Mitochondrial DNA Fragments Research Articles

DIVERSITY OF Wasmannia auropunctata (HYMENOPTERA: FORMICIDAE) AND THE USE OF MITOCHONDRIAL INTERGENIC SPACER AND LEUCINE tRNA FOR ITS IDENTIFICATION

Formigas da espécie Wasmannia auropunctata (Roger, 1863) tem sido o foco de muitos estudos abordando o seu status de invasora, o seu sistema reprodutivo e o seu controle. Porém, poucos estudos têm analisado sua diversidade genética ou estudado novas técnicas para a identificação da espécie. No presente artigo foi estudada a diversidade de W. auropunctata em duas de suas regiões nativas, Brasil e Colômbia, testando novos métodos moleculares de identificação. Para tal, foi sequenciado o gene citocromo c oxidase subunidade I (COI), o espaçador intergênico (IGS), o RNA transportador (tRNA) e o gene citocromo c oxidase subunidade II (COII) de operárias de 31 localidades. Os resultados obtidos mostraram que 80% dos mitótipos identificados nunca haviam sido registrados anteriormente e que as amostras apresentaram uma alta diversidade haplotípica. A análise dos dados e comparação com diferentes estudos publicados indicam que o tRNA entre o IGS e o gene COII foi similar para todas as formigas dessa espécie, enquanto o IGS entre o gene COI e o tRNA variou em comprimento. Sendo assim, tais padrões poderiam contribuir para a identificação inter e intraespecífica, respectivamente. O presente trabalho demonstra a importância de mais estudos moleculares na área, uma vez que podem oferecer novas perspectivas sobre a diversidade da espécie, assim como métodos de identificá-la.

Plasma mtDNA Analysis Aids in Predicting Pancreatic Necrosis in Acute Pancreatitis Patients: A Pilot Study.

Specific plasma biomarkers in predicting pancreatic necrosis (PNec) are needed in treating acute pancreatitis (AP). To investigate the prognostic value of plasma mitochondrial DNA fragments (mtDNA) in patient with AP for PNec. AP patients with symptoms onset within 72h were prospectively enrolled from June 2015 through June 2017 and were assessed for PNec using contrast-enhanced CT scan. Plasma mtDNA concentration (specific mitochondrial gene ND1) was measured using qRT-PCR. Of the 74 AP patients included, significant higher median level of plasma mtDNA was found in severe AP patients than in mild AP patients and healthy controls, but not in moderately severe AP patients. Patients with PNec had higher level of plasma mtDNA than those without PNec (774.2 [IQR 397.6-2205.0] vs. 169.5 [IQR 73.6-683.4] pg/ml, P < 0.05). The area under the receiver operator characteristic curve (ROC-AUC) of mtDNA for predicting PNec was higher than that of CRP (0.813 [95% CI 0.705-0.895] vs. 0.678 [95% CI 0.558-0.783]). Using a cutoff value of 302.5pg/ml, the sensitivity and specificity for diagnosing PNec were 90.9 and 68.3%, respectively. Finally, plasma mtDNA levels decreased significantly after continuous renal replacement therapy (717.7 [IQR 307.00-1370.00] vs. 237.5 [IQR 117.20-464.80] pg/ml, P < 0.01). Elevated plasma mtDNA content in AP patients may be used as a more accurate early predictor of PNec in contrast to traditional CRP.

An integrative description of a new tardigrade species Mesobiotus romani sp. nov. (Macrobiotidae: harmsworthi group) from the Ecuadorian Pacific coast.

In a mixed moss and lichen sample collected in Esmeraldas Province in north-western Ecuador, 20 tardigrades and 11 eggs, belonging to a new species of the genus Mesobiotus, were found. In addition to the traditional taxonomic description with morphometrics, light and scanning microscopy imaging, we also provide nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragments of the new species. Based on the egg chorion morphology, Mesobiotus romani sp. nov. is the most similar to: M. binieki, M. coronatus, M. dimentmani, M. patiens, M. perfidus, M. philippinicusi, M. pseudoblocki, M. pseudocoronatus, M. pseudopatiens, M. radiatus, M. rigidus, M. simulans and M. wuzhishanensis, but differs mainly by some specific characters of both egg and adult morphology, and morphometrics.

Ethanolic leaf extract of Coccinia grandis is effective against both drug resistant and drug sensitive clinical isolates of Indian Kala-azar.

The emergence of resistance to the current available drugs used for treatment against Indian Kala-azar (KA) or Visceral Leishmaniasis makes the control strategy inadequate for the disease. This grave epidemiological situation directed researches towards alternative treatments including herbal therapy. In this background, the aim of the present study was to evaluate the antileishmanial activity of the leaves of Coccinia grandis (a tropical vine) against both the Sodium Stibo Gluconate (SSG) sensitive and resistant as well as Miltefosine (MIL) sensitive and resistant field isolates of Leishmania donovani. The cytotoxicity effect of ethanolic extract of leaves of C. grandis (Cg-LE) against the clinical isolates of L. donovani was checked both in promastigotes and intracellular amastigotes stages. In both sensitive and resistant promastigotes, Cg-LE stimulated reactive oxygen species generation and apoptosis. Parasites infected macrophages showing enhanced nitric oxide production after Cg-LE treatment suggested the leishmanicidal activity of the leaf extract. Furthermore, Cg-LE treatment led to mitochondrial membrane damage and DNA fragmentation in promastigotes. The present study is very encouraging for the fact that Cg-LE showed promising antileishmanial activity against both SSG and MIL drug resistant clinical isolates of Indian KA.

Trichlorfon inhibits proliferation and promotes apoptosis of porcine trophectoderm and uterine luminal epithelial cells

Trichlorfon is an organophosphate insecticide widely used in agriculture. Additionally, it is applied to pigs for control of endo- and ectoparasites. Previous studies have shown the effects of trichlorfon in pigs during late stages of gestation; however, little is known about its effects during early pregnancy, including implantation and placentation. We investigated whether trichlorfon affects proliferation and apoptosis of porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. Trichlorfon inhibited the proliferation of pTr and pLE cells, as evidenced by cell cycle arrest, and altered the expression of proliferation-related proteins. In addition, trichlorfon induced cell death and apoptotic features, such as loss of mitochondrial membrane potential and DNA fragmentation, in pTr and pLE cells. Moreover, trichlorfon treatment decreased concentrations of Ca2+ in the cytoplasm in both cell lines and increased concentrations of Ca2+ in mitochondria of pTr cells. Trichlorfon inhibited the activation of phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase signaling pathways in pTr and pLE cells. Therefore, we suggest that trichlorfon-treated pTr and pLE cells exhibited abnormal cell physiology which might lead to early pregnancy failure.

Sperm quality in frozen beef and dairy bull semen

BackgroundThere is speculation that beef bull semen quality is inferior to that of dairy bulls although few scientific studies are available in the literature. The aim of this study was to evaluate sperm quality in beef bull semen and to determine which parameters could be indicative of fertility after insemination. Sperm quality, assessed by computer assisted sperm motility analysis and flow cytometric evaluation of membrane integrity, levels of reactive oxygen species, mitochondrial membrane potential, acrosome status and DNA fragmentation index, was evaluated in beef and dairy bull semen.ResultsFor beef bulls, normal morphology (r = 0.62, P < 0.05) and WOBBLE (r = 0.57, P < 0.05) were significantly correlated with 56-day non-return rate, whereas sperm quality was not significantly correlated with the fertility index score for dairy bulls. Membrane integrity (46 ± 8.0% versus 40 ± 11%, P < 0.05), normal morphology (87 ± 6% versus 76 ± 8%; P < 0.05), and high respiratory activity (52 ± 13 versus 12 ± 4%; P < 0.001) were higher for dairy bulls than for beef bulls. The DNA fragmentation index was lower for dairy bull spermatozoa than beef (3.8 ± 1.1% versus 6.1 ± 2.9%; P < 0.01), whereas some sperm kinematics were higher. Multivariate analysis indicated that type of bull (beef versus dairy) had an impact on sperm quality.ConclusionsDifferent assays of sperm quality may be needed for appropriate analysis of beef and dairy bull semen. These finding could be important for cattle breeding stations when evaluating semen quality.

Abstract 1634: BRCA-like phenotype constitutes hallmark of osteosarcoma

Abstract Background: Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which limits treatment options in patients who do not respond to standard chemotherapy. Methods: We sequenced 104 exomes and low-coverage genomes from 14 time-series consisting of chemotherapy-naïve biopsies, resection specimens, locally recurring tumours and/or distant metastases. We then identified cancer driver mutations, signatures of mutation processes and reconciled clonal structures of tumours with phylogenetic trees, thus building and evolutionary history of each tumour. Where possible, we searched for the evidence of selection acting on the tumours and molecular hallmarks that could be therapeutically exploited. Results: Interrogating genomic data from time series, we identified pathogenic mutations in at least 10 cancer genes, but there was no universal cancer driver event present in the majority of tumours at any given time point. However, &amp;gt;80% of osteosarcomas exhibited specific combination of single-base substitutions, LOH and large-scale genome instability characteristic of BRCA1/2-deficient tumours. In osteosarcoma, "BRCAness" developed recurrently through independent BRCA1/2 mutations and mutations in their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib. In combination with temozolomide, talazoparib led to the to good response of osteosarcoma cell lines, activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation and caspase-dependent cell death. Conclusion: The acquisition of a BRCA-like phenotype in osteosarcoma has implications for the development of novel treatment strategies using PARP inhibitors alone, or together with standard chemotherapy. Citation Format: Michal Kovac, Sebastian Ribi, Claudia Blattmann, Michaela Nathrath, Daniel Baumhoer. BRCA-like phenotype constitutes hallmark of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1634.

Several Subspecies or Phenotypic Plasticity? A Geometric Morphometric and Molecular Analysis of Variability of the Mayan CichlidMayaheros urophthalmusin the Yucatan

The Mayan Cichlid (Mayaheros urophthalmus) is usually considered to be a complex of 18 subspecies, most of which are endemic to the Yucatan Peninsula and were diagnosed by phenetic analyses based on traditional morphometrics and color pattern. However, morphological differences can be due to environmental conditions rather than taxonomic distinctiveness. We evaluated, by means of a geometric morphometric analysis, two hypotheses for shape differences in 20 natural populations of M. urophthalmus, including five subspecies recently raised to species status: M. alborus, M. cienagae, M. conchitae, M. mayorum, and M. zebra. The geographical distribution and three types of aquatic environment (River, Lagoon or Pond, and Cenote) were used as classificatory variables. In addition, a molecular analysis of two concatenated fragments of mitochondrial DNA (mtDNA), cytochrome b (cytb) and cytochrome oxidase subunit I (COI), showed genetic differentiation among some populations (FST = 0.36). Whereas the geometric morph...

A phylogenetic assessment of the meadow lizard Darevskia praticola(Eversmann, 1834) from Iran.

The meadow lizard Darevskia praticola (Eversmann, 1834) displays a wide distribution range from the Western Balkans and Caucasus to the Hyrcanian forest in Iran. Based on previous studies, four subspecies have been described (D. p. praticola, D. p. pontica, D. p. hyrcanica and D. p. loriensis), but molecular evidence suggests two main phylogenetic lineages, Balkan and Caucasus. In this study we incorporated geographic records and analysed new samples from Alborz Mountains (Gilan Province, Iran), exploring their phylogenetic affinities using two mitochondrial markers (Cyt b and ND4). For the first time, Iranian samples are included in a molecular phylogeny. Our results meet the previous suggestions regarding the presence of a nuclear mitochondrial DNA fragment (numt) in Cyt b, causing misleading phylogenetic inferences since it renders the species paraphyletic. In contrast, the ND4 genealogy groups the species into a monophyletic clade, as expected. Samples attributed to D. p. hyrcanica (Azerbaijan and Iran) and D. p. loriensis (Armenia) are settled in a single clade with minute genetic distance, suggesting synonymisation.

Effect of dietary supplementation with a highly pure and concentrated docosahexaenoic acid (DHA) supplement on human sperm function

The aim of this study was to evaluate the possible beneficial effects of diet supplementation with a highly concentrated and purified docosahexaenoic acid (DHA) formula on human sperm function. We performed a prospective, randomized, double blind, placebo-controlled intervention study. One-hundred eighty human semen samples from sixty infertile patients recruited in a private assisted reproduction center were included. All samples were examined according to World Health Organization guidelines. We analyzed macroscopic and microscopic sperm parameters, oxidative stress, apoptosis, lipid peroxidation, mitochondrial membrane potential and DNA fragmentation before and after supplementation with different DHA daily doses (0.5, 1 and 2 g) or placebo for 1 and 3 months. No differences were found in traditional sperm parameters except for progressive sperm motility, with a significant increase after DHA ingestion after the first month with 1 or 2 g doses and after 3 months with 0.5 g of DHA. This effect was more evident in asthenozoospermic patients. No differences were found in any molecular semen parameter except oxidative stress, in which a slight benefit was observed after DHA treatment. In conclusion, this study support previous indications that highlight the importance of DHA supplementation as a means of improving sperm quality in asthenozoospermic men.

Mitochondrial diversity and phylogeographic structure of native cattle breeds from Yunnan, Southwestern China

Little is so far known about genetic diversity and phylogeographic structure of native cattle breeds from Yunnan in Southwestern China, a frontier zone that connects China with South and Southeast Asia. Herein we analyzed a 910-bp fragment of mitochondrial DNA (mtDNA) D-loop region sequences of 280 individuals from 6 Yunnan native cattle breeds, of which 251 sequences were newly determined. There were 93 variable sites that defined 117 haplotypes among all sequences. Phylogenetic analysis of all haplotypes revealed that Yunnan native cattle had two distinct mtDNA lineages - taurine and zebu. The taurine sequences fell into four haplogroups T1a, T2, T3 and T4, whereas the zebu sequences grouped into two haplogroups I1 and I2. In addition, our results revealed patterns of gradient changes in frequencies of taurine and zebu mtDNA lineages across different geographic regions of Yunnan. Furthermore, three kinds of methods (haplotype network, analysis of molecular variance, and Mantel tests) consistently showed no significant phylogeographic structuring among Yunnan native cattle breeds. This might be attributed to historical strong gene flow and genetic admixture among native cattle breeds in different geographic regions.

Introduction history and genetic diversity of the invasive ant Solenopsis geminata in the Galápagos Islands

The Galapagos Islands constitute one of the most pristine tropical systems on Earth. However, the complex and fragile equilibrium of native species is threatened by invasive species, among which is one of the most successful ants in the world, the tropical fire ant, Solenopsis geminata. We characterized the genetic structure and diversity of populations of S. geminata in the Galapagos Islands and unravelled the archipelago colonization by combining Bayesian clustering methods and coalescent-based scenario testing. Using 12 microsatellite markers and one mitochondrial DNA fragment (COI), we analysed individuals collected in all main invaded islands of the archipelago and from the native areas in Costa Rica and mainland Ecuador. We also used mitochondrial DNA to infer evolutionary relationships of samples collected in Galapagos Islands, Ecuador, Costa Rica and other Latin American countries. Our results showed that genetic diversity was significantly lower in Galapagos Islands and mainland Ecuador populations when compared to Costa Rican populations, and that samples from Galapagos Islands and mainland Ecuador (Guayaquil) clustered in a single group and all share a single mtDNA haplotype. Approximate Bayesian Computation favoured a scenario assuming that populations from Galapagos Islands diverged from mainland Ecuador. The city of Guyaquil, an obligatory hub for tourism and trade, could act as a bridgehead.

In Vitro and In Vivo Evaluation of Antitumor Activity of Ligustrum robustum, A Chinese Herbal Tea.

To examine the effect of the aqueous extract of Ligustrum robustum on tumor growth in vitro and in vivo and explore the possible molecular mechanisms. In in vitro study, cell viabilities of human cervical carcinoma cells (HeLa), human breast cancer cells (MCF-7), human prostate cancer cells (PC-3), human hepatoma cells (7721) and human colon carcinoma cells (SW480) were evaluated with cell counting kit-8. For L. robustum-treated Hela cells, early or late apoptosis were evaluated by annexin V/PI staining. Mitochondrial membrane potential was measured by staining cells with JC-1. Apoptosis was monitored by nuclear morphology based on chromatin condensation and fragmentation by 4',6-diamidino-2-phenylinole (DAPI) staining. Caspase-3 and -8 activity levels were measured by a colorimetric assay. In vivo, to evaluate the possible mechanism of L. robustum-mediated antitumor effect, nude mouse xenograft study was also conducted. In in vitro study, L. robustum was found to be toxic to HeLa, MCF-7, PC-3, 7721, SW480, with an half maximal inhibitory concentration value of 2-5 mg/mL (P<0.05). Moreover, externalization of phosphatidylserine, loss of mitochondrial membrane potential, DNA fragmentation and activation of caspase-3 and -8 were detected in L. robustum-treated Hela cells. Using a nude mouse model bearing Hela xenografts, we found that L. robustum reduced tumor volume and tumor weight (P<0.05), but had no effect on body weight and histological damage of important organs. Intraperitoneal injection of L. robustum caused a significant reduction in serum aspartate transaminase and alanine transaminase levels (P<0.05). Furthermore, cleaved caspase-3-positive and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells were observed in L. robustum-treated tumor tissues. L. robustum inhibits tumor cell growth both in vitro and in vivo by inducing apoptosis in a caspase-dependent way without apparent hepatic toxicity and histological damage, which may offer partial scientific support for the ethnopharmacological claims of L. robustum as a herbal tea for its antitumor activity.

First morphological and molecular analysis of Eucoleus boehmi like eggs in dogs from Argentina.

The canid parasites Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi) parasitize the lower and the upper respiratory tract, respectively. Reports and descriptions of these nematodes are scarce in Argentina, possibly due to misdiagnosis of morphologically similar trichuroids eggs, and the lack of knowledge about the species of Eucoleus in this geographical area. Scanning electron microscopy is a useful tool for identification of E. boehmi eggs based on the characteristics of the shell structure which differentiate between species. Molecular analysis complements morphological identification. Until now, there are no studies based on the analysis of E. boehmi eggs in Argentina. The aim of the present work was to study by morphological, morphometric, and molecular analysis, eggs attributable to E. boehmi isolated from dogs naturally infected in Mar del Plata city, Argentina. Eggs isolated from two dog fecal samples were analyzed by light and scanning electron microscopy. A fragment of mitochondrial DNA (mtDNA) of the cytochrome c oxidase subunit I gene (cox1) from eggs was sequenced, and phylogenetic analysis was performed in this study. According to morphological results based on the wall surface ultrastructure, the eggs studied were assigned to E. boehmi. Molecular analysis supported the morphological identification. The divergence of 9-12% with the European isolated could suggest a new geographical genetic variation of E. boehmi, but also question the possible existence of cryptic species. This is the first characterization of E. boehmi eggs in dogs from Argentina.

Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA

Environmental DNA (eDNA) is increasingly used to monitor aquatic macrofauna. Typically, short mitochondrial DNA fragments are targeted because these should be relatively more abundant in the environment as longer fragments will break into smaller fragments over time. However, longer fragments may permit more flexible primer design and increase taxonomic resolution for eDNA metabarcoding analyses, and recent studies have shown that long mitochondrial eDNA fragments can be extracted from environmental water samples. Nuclear eDNA fragments have also been proposed as targets, but little is known about their persistence in the aquatic environment. Here we measure the abundance of mitochondrial eDNA fragments of different lengths and of short nuclear eDNA fragments, originating from captive fish in experimental tanks, and we test whether longer mitochondrial and short nuclear fragments decay faster than short mitochondrial fragments following fish removal. We show that when fish are present, shorter mitochondrial fragments are more abundant in water samples than both longer mitochondrial fragments and short nuclear eDNA fragments. However, the rate of decay following fish removal was similar for all fragment types, suggesting that the differences in abundance resulted from differences in the rates at which different fragment types were produced rather than differences in their decay rates.

Ceragenin CSA-13 as free molecules and attached to magnetic nanoparticle surfaces induce caspase-dependent apoptosis in human breast cancer cells via disruption of cell oxidative balance.

Natural antimicrobial peptides and ceragenins, as non-peptide amphipathic mimics, have been proposed as anti-cancer agents. To date, it has been confirmed that cathelicidin LL-37 and ceragenin CSA-13, both in free form and immobilized on the surface of magnetic nanoparticles (MNP@LL-37, MNP@CSA-13) induce apoptosis in colon cancer cells. Nevertheless, the question remains whether ceragenins, as synthetic analogs of LL-37 peptide and mimicking a number of its properties, act as antineoplastic agents in breast cancer cells, where LL-37 peptide stimulates oncogenesis. Considering potential anticancer activity, we determined whether CSA-13 and MNP@CSA-13 might be effective against breast cancer cells. Our study provides evidence that both CSA-13 and MNP@CSA-13 decreased viability and inhibit proliferation of MCF-7 and MDA-MB-231 cells despite the protumorigenic properties of LL-37 peptide. Flow cytometry-based analyses revealed that ceragenin treatment results in increases in dead and PI-negative/low-viability cells, which was associated with glutathione (GSH) depletion and increased reactive oxygen species (ROS) generation followed by mitochondrial membrane depolarization, caspase activation, and DNA fragmentation. These findings demonstrate that both CSA-13 and MNP@CSA-13 cause disruption of the oxidative balance of cancer cells. This novel mechanism of ceragenin-mediated eradication of cancer cells suggest that these agents may be developed as a possible treatment of breast cancer.

Synthesis and Biological Evaluation of Novel Thiazol-2yl-amine Derivatives as Potential Anticancer Agents

Background: Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that can occur in any age group but often seen in adults and contributing for about 20% of adult leukemias and it may contribute up to 15% of all types of leukemias threatening the globe. Therefore, treatment of CML remains as a major challenge in cancer therapeutics. Methods: We synthesized a library of novel 2-amino-4-(4-substituted phenyl) thiazole derivatives and evaluated their anti-leukemic activity by trypan blue and MTT assay. 4-(4'-phenoxybiphenyl-4-yl) thiazol- 2-amine (compound 3m) was identified as a lead anticancer agent and further, the effect of 3m on CML cells (K562) was investigated by flow cytometry, annexin V-FITC-propidium iodide staining, measuring the mitochondrial membrane potential (JC-1 staining) and DNA fragmentation assay. Results: MTT and trypan blue dye exclusion assay results presented 3m as the lead anticancer agent. Flow cytometric analysis revealed the accumulation of K562 cells in subG1phase in a time- and dosedependent manner. Annexin-V-FITC-PI staining demonstrated the increase in percentage of apoptotic cells on treatment with 3m. Furthermore, 3m also induced DNA fragmentation and disrupted mitochondrial membrane potential in K562 cells in dose-dependent manner. In addition, apoptosis inducing effect of 3m was reconfirmed by live-dead assay and confocal microscopic studies. Conclusion: The present study suggests that compound 3m has the potential to be a promising candidate for the treatment of CML. Keywords: Anticancer agent, chemotherapeutics, hantsch reaction, K562, suzuki coupling, thiazole.

Optimizing the genetic management of reintroduction projects: genetic population structure of the captive Northern Bald Ibis population

Many threatened species are bred in captivity for conservation purposes and some of these programmes aim at future reintroduction. The Northern Bald Ibis, Geronticus eremita, is a Critically Endangered bird species, with recently only one population remaining in the wild (Morocco, Souss Massa region). During the last two decades, two breeding programs for reintroduction have been started (in Austria and Spain). As the genetic constitution of the founding population can have strong effects on reintroduction success, we studied the genetic diversity of the two source populations for reintroduction (‘Waldrappteam’ and ‘Proyecto eremita’) as well as the European zoo population (all individuals held ex situ) by genotyping 642 individuals at 15 microsatellite loci. To test the hypothesis that the wild population in Morocco and the extinct wild population in the Middle East belong to different evolutionary significant units, we sequenced two mitochondrial DNA fragments. Our results show that the European zoo population is genetically highly structured, reflecting separate breeding lines. Genetic diversity was highest in the historic samples from the wild eastern population. DNA sequencing revealed only a single substitution distinguishing the wild eastern and wild western population. Contrary to that, the microsatellite analysis showed a clear differentiation between them. This suggests that genetic differentiation between the two populations is recent and does not confirm the existence of two evolutionary significant units. The European zoo population appears to be vital and suitable for reintroduction, but the management of the European zoo population and the two source populations for reintroductions can be optimized to reach a higher level of admixture.

Sperm quality biomarkers complement reproductive and endocrine parameters in investigating environmental contaminants in common carp (Cyprinus carpio) from the Lake Mead National Recreation Area

Lake Mead National Recreational Area (LMNRA) serves as critical habitat for several federally listed species and supplies water for municipal, domestic, and agricultural use in the Southwestern U.S. Contaminant sources and concentrations vary among the sub-basins within LMNRA. To investigate whether exposure to environmental contaminants is associated with alterations in male common carp (Cyprinus carpio) gamete quality and endocrine- and reproductive parameters, data were collected among sub-basins over 7 years (1999–2006). Endpoints included sperm quality parameters of motility, viability, mitochondrial membrane potential, count, morphology, and DNA fragmentation; plasma components were vitellogenin (VTG), 17ß-estradiol, 11-keto-testosterone, triiodothyronine, and thyroxine. Fish condition factor, gonadosomatic index, and gonadal histology parameters were also measured. Diminished biomarker effects were noted in 2006, and sub-basin differences were indicated by the irregular occurrences of contaminants and by several associations between chemicals (e.g., polychlorinated biphenyls, hexachlorobenzene, galaxolide, and methyl triclosan) and biomarkers (e.g., plasma thyroxine, sperm motility and DNA fragmentation). By 2006, sex steroid hormone and VTG levels decreased with subsequent reduced endocrine disrupting effects. The sperm quality bioassays developed and applied with carp complemented endocrine and reproductive data, and can be adapted for use with other species.

Abstract A06: BRCA-like phenotype as a hallmark of osteosarcoma's converging evolution

Abstract Background: Osteosarcomas are aggressive bone tumors with a high degree of genetic heterogeneity, which has historically limited treatment options in patients who do not respond to standard chemotherapy. Methods: We sequenced exomes and genomes of 32 pretherapeutic osteosarcomas, 6 time-series consisting of chemotherapy-naïve biopsies, resection specimens, recurring tumors and/or metastases, and 15 unpaired metastases from (mainly) poor responders. We then interrogated signatures of mutation processes in cancer genomes and reconciled clonal maps with phylogenetic trees, thus building an evolutionary history of each tumor. Where possible, we searched for evidence of selection acting on the tumors and hallmarks that could be therapeutically exploited. Results: We identified pathogenic mutations in at least 14 cancer drivers, some of which were formerly unknown in the context of osteosarcoma. However, there was no single cancer driver gene that could explain the majority of cases. Instead, &amp;gt;80% of osteosarcomas exhibited a specific combination of single-base substitutions, LOH, or large-scale genome instability characteristic of BRCA1/2-deficient tumors. The BRCA-like phenotype developed through parallel evolution by deleterious mutations affecting the BRCA1/2 genes and their binding partners such as PALB2 or CHEK2. We therefore investigated the sensitivity of a panel of osteosarcoma cell lines to the poly(ADP)-ribose polymerase inhibitor talazoparib alone and in combination with chemotherapeutic drugs. We identified an association between homologous recombination repair deficiency and the response of osteosarcoma cell lines to talazoparib. In combination with temozolomide, talazoparib led to the activation of BAX and BAK, loss of mitochondrial membrane potential, DNA fragmentation, and caspase-dependent cell death. Conclusion: The acquisition of a BRCA-like phenotype through converging evolution has important implications for the development of novel treatment strategies in osteosarcoma using PARP inhibitors alone or together with standard chemotherapy. Citation Format: Michal Kovac, Sebastian Ribi, Claudia Blattmann, Michaela Nathrath, Daniel Baumhoer. BRCA-like phenotype as a hallmark of osteosarcoma's converging evolution [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A06.