Benchmark Dose Assessment for Coke Oven Emissions-Induced Mitochondrial DNA Copy Number Damage Effects

Objective The study aimed to estimate the benchmark dose(BMD)of coke oven emissions(COEs)exposure based on mitochondrial damage with the mitochondrial DNA copy number(mtDNAcn)as a biomarker.Methods A total of 782 subjects were recruited,including 238 controls and 544 exposed workers.The mtDNAcn of peripheral leukocytes was detected through the real-time fluorescence-based quantitative polymerase chain reaction.Three BMD approaches were used to calculate the BMD of COEs exposure based on the mitochondrial damage and its 95%confidence lower limit(BMDL).Results The mtDNAcn of the exposure group was lower than that of the control group(0.60±0.29 vs.1.03±0.31;P<0.001).A dose-response relationship was shown between the mtDNAcn damage and COEs.Using the Benchmark Dose Software,the occupational exposure limits(OELs)for COEs exposure in males was 0.00190 mg/m^(3).The OELs for COEs exposure using the BBMD were 0.00170 mg/m^(3)for the total population,0.00158 mg/m^(3)for males,and 0.00174 mg/m^(3)for females.In possible risk obtained from animal studies(PROAST),the OELs of the total population,males,and females were 0.00184,0.00178,and 0.00192 mg/m^(3),respectively.Conclusion Based on our conservative estimate,the BMDL of mitochondrial damage caused by COEs is0.002 mg/m^(3).This value will provide a benchmark for determining possible OELs.

Late-onset mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes syndrome with mitochondrial DNA 3243A>G mutation masquerading as autoimmune encephalitis:A case report

BACKGROUND Here,we present a unique case of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes(MELAS)syndrome,which initially appeared to be autoimmune encephalitis and was ultimately confirmed as MELAS with the mitochondrial DNA 3243A>G mutation.CASE SUMMARY A 58-year-old female presented with acute-onset speech impediment and auditory hallucinations,symmetrical bitemporal lobe abnormalities,clinical and laboratory findings,and a lack of relevant prodromal history,which suggested diagnosis of autoimmune encephalitis.Further work-up,in conjunction with the patient’s medical history,family history,and lactate peak on brain lesions on magnetic resonance imaging,suggested a mitochondrial disorder.Mitochondrial genome analysis revealed the m.3243A>G variant in the MT-TL1 gene,which led to a diagnosis of MELAS syndrome.CONCLUSION This case underscores the importance of considering MELAS as a potential cause of autoimmune encephalitis even if patients are over 40 years of age,as the symptoms and signs are atypical for MELAS syndrome.

Origin and phylogenetic analysis of Tibetan Mastiff based on the mitochondrial DNA sequence

At present, the Tibetan Mastiff is the oldest and most ferocious dog in the world. However, the origin of the Tibetan Mastiff and its phylogenetic relationship with other large breed dogs such as Saint Bernard are unclear. In this study, the primers were designed accord- ing to the mitochondrial genome sequence of the domestic dog, and the 2,525 bp mitochondrial sequence, containing the whole sequence of Cytochrome b, tRNA-Thr, tRNA-Pro, and control region of the Tibetan Mastiff, was obtained. Using grey wolves and coyotes as out- groups, the Tibetan Mastiff and 12 breeds of domestic dogs were analyzed in phylogenesis. Tibetan Mastiff, domestic dog breeds, and grey wolves were clustered into a group and coyotes were clustered in a group separately. This indicated that the Tibetan Mastiff and the other domestic dogs originated from the grey wolf, and the Tibetan Mastiff belonged to Carnivora, Canidae, Canis, Canis lupus, Canis lupus familiaris on the animal taxonomy. In domestic dogs, the middle and small breed dogs were clustered at first; German Sheepdog, Swedish Elkhound, and Black Russian Terrier were clustered into one group, and the Tibetan Mastiff, Old English Sheepdog, Leonberger, and Saint Bernard were clustered in another group. This confirmed the viewpoint that many of the famous large breed dogs worldwide such as Saint Bernard possibly had the blood lineage of the Tibetan Mastiff, based on the molecular data. According to the substitution rate, we concluded that the approximate divergence time between Tibetan Mastiff and grey wolf was 58,000 years before the present （YBP）, and the approximate divergence time between other domestic dogs and grey wolf was 42,000 YBP, demonstrating that the time of origin of the Tibetan Mastiff was earlier than that of the other domestic dogs.

Mitochondrial DNA Mutations Associated with Aminoglycoside Ototoxicity

The mitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and non-syndromic hearing loss. Of all the mutations, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region in the 12S rRNA have been associated with aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. The A1555G or C1494T mutation is expected to form novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the secondary structure of this RNA more closely resemble the corresponding region of bacterial 16S rRNA. Thus, the new U-A or G-C pair in 12S rRNA created by the C1494T or A1555G transition facilitates the binding of aminoglycosides, thereby accounting for the fact that the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying these mutations. Furthermore, the growth defect and impairment of mitochondrial translation were observed in cell lines carrying the A1555G or C1494T mutation in the presence of high concentration of aminoglycosides. In addition, nuclear modifier genes and mitochondrial haplotypes modulate the phenotypic manifestation of the A1555G and C1494T mutations. These observations provide the direct genetic and biochemical evidences that the A1555G or C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and nonsyndromic hearing loss. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

Differentiation of coral trout (Plectropomus leopardus) based on an analysis of morphology and complete mitochondrial DNA: Are cryptic species present?

Two morphotypes of Plectropomus leopardus have been identified; morphometric and meristic analyses show that there is no diagnostic difference between them. A difference in color pattern was the most ap- propriate phenotypic character with which to distinguish between the two morphotypes. Complete mito- chondrial DNA sequencing, however, indicated a clear difference between the two morphotypes. Barcoding analysis revealed no significant difference （P〉0.05） in CO1 or ND2 divergence among intramorphotypic individuals, even between geographically separated populations, whereas the intermorphotypic CO1 and ND2 divergences were large enough （averaging 0.95% for CO1 and 1.37% for ND2） to clearly discriminate between the two morphotypes. The color pattern difference, geographical distribution, together with the mtDNA and barcode sequencing data, suggest that the two morphotypes should be of two subspecies or even two species.

Protective Roles of α-lipoic Acid in Rat Model of Mitochondrial DNA4834bp Deletion in Inner Ear

The protective roles of α-lipoic acid in the rat model of mitochondrial DNA (mtDNA) 4834bp deletion in inner ear were investigated. Forty female Wistar rats at 4 weeks of age were divided into four groups: group A (D-galactose group, n=10), group B (D-galactose+α-lipoic acid group, n=10), group C (α-lipoic acid group, n=10), and group D (control group, n=10). Auditory brainstem response (ABR) was used to detect the hearing threshold. Colorimetry was used to analyze activity of superoxide dismutase (SOD) and concentration of malondialdehyde (MDA). The percentage of mtDNA4834bp deletion in inner ear was identified by real-time PCR. There was no significant difference in ABR threshold shift among all groups. The percentage of mtDNA4834bp deletion in group A was higher than that in other groups, but there was no significant difference in percentage of mtDNA4834bp deletion among groups B, C, and D. The activity of SOD in group A was lower than that in other groups. The concentration of MDA in group A was higher than that in other groups. It was concluded that there was no significant hearing loss when the percentage of mtDNA4834bp deletion was lower than 12.5%. α-Lipoic acid could prevent the reactive oxygen species (ROS)-induced mtDNA4834bp deletion in inner ear of rats.

Mitochondrial DNA haplogroup associated with sperm motility in the Han population

In this study, we aimed to determine whether the main mitochondrial DNA （mtDNA） haplogroups of the Han people have an impact on spermatozoa motility, We recruited 312 men who were consecutively admitted to two affiliated hospitals of College of Medicine, Zhejiang University from May 2011 to April 2012 as part of fertility investigations. Semen and whole blood samples were collected from the men. We determined the mtDNA haplogroups by analysing the sequences of mtDNA hypervariable segment I and testing diagnostic polymorphisms in the mtDNA coding region with DNA probes, No significant differences were found in the clinical characteristics of the mtDNA haplogroup R and non-R （P〉0.05）. Our results suggest that mtDNA haplogroup R is a strong independent predictor of sperm motility in the Han population, conferring a 2.97-fold （95% confidence interval： 1.74-4.48, P〈0.001） decreased chance of asthenozoospermia compared with those without haplogroup R.

The Mitochondrial DNA Mutation at Position 11778 in Chinese Families with Leber's Hereditary Optic Neuropathy

We amplified the 340 bp of mitochondrial DMA (mtDNA) by PCR including the recognized sequence of restriction enzyme of SfaN I . After amplification and digestion of SfaN I , two bands of 190 bp and 150 bp appeared in the mtDNA of four normal individuals but only one band of 340 bp appeared in the mtDNA with the mutation of G to A at the site of the nucleotide 11778 because such mutation destroyed the recognized sequence of SfaN I . We studied the mtDNAs of the patients with Leber's hereditary optic neur...

Simulated aeromedical evacuation exacerbates burn induced lung injury:targeting mitochondrial DNA for reversal

Background:Aeromedical evacuation of patients with burn trauma is an important transport method in times of peace and war,during which patients are exposed to prolonged periods of hypobaric hypoxia;however,the effects of such exposure on burn injuries,particularly on burn-induced lung injuries,are largely unexplored.This study aimed to determine the effects of hypobaric hypoxia on burn-induced lung injuries and to investigate the underlying mechanism using a rat burn model.Methods:A total of 40 male Wistar rats were randomly divided into four groups(10 in each group):sham burn(SB)group,burn in normoxia condition(BN)group,burn in hypoxia condition(BH)group,and burn in hypoxia condition with treatment intervention(BHD)group.Rats with 30%total body surface area burns were exposed to hypobaric hypoxia(2000 m altitude simulation)or normoxia conditions for 4 h.Deoxyribonuclease I(DNase I)was administered systemically as a treatment intervention.Systemic inflammatory mediator and mitochondrial deoxyribonucleic acid(mtDNA)levels were determined.A histopathological evaluation was performed and the acute lung injury(ALI)score was determined.Malonaldehyde(MDA)content,myeloperoxidase(MPO)activity,and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3)inflammasome level were determined in lung tissues.Data among groups were compared using analysis of variance followed by Tukey’s test post hoc analysis.Results:Burns resulted in a remarkably higher level of systemic inflammatory cytokines and mtDNA release,which was further heightened by hypobaric hypoxia exposure(P<0.01).Moreover,hypobaric hypoxia exposure gave rise to increased NLRP3 inflammasome expression,MDA content,and MPO activity in the lung(P<0.05 or P<0.01).Burn-induced lung injuries were exacerbated,as shown by the histopathological evaluation and ALI score(P<0.01).Administration of DNase I markedly reduced mtDNA release and systemic inflammatory cytokine production.Furthermore,the NLRP3 inflammasome level in lung tissues was decreased and burn-induced lung injury was ameliorated(P<0.01).Conclusions:Our results suggested that simulated aeromedical evacuation further increased burn-induced mtDNA release and exacerbated burn-induced inflammation and lung injury.DNase I reduced the release of mtDNA,limited mtDNA-induced systemic inflammation,and ameliorated burn-induced ALI.The intervening mtDNA level is thus a potential target to protect from burn-induced lung injury during aeromedical conditions and provides safer air evacuations for severely burned patients.

Identification of Two Novel Mitochondrial DNA Deletions Induced by Ionizing Radiation

Abstract Objective We identify ionizing radiation-induced mitochondrial DNA （mtDNA） deletions in human lymphocytes and their distribution in normal populations. Methods Long-range polymerase chain reactions （PCR） using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy 6~Co y-rays. Limited-condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long-range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy ^60Co y-rays, and real-time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated. Results Two mtDNA deletions, a 7455-bp deletion （nt475-nt7929 in heavy strand） and a 9225-bp deletion （nt7714 -nt369 in heavy strand）, occurring between two 8-bp direct repeats, were identified in lymphoblastoid cells using long-range PCR, limited-condition PCR and sequencing. These results were also observed for ^60Co y-rays irradiated human peripheral blood cells. Conclusion Two novel mtDNA deletions, a 7455-bp deletion and a 9225-bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors＇ age, but was independent of gender.

Molecular Taxonomy of Conogethes punctiferalis and Conogethes pinicolalis(Lepidoptera: Crambidae) Based on Mitochondrial DNA Sequences

Conogethes punctiferalis（Guenée）（Lepidoptera： Crambidae） was originally considered as one species with fruit-feeding type（FFT） and pinaceae-feeding type（PFT）, but it has subsequently been divided into two different species of Conogethes punctiferalis and Conogethes pinicolalis. The relationship between the two species was investigated by phylogenetic reconstruction using maximum-likelihood（ML） parameter estimations. The phylogenetic tree and network were constructed based upon sequence data from concatenation of three genes of mitochondrial cytochrome c oxidase subunits I, II and cytochrome b which were derived from 118 samples of C. punctiferalis and 24 samples of C. pinicolalis. The phylogenetic tree and network showed that conspecific sequences were clustering together despite intraspecific variability. Here we report the results of a combined analysis of mitochondrial DNA sequences from three genes and morphological data representing powerful evidence that C. pinicolalisand C. punctiferalis are significantly different.

Taxonomic Status of the Spot-legged Treefrog in Southern Yunnan,Inferred from Mitochondrial DNA Sequences

Populations of the spot-legged treefrogs(Polypedates megacephalus) in China show significant morphological variation,but no has yet been conducted to investigate the correlation between morphological variation and genetic/ecological divergence.In this study,mitochondrial DNA sequences from the 12S rRNA gene(374 bp) were amplified from 25 individual spot-legged treefrogs from southern Yunnan,China.The phylogenetic analysis using Bayesian Inference determined two haplotype clades,different from those detected by Richards and Moore(1998).Our results suggest that the phylogenetic lineages reconstructed in this study are not correlated with morphology,thus indicating that the populations in southern Yunnan may be P.leucomystax rather than P.megacephalus.

Discrimination of mitochondrial DNA 10400 locus by SNP-operated on/off Switch

Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled and 3 ＇ phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3 ＇ exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results： The unmodified primers were extended by both exo and exo^＋ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3＇ phosphorothioate-modified primers with a terminal mismatch triggered an ＂off-switch＂ of exo~ polymerase when compared to exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified primers with exo^＋ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening.

Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status

BACKGROUND Acute ischemic stroke(AIS)is one of the major causes of the continuous increasing rate of global mortality due to the lack of timely diagnosis,prognosis,and management.This study provides a primitive platform for non-invasive and cost-effective diagnosis and prognosis of patients with AIS using circulating cellfree mitochondrial DNA(cf-mtDNA)quantification and validation.AIM To evaluate the role of cf-mtDNA as s non-invasive,and affordable tool for realtime monitoring and prognosticating AIS patients at disease onset and during treatment.METHODS This study enrolled 88 participants including 44 patients with AIS and 44 healthy controls with almost similar mean age group at stroke onset,and at 24 h and 72 h of treatment.Peripheral blood samples were collected from each study participant and plasma was separated using centrifugation.The cf-mtDNA concentration was quantified using nanodrop reading and validated through real-time quantitative polymerase chain reaction(RT-qPCR)of NADH-ubiquinone oxidoreductase chain 1(ND1)relative transcript expression levels.RESULTS Comparative analysis of cf-mtDNA concentration in patients at disease onset showed significantly increased levels compared to control individuals for both nanodrop reading,as well as ND1 relative expression levels(P<0.0001).Intergroup analysis of cf-mtDNA concentration using nanodrop showed significantly reduced levels in patients at 72 h of treatment compared to onset(P<0.01).However,RT-qPCR analysis showed a significant reduction at 24 h and 72 h of treatment compared to the disease onset(P<0.001).The sensitivity and specificity were relatively higher for RT-qPCR than nanodrop-based cfmtDNA quantification.Correlation analysis of both cf-mtDNA concentration as well as ND1 relative expression with National Institute of Health Stroke Scale score at baseline showed a positive trend.CONCLUSION In summary,quantitative estimation of highly pure cf-mtDNA provides a simple,highly sensitive and specific,non-invasive,and affordable approach for real-time monitoring and prognosticating AIS patients at onset and during treatment.

Population Genetic Analysis of Sillago nigrofasciata (Perciformes:Sillaginidae) Along the Coast of China by Sequencing Mitochondrial DNA Control Region

Sillago nigrofasciata, a small to moderate size nearshore species, is newly found along the eastern and southern coasts of China. The present study is carried out in order to analyze the population genetics of the S. nigrofasciata. The control region sequence of mitochondrial DNA revealing 73 haplotypes were obtained from 162 individuals collected at 8 locations along the coast of China. The whole S. nigrofasciata population along the coast of China showed a low nucleotide diversity(0.012) and a high population diversity(haplotype diversity)(0.943), and all the 8 local populations showed low nucleotide diversities(0.014 – 0.001), suggesting the protective measures are effective. The haplotypes of the 8 local populations were widely distributed in haplotype network diagram and neighbor-joining phylogenetic tree, while no branch associating with sampling locations was detected. Recent gene flow analysis showed asymmetric gene exchanges among local populations. The pairwise FST values and unweighted pair-group method with arithmetic mean(UPGMA) tree revealed a certain amount of genetic difference among local populations. Moreover, analysis of molecular variance(AMOVA) reflected genetic differences between hypothetical subdivision groups. Neutral test and mismatch distribution of pairwise nucleotide suggested S. nigrofasciata may have experienced recent population expansion events. The historical geographic events associating with ice age may be the main explanation to the heterogeneity among local populations with short geographic distances, and the homogeneity among local populations with long geographic distances.

Investigation of mitochondrial DNA genetic diversity and phylogeny of goats worldwide

Genetic diversity,population structure,and population expansion of goats worldwide(4165 individuals from 196 breeds)were analyzed using published mitochondrial DNA(mtDNA)D_loop hypervariable region sequences.Results showed that 2409 haplotypes and 301 polymorphic sites were present within the 401-bp length D_loop region,the nucleotide diversity(Pi)was 0.03471,and the haplotype diversity(Hd)was 0.9983.Phylogenetic analysis revealed that 98.92%of haplotypes were divided into six obvious clusters,consistent with the classification of the known mitochondrial haplogroups of goats.Haplogroup A accounted for the largest proportion(86%).Interestingly,two unknown divisions(Unknown I and Unknown II)were discovered from goats in Southwest China,suggesting that Southwest China has unique maternal haplogroups.Analysis of molecular variance(AMOVA)and the average number of pairwise differences between populations(PiXY)indicated that geographical variation was small but significant.Neutrality tests(Tajima’s D and Fu’s FS tests)and mismatch distribution showed that haplogroups B,C,and G had expansion histories.In addition,the phylogenetic relationship between domestic and wild goats suggested that Capra aegagrus is the most likely wild ancestor and may have participated in the domestication of ancestral populations of A,B,C,and F haplogroups.A meta-analysis on the mtDNA sequences of goats from international databases was conducted to analyze goats’genetic diversity,population structure,and matrilineal system evolution worldwide.The results may help further understand the domestication history and gene flow of goats worldwide.

Genetic variation of the small yellow croaker(Larimichthys polyactis)inferred from mitochondrial DNA provides novel insight into the fluctuation of resources

The small yellow croaker(Larimichthys polyactis)belongs to the family Sciaenidae,which is an offshore warm fish species and widely distributed in the western Pacific.In this study,the variation of genetic diversity and genetic differentiation among L.polyactis populations was analyzed by mitochondrial DNA control region.A total of 110 polymorphic sites were checked,which defined 134 haplotypes.High level of haplotype diversity(h=0.993±0.002)was detected in the examined range.Population genetic structure analyse(analysis of molecular variance,Fst)showed there were high gene flow among L.polyactis populations.The result showed that there were relatively high genetic diversity and low genetic differentiation among the Yellow Sea and the East China Sea populations,which can be attributed to diverse habitats,wide distribution range and high mutation rate of control region.Using phylogenetic methods,coalescent analyses(neutrality tests,mismatch distribution analysis,Bayesian skyline analyses)and molecular dating interpreted in conjunction with paleoclimatic and physiographic evidence,we inferred that the genetic make-up of extant populations of L.polyactis was shaped by Pleistocene environmental impacts on the historical demography of this species.Besides,relatively constant genetic diversity and larger effective population size were detected in recent L.polyactis population.The result showed that the fishing policy certainly,such as the summer closed fishing,played a role in protecting resources of L.polyactis.This study can offer a wealth of biological novelties which indicates genetic structure of L.polyactis population and provides the foundation for resources protection and policy setting.

Mutations in the D-loop region of mitochondrial DNA in gastric cancer

Objective: To investigate the mutati ons in the D-loop region of mitochondrial DNA (mtDNA) in gastric cancer. Methods: The mtDNA of D-loop region was amplified by PCR and sequence d in 20 samples from gastric cancer tissue and adjacent normal membrane. Results: There were 7/20(35%) mutations in the mtDNA of D-loop regio n in gastric cancer patients. There were four microsatellite instabilities among the 18 mutations. Nine new polymorphisms were identified in 20 patients. Conclusion: The mtDNA of D-loop region might be highly polymorphoric and the mutation rate is high in patients with gastric cancer.

Mitochondria Dynamically Transplant into Cells in Vitro and in Mice and Rescue Aerobic Respiration of Mitochondrial DNA-Depleted Motor Neuron NSC-34

It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 ρ° cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 ρ° cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 ρ° cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 ρ° cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.

Complete Nucleotide Sequence of the Mitochondrial DNA of <i>Halyomorpha halys</i>(Stal) (Hemiptera: Pentatomidae) Specimens Collected Across Georgia

The brown marmorated stink bug, Halyomorpha halys (Stal) (Hemiptera: Pentatomidae) is an invasive species native to East Asia that has spread across Asia, Europe, and North America. H. halys causes damages to various grains, fruits, and vegetables, which is exemplified by the significant damage to the hazelnut harvest in Georgia (during 2016). This report describes the first attempted genetic study of the spread of H. halys in Georgia. The first main goal of this research was to identify the haplotype of an invasive population in Georgia. For this purpose, the mitochondrial cytochrome c oxidase I subunit (COI) gene fragment from 65 samples of H. halys collected from different regions across Georgia was sequenced on an Applied Biosystems 3100 or 3700 genetic analyzer. In all cases, only the H1 haplotype, which is native to China, was identified. The second goal of this research was to determine the complete mitochondrial DNA sequence of H. halys (Stal) specimens collected across Georgia. The complete mitochondrial DNA of H1 haplotype sequenced on an Illumina MiSeq platform. The mitochondrial DNA of the Georgian H1 haplotype has a length of 15,478 base pairs. Using the sequence of the H22 haplotype of H. halys (native to Korea) as a reference, 62 single nucleotide polymorphisms (SNPs), three inversions, and four single T insertions were identified. Furthermore, 60 SNPs and four insertions in two tRNA and one rRNA genes were identified among 18 mitochondrial genes from the Georgian H1 haplotype. Nine of these SNPs resulted in amino acid substitutions. Furthermore, the detection of SNPs revealed many other polymorphic sites beyond the COI gene, which can be used to detect new haplotypes.