitochondrial disorders are by far the most genetically heterogeneous group of diseases, involving two genomes, the 16.6 kb mitochondrial genome and ~ 1500 genes encoded in the nuclear genome. For maternally inherited mitochondrial DNA disorders, a complete molecular diagnosis requires several different methods for the detection and quantification of mtDNA point mutations and large deletions. For mitochondrial disorders caused by autosomal recessive, dominant, and X-linked nuclear genes, the diagnosis has relied on clinical, biochemical, and molecular studies to point to a group of candidate genes followed by stepwise Sanger sequencing of the candidate genes one-by-one. The development of Next Generation Sequencing (NGS) has revolutionized the diagnostic approach. Using massively parallel sequencing (MPS) analysis of the entire mitochondrial genome, mtDNA point mutations and deletions can be detected and quantified in one single step. The NGS approach also allows simultaneous analyses of a group of genes or the whole exome, thus, the mutations in causative gene(s) can be identified in one-step. New approaches make genetic analyses much faster and more efficient. Huge amounts of sequencing data produced by the new technologies brought new challenges to bioinformatics, analytical pipelines, and interpretation of numerous novel variants. This article reviews the clinical utility of next generation sequencing for the molecular diagnoses of complex dual genome mitochondrial disorders.     

Genetic structure of expanding wolf (Canis lupus) populations in Italy and Croatia,and the early steps of the recolonization of the Eastern Alps

After centuries of range contraction and demographic declines wolves are now expanding in Europe, colonizing regions from where they have been absent for centuries. Wolf colonizing the western Alps originate by the expansion of the Italian population. Vagrant wolves of Italian and Dinaric-Balkan origins have been recently observed in the Eastern Alps. In this study we compared the genetic structure of wolf populations in Italy and Croatia, aiming to identify the sources of the ongoing recolonization of the Eastern Alps. DNA samples, extracted from 282 Italian and 152 Croatian wolves, were genotyped at 12 autosomal microsatellites (STR), four Y-linked STR and at the hypervariable part of the mitochondrial DNA control-region (mtDNA CR1). Wolves in Croatia and Italy underwent recent demographic bottlenecks, but they differ in genetic diversity and population structure. Wolves in Croatia were more variable at STR loci (N_A = 7.4, H_O = 0.66, H_E = 0.72; n = 152) than wolves in Italy (N_A = 5.3, H_O = 0.57, H_E = 0.58; n = 282). We found four mitochondrial DNA (mtDNA CR1) and 11 Y-STR haplotypes in Croatian wolves, but only one mtDNA CR1 and three Y-STR haplotypes in Italy. Wolves in Croatia were subdivided into three genetically distinct subpopulations (in Dalmatia, Gorski kotar and Lika regions), while Italian wolves were not sub-structured. Assignment testing shows that the eastern and central Alps are recolonized by wolves dispersing from both the Italian and Dinaric populations. The recolonization of the Alps will predictably continue in the future and the new population will be genetically admixed and very variable with greater opportunities for local adaptations and survival.     

Crossing the border? Structure of the red deer (Cervus elaphus) population from the Bavarian–Bohemian forest ecosystem

Anthropogenic impact such as overhunting and habitat fragmentation has reduced the total red deer population (Cervus elaphus) across Europe. In Germany remaining subpopulations are even confined to designated areas with limited or no gene flow among them. Red deer populations inhabiting the Bavarian–Bohemian forest ecosystem had been divided by a fortified State border between Germany and former Czechoslovakia. To assess red deer genetic diversity more than two decades after the removal of the fortifications, we analysed a population from the Bavarian Forest National Park, Germany, and one from the National Park ?umava, Czech Republic, using 11 microsatellite loci and a 910 bp long section of the mitochondrial control region (mtDNA). Bayesian analyses of microsatellite allele frequencies favoured the presence of a single population in the Bavarian-Bohemian forest ecosystem over other population genetic structures. This admixture was supported by a lack of population pairwise differentiation between German and Czech red deer microsatellite genotypes in the analysis of molecular variance (AMOVA, F_ST = 0.009, p = 0.383). Contrastingly, AMOVA revealed a highly significant matrilinear differentiation of mtDNA between the two samples (Φ_ST = 0.285, p = 0.002), whereby German red deer belonged predominantly to haplogroup A (western Europe) and Czech red deer predominantly to haplogroup C (eastern Europe). In combination, these findings indicated a high degree of philopatry by does and extensive gene flow across the former border mediated by stags. They also identified the Bavarian–Bohemian forest ecosystem as part of a suture zone between western and eastern European red deer matrilines.     

Molecular genetic studies in Saudi population; identified variants from GWAS and meta-analysis in stroke

IntroductionStroke is a multifactorial and heterogeneous disorder, correlates with heritability and considered as one of the major diseases. The prior reports performed the variable models such as genome-wide association studies (GWAS), replication, case-control, cross-sectional and meta-analysis studies and still, we lack diagnostic marker in the global world. There are limited studies were carried out in Saudi population, and we aim to investigate the molecular association of single nucleotide polymorphisms (SNPs) identified through GWAS and meta-analysis studies in stroke patients in the Saudi population.MethodsIn this case-control study, we have opted gender equality of 207 cases and 207 controls from the capital city of Saudi Arabia in King Saud University Hospital. The peripheral blood (5 ml) sample will be collected in two different vacutainers, and three mL of the coagulated blood will be used for lipid analysis (biochemical tests) and two mL will be used for DNA analysis (molecular tests). Genomic DNA will be extracted with the collected blood samples, and specific primers will be designed for the opted SNPs (SORT1-rs646218 and OLR1-rs11053646 polymorphisms) and PCR-RFLP will be performed and randomly DNA sequencing will be carried out to cross check the results.ResultsThe rs646218 and rs11053646 polymorphisms were significantly associated with allele, genotype and dominant models with and without crude odds ratios (OR’s) and Multiple logistic regression analysis (p < 0.05). Correlation between lipid profile and genotypes has confirmed the significant relation between triglycerides and rs646218 and rs1105364 6polymorphisms. However, rs11053646 polymorphism was correlated with HDLC (p = 0.04). Genotypes were examined in both males' vs. males and females' vs. females in cases and control and we concluded that in rs11053646 polymorphisms with male subjects compared between cases and controls found to be associated with dominant model heterozygote genotypes (p < 0.05).ConclusionThe results of the current study confirmed the SORT1 and OLR1 SNPs were associated in the Saudi population. The current results were in the association with the prior study results documented through GWAS and meta-analysis association. However, other ethnic population studies should be performed to rule out in the human hereditary diseases.     

Detection of an East European wolf haplotype puzzles mitochondrial DNA monomorphism of the Italian wolf population

Southern European wolves suffered from reiterated population declines during glacial periods and historically due to human persecution. Differently from other European wolf populations, a single mitochondrial DNA (mtDNA) control region haplotype (W14) has been so far described in the Italian wolves, although no intensive genetic sampling has ever been conducted in historical source populations from central and southern Italy. Using non-invasive genetic techniques, we report the occurrence of an unexpected mtDNA haplotype (W16) in the wolf population of the Abruzzo, Lazio and Molise National Park (PNALM), central Italy. This haplotype, detected in three out of 90 faecal samples from the PNALM, was previously reported in wolves from the North Carpathians, Slovakia and the Balkans only. Microsatellite analysis and molecular sex determination confirmed that the W16 samples belonged to three distinct wolves. Although alternative explanations can be formulated for the origin of this mtDNA haplotype in the otherwise monomorphic Italian wolf population, assignment procedures indicated the likely admixed ancestry of one W16 sample with East European wolves. Anthropogenic introgression with dogs has been detected in the Italian wolf population using nuclear DNA microsatellites, but no population-wide genetic survey had previously reported a mtDNA control region variant in Italian wolves. Our findings strongly suggest that, in addition to wolf × dog hybridization, captive-released wolves or wolf × dog hybrids may successfully interbreed with wolves in the wild, and that human-mediated introgression may occur even in well established protected areas.     

Genealogical surveys show a high rate of non-paternal surname transmission with regional differences in Argentina

Surnames are a vertically transmitted cultural trait that in Argentina follows the paternal line of descent when the paternity is known. There was a lack of empirical information regarding non-paternal surname transmissions among the general population, so we performed 2,550 genealogical interviews, which included 6,954 surname passes, in different regions of this country. We compared the proportion of non-paternal transmissions between the propositus and parental generation and found no significant difference between them (p < 0.01). Inter-population comparisons allowed us to describe 4 regional groups. We also drew models and simulations to estimate how many generations it would take to find that only half of the population maintained the paternal transmission. The lowest proportion of non-paternal transmission was 7.3%, estimating 9 generations (between 225 and 315 years) to find that, at most, half its population keeps following the paternal transmission; the highest proportion was 23%, taking 3 generations (75–105 years). Our results show a high proportion of unrecognized paternities among the general population, a very quick loss of association between male lineages and surnames, and regional proportions with significant differences between each other.     

Decreased arylesterase activity of paraoxonase-1 (PON-1) might be a common denominator of neuroinflammatory and neurodegenerative diseases

High-density lipoprotein (HDL)-bound paraoxonase-1 (PON-1) is mechanistically related to oxidative stress, inflammation and atherosclerosis and this multirole nature positions the enzyme as potential pathogenic player and candidate biomarker for many diseases. Our previous work suggests that decline in serum PON-1 activities, i.e. arylesterase and paraoxonase, might be associated with the occurrence of mild cognitive impairment (MCI) to late onset Alzheimer’s disease (LOAD) or vascular dementia (VAD). The present study aimed to: (1) expand our previous findings in a larger and different population, including patients with LOAD-VAD mixed dementia (MD); (2) explore a possible association between PON-1 and multiple sclerosis (MS); (3) evaluate if cerebrospinal fluid (CSF) levels of PON-1 activities might be useful biomarkers for MS. We found that serum arylesterase, but not paraoxonase, levels of PON-1 were significantly lower in patients affected by MCI (n = 232), VAD (n = 65), LOAD (n = 175), MD (n = 88) as well as those with MS (n = 104) as compared to healthy controls. Notably, the most pronounced decline in this activity was shown by MD (−18%, p < 0.01) and MS (−23%, p < 0.001), while the lowest changes were detected in the MCI group (11%, p < 0.05). Only arylesterase was detectable in the CSF of MS patients and the levels were not significantly different from those detected in the other two neurological control groups. Overall our data suggest that a depressed arylesterase activity could be a common denominator of different neurological diseases which, independently of their peculiar ethiopathogenesis and pathophysiology, appear to be all characterized by an altered systemic redox balance.     

Synthesis and in vitro evaluation of fluorinated diphenyloxide derivatives and sulfur analogs as serotonin transporter ligands

As the serotonin transporter (SERT) is involved in several neurodegenerative and psychiatric disorders; radiopharmaceuticals to image the SERT by PET would be valuable in studying these diseases. To this end we synthesized diphenyloxide derivatives and sulfide analogs, as new tracers, incorporating a fluorine or oxyalkyl fluorinated group on 4′ or 5′-position on phenyl ring B. Three of these exhibited good to high in vitro affinity (7 < K_i < 8 nM) and selectivity for the SERT over the other monoamine transporters.     

Mitochondrial copy number and risk of breast cancer: A pilot study

It has been proposed that the copy number of mitochondria DNA (mtDNA) per cell reflects gene–environment interactions between unknown hereditary factors and exposures affecting levels of oxidative stress. However, whether copy number of mtDNA could be a risk predictor of oxidative stress-related human cancers, such as breast cancer, remains to be determined. To explore the role of mtDNA copy number in breast cancer etiology, we analyzed mtDNA copy number in whole blood from 103 patients with breast cancer and 103 matched control subjects and examined in relation to endogenous antioxidants. Case patients with breast cancer had a statistically significantly higher mtDNA copy number than control subjects (median: 1.29 vs. 0.80, P < 0.01). High mtDNA copy number (above the median in controls) was associated with a statistically significantly increased risk of breast cancer, compared with low copy number (Odds ratio (OR) = 4.67, 95% CI: 2.45–8.92), with a statistically significant dose–response relationship in trend analysis (P < 0.01). Moreover, mtDNA copy number was significantly inversely associated with several important endogenous oxidants and antioxidants in blood in either the cases (total glutathione, CuZn-SOD activity and myeloperoxidase (MPO)) or the controls (catalase (CAT) activity). These results suggest the mtDNA copy number could be associated with risk of breast cancer, perhaps through an oxidative stress mechanism.     

The significance and occurrence of TNF receptor polymorphisms in the Saudi population

Background and objective: On the basis that the inflammatory effects of TNF (tumour necrosis factor) are predominantly mediated through interaction with the TNF receptor-1 (TNFRSF1A), the current study was designed to establish the prevalence of the mutations, R92Q and P46L TNFRSF1A polymorphisms both in the general healthy Saudi population, and in Saudi patients carrying inflammatory diseases such as atherosclerosis or rheumatoid arthritis. We felt it important to report the frequency of the mutations, R92Q and P46L TNFRSF1A polymorphisms in healthy Saudi individuals, and those with inflammatory conditions, as well as to describe the pattern of immunological factors in individuals expressing R92Q or P46L TNFRSF1A. Patients and methods: We collected in PAX gene blood RNA tubes (for RT-PCR and sequencing) 500 blood samples from normal healthy individuals from the West and Center of Saudi Arabia, as well as 100 from patients with atherosclerosis, and 100 patients diagnosed with rheumatoid arthritis. All were screened for the levels of soluble TNF, C-reactive protein (CRP), interleukin6 (IL-6) and sTNFR1. In addition, they were screened for R92Q and P46L TNFRSF1A by RT-PCR. Moreover, phenotype and expression of peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry (FACS). Results: Across 500 normal individuals, 8 (1.6%) expressed both R92Q and P46L mutations. By contrast, of the 100 patients in our study with atherosclerosis, 34% expressed both the R92Q and P46L mutations, whilst 42% of patients with rheumatoid arthritis expressed both mutations R92Q and P46L. No significant differences were observed between cell markers of normal individuals (CD3, 4, 8, 16, 56, 19, 25, ICAM-1, VLA-4 & l-selectin) and patients with atherosclerosis. There were significantly high values of cell markers in patients with rheumatoid arthritis compared with normal individuals both in terms of percentage and absolute counts (p < 0.05). Soluble IL-6 and sTNFR1 showed significant decreases in atherosclerosis and rheumatoid arthritis when compared with controls (p < 0.05). In addition, CRP and sTNF showed significant increases in the atherosclerosis and rheumatoid arthritis groups when compared to controls (p < 0.05). Conclusion: Our findings reasonably anticipate the presence of TRAPS disease (low penetrance mutations) amongst the Saudi population although further studies are needed to confirm these results.     

Selenium nanoparticles from Lactobacillus paracasei HM1 capable of antagonizing animal pathogenic fungi as a new source from human breast milk

The current study was performed to develop a simple, safe, and cost-effective technique for the biosynthesis of selenium nanoparticles (SeNPs) from lactic acid bacteria (LAB) isolated from human breast milk with antifungal activity against animal pathogenic fungi. The LAB was selected based on their speed of transforming sodium selenite (Na₂SeO₃) to SeNPs. Out of the four identified LAB isolates, only one strain produced dark red color within 32 h of incubation, indicating that this isolate was the fastest in transforming Na₂SeO₃ to SeNPs; and was chosen for the biosynthesis of LAB-SeNPs. The superior isolate was further identified as Lactobacillus paracasei HM1 (MW390875) based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and phylogenetic tree analysis of 16S rRNA sequence alignments. The optimum experimental conditions for the biosynthesis of SeNPs by L. paracasei HM1 were found to be pH (6.0), temperature (35˚C), Na₂SeO₃ (4.0 mM), reaction time (32 h), and agitation speed (160 rpm). The ultraviolet absorbance of L. paracasei-SeNPs was detected at 300 nm, and the transmission electron microscopy (TEM) captured a diameter range between 3.0 and 50.0 nm. The energy-dispersive X-ray spectroscopy (EDX) and the Fourier-transform infrared spectroscopy (FTIR) provided a clear image of the active groups associated with the stability of L. paracasei-SeNPs. The size of L. paracasei-SeNPs using dynamic light scattering technique was 56.91 ± 1.8 nm, and zeta potential value was −20.1 ± 0.6 mV in one peak. The data also revealed that L. paracasei-SeNPs effectively inhibited the growth of Candida and Fusarium species, and this was further confirmed by scanning electron microscopy (SEM). The current study concluded that the SeNPs obtained from L. paracasei HM1 could be used to prepare biological antifungal formulations effective against major animal pathogenic fungi. The antifungal activity of the biologically synthesized SeNPs using L. paracasei HM1 outperforms the chemically produced SeNPs. In vivo studies showing the antagonistic effect of SeNPs on pathogenic fungi are underway to demonstrate the potential of a therapeutic agent to treat animals against major infectious fungal diseases.     

Anuran species richness,complementarity and conservation conflicts in Brazilian Cerrado

Broad-scale correlations between species richness and human population suggest that processes driving species richness, mainly related to high ecological productivity, may also drive human populations. However, it is still under debate if this coincidence implies conflicts between biodiversity conservation and human development. In this paper, we analyzed the relationships among human population size, species richness and irreplaceability in Brazilian Cerrado. We analyzed a dataset with 131 species of anurans distributed in 181 cells with 1° of spatial resolution covering the biome. We found a positive correlation between human population size and anuran species richness (r = 0.46; P = 0.033 with 19.5 geographically effective degrees of freedom, v*), but the irreplaceability of each cell was poorly correlated with human population size (r = 0.075; P = 0.323; v* = 173.9). The 17 cells in the 97 optimal reserve networks contained a total human population ranging from 2942,195 to 4319,845 people, representing on average 11.8% of the human population in the entire Cerrado grid. The comparison of these observed values with 10,000 values from randomly generated networks suggests a relatively high flexibility in optimal complementarity sets for reserve selection. Our results indicated that correlation between richness and human population does not necessarily result in conflicts, given the opportunities for conciliating conservation and development. However, the analyses performed here are initial explorations within the framework of conservation biogeography, so more detailed studies are necessary to establish conservation planning at regional and local scales.     

First mitochondrial DNA analysis of the spectacled porpoise (Phocoena dioptrica) from Tierra del Fuego,Argentina

The spectacled porpoise (Phocoena dioptrica, Lahille, 1912) is one of the cetacean species about which least is known. Most information on the biology and ecology of this species has been obtained from stranded specimens and sightings at sea. In this study, analysis of 380 bp fragment of mitochondrial DNA (mtDNA) control region sequences (N = 50) was performed to provide a preliminary assessment of the genetic variation in spectacled porpoise specimens found stranded or by caught on the coast of Tierra del Fuego, Argentina. Results showed high levels of mtDNA diversity, as expected in large size and stable populations, and similar to other species of porpoises. The star-like shape phylogeny of haplotypes indicates a recent population expansion. This is the first report on the genetic variation of this species. Other lines of evidence (microsatellite loci, single-nucleotide polymorphism (SNPs)) are needed to improve our knowledge on the molecular biology of the spectacled porpoise.     

Association of mitochondrial haplogroup D and risk of esophageal cancer in Taihang Mountain and Chaoshan areas in China

Both the Taihang Mountain area in north-central China and Chaoshan area in the southeastern littoral of China are areas with high risk of esophageal cancer (EC). Our previous study confirmed that populations from the two areas might share similar matrilineal backgrounds and found that mitochondrial DNA (mtDNA) haplogroup D, especially subhaplogroups D4a and D5a, might be genetic background markers of EC in Chaoshan area. Here, to further determine whether D4a, D5a, and D might be susceptibility markers for EC in the two high-risk areas, we performed a case–control study with larger samples and analyzed the distributions of these three haplogroups in subjects (controls [n = 898] and patients [n = 768]) from the two areas. D4a haplogroup was significantly associated with increased risk of EC in Taihang Mountain subjects, especially women. D5 haplogroup was associated with EC at the general population level in the Taihang Mountain area and in subjects ≤ 60 years, especially women ≤ 60 years, in the Chaoshan area. D haplogroup was associated with EC only in subjects ≤ 60 years, especially men ≤ 60 years, in the Chaoshan area. D4a and D5 showing positive association with EC in the Taihang Mountain area became the predominant subhaplogroups of D in Chaoshan controls. In conclusion, D, D4a, and D5 haplogroups might be susceptibility markers for EC in the two high-risk areas in China, particularly D4a and D5 for the Taihang Mountain area and D and D5 for the Chaoshan area.     

Reference database of the gait cycle for young healthy Tunisian adults

BackgroundQuantified gait analysis is a rising technology used increasingly to assess motor disorders. Normal reference data are required in order to evaluate patients, but there are no reference data available for the Tunisian healthy population.AimTo assess the features of normal Tunisian gait pattern, and examine the intrinsic reliability of spatio-temporal, kinematic and kinetic parameters within a new specific reference database.MethodsEighteen healthy active-young adults (age: 23.30 ± 2.54 years, height: 1.78 ± 0.04 m and, weight: 70.00 ± 4.80 kg) have participated to five trials of step gait where the dominant lower limb were recorded. Two over the five trials were randomly selected to be further analyzed. Twenty-three spatio-temporal, kinematic and kinetic parameters determined from 3-dimensional gait analysis. The intrinsic reliability was examined for each variable and our results were compared with those available in the literature.ResultsTwelve over 23 parameters have an excellent intrinsic reliability (P > 0.05, ICC > 0.9 and SEM < 5% of the grand mean). There are similarities with other studies (P < 0.05) but we noticed the existence of some specificity (the height of hip extension peak and the low cadence of gait) that could characterize the Tunisian population.ConclusionA specific reference database of the gait cycle has been established for healthy Tunisian active-young adults and excellent inter-trial reliability may be observed for different variables.