Field assessment of horse-associated genetic markers HoF597 and mtCytb for detecting the source of contamination in surface waters.

We investigated the specificity and sensitivity of two horse-associated markers, HoF597 and Horse mtCytb, and 12 mitochondrial and bacterial markers of six animal species (human, cow, pig, bird, dog, chicken) in the faecal samples of 50 individual horses. Both horse markers were detected in 48 (96%) faecal samples. Cross-reactivity with dog (BacCan545) and pig (P23-2) occurred in 88% and 72% of horse faecal samples, respectively. Several other bacterial and mitochondrial markers of non-target hosts were also detected; however, their specificities were >80%. Analyses of samples from surface waters (n = 11) on or adjacent to properties from which horse faecal samples had been collected showed only the presence of HoF597 but not horse mitochondrial marker. Our data suggest that while bacterial and (or) mitochondrial markers of other animal species may be present in horse faeces, dog and pig markers may predominantly be present in horse faecal samples, which points to their nonspecificity as markers for microbial source tracking. Although HoF597 and Horse mtCytb are highly sensitive and specific for the detection of horse faecal pollution, because of their low numbers, mitochondrial (mtDNA) markers may not be robust for screening surface waters.

A Natural mtDNA Polymorphism in Complex III Is a Modifier of Healthspan in Mice.

In this study, we provide experimental evidence that a maternally inherited polymorphism in the mitochondrial cytochrome b gene (mt-Cytb; m.15124A>G, Ile-Val) in mitochondrial complex III resulted in middle-aged obesity and higher susceptibility to diet-induced obesity, as well as age-related inflammatory disease, e.g., ulcerative dermatitis, in mice. As a consequence of the gene variation, we observed alterations in body composition, metabolism and mitochondrial functions, i.e., increased mitochondrial oxygen consumption rate and higher levels of reactive oxygen species, as well as in the commensal bacterial composition in the gut, with higher abundance of Proteobacteria in mice carrying the variant. These observations are in line with the previously described links of the mitochondrial complex III gene with obesity and metabolic diseases in humans. Given that these functional changes by the G variant at m.15124 in the mt-Cytb are already present in young mice that were kept under normal condition, it is plausible that the m.15124A>G variant is a disease susceptibility modifier to the diseases induced by additional stressors, i.e., dietary and/or aging stress, and that the variant results in the higher incidence of clinical diseases presentation in C57BL/6J-mt129S1/SvlmJ than C57BL/6J mice. Thus, mtDNA variants could be potential biomarkers to evaluate the healthspan.

LncRNA MFRL regulates the phenotypic switch of vascular smooth muscle cells to attenuate arterial remodeling by encoding a novel micropeptide MFRLP.

BACKGROUND: Arterial remodeling is a common pathophysiological change in the pathogenesis of cardiovascular diseases in which the phenotypic switch of vascular smooth muscle cells (VSMC) plays an important role. Recently, an increasing number of long non-coding RNAs(lncRNAs) have been shown to encode micropeptides that play biological roles and have great clinical transformation potential. However, the role of micropeptides encoded by lncRNAs in arterial remodeling has not been well studied and requires further exploration. METHODS AND RESULTS: Through bioinformatic analysis and experimental verification, we found that a new lncRNA, the mitochondrial function-related lncRNA (MFRL), encodes a 64-amino acid micropeptide, MFRLP. MFRL and MFRLP play important roles in the phenotypic switch of VSMC. Further experiments showed that MFRLP interacts with mitochondrial cytochrome b to reduce accumulation of reactive oxygen species, suppress mitophagy and inhibit the VSMC switch from contractile to synthetic phenotype. CONCLUSIONS: LncRNA MFRL encodes the micropeptide MFRLP, which interacts with mitochondrial cytochrome b to inhibit the VSMC switch from contractile to synthetic phenotype and improve arterial remodeling.

Phylogeographic Substructuring in the Southernmost Refugium of the European Common Frog Rana temporaria.

Rana temporaria is one of the most widespread Palearctic brown frogs. We aimed to clarify distribution pattern of two main genetic clades in the understudied Balkan peninsula by using 16SrRNA and MT-CYTB sequences, already widely applied in analyses of populations from other parts of Europe, while focusing on the broad area along the Morava river (central Balkans) as a known gap in the species distribution. Additionally, we were interested in revealing the extent of haplotype diversity within the main genetic clades in the Balkans, particularly around the supposed suture zone. The results revealed a suture zone between the Western and Eastern Clades in the central part of the Balkan Peninsula. This indicated the existence of a historical barrier between the Balkan Mountain Belt and geographically close mountains surrounding the Vlasina Plateau (Rhodope/Serbian-Macedonian Massif). The overall observed haplotype diversity in populations of R. temporaria from the Balkan Peninsula seems high. Harboring both main genetic clades of R. temporaria qualifies the Balkan Peninsula as another important center of species' genetic diversity, as well as rich in unique haplotypes. This points out the necessity of applying conservation measures focused on the common European frog populations and habitats in this part of the species' distribution area.

A Cryptic Subterranean Mammal Species, the Lesser Blind Mole Rat (Nannospalax leucodon syrmiensis)-Retreated but Not Extinct.

Blind mole rats (genus Nannospalax) attract a great deal of attention because of their cancer resistance and longevity. Due to the high rate of chromosome rearrangements, 74 Nannospalax chromosomal forms have been discovered. The convergence of their external morphology complicates their taxonomy, and many cryptic species remain unrecognized. Thus, the European N. leucodon supersp. is listed in the IUCN Red List of Threatened Species with "Data Deficient" status. It is crucial for the conservation of biodiversity to clarify its taxonomy, to recognize each cryptic species, and assign to them the correct conservation status. Of the more than 20 chromosomal forms described within N. leucodon, five cryptic species occur in Serbia. The most threatened among them-N. l. syrmiensis, described and named 50 years ago in the regions of Srem, Belgrade and Macva-has been declared extinct in the literature, which may have negative consequences for the conservation of wildlife genetic diversity. Through five years of fieldwork and comparison of 16SrRNA and MT-CYTB gene segments between old, archived teeth and recently collected material, we show that N. l. syrmiensis is not extinct. However, its habitat has been fragmented and reduced, owing primarily to anthropogenic impact. Therefore, detailed surveillance, population-structure studies, risk assessment, and appropriate conservation measures are needed.

Biogeographic determinants of genetic diversification in the mouse opossum Gracilinanus agilis (Didelphimorphia: Didelphidae).

The genetic variation of Brazilian populations of the mouse opossum Gracilinanus agilis was analyzed on the basis of the mitochondrial Cytochrome b gene (mt-Cytb) and the exon 28 of the nuclear Von Willenbrand factor (e28-vWF). The radiation of Gracilinanus was dated at 4.80 Ma, with the appearance of G. agilis around 1.93 Ma. Gracilinanus aceramarcae appeared as the first offshoot of the genus, followed by Gracilinanus emiliae and Gracilinanus microtarsus, which composed a sister clade of G. agilis. Phylogeographic analyses and genetic distance estimates indicate G. agilis as a single species, with haplotypes grouping in three well-supported clades, one from midwestern Brazil, a second one from northeastern Brazil, and a third one from eastern Brazil. Phylogeographic patterns in G. agilis were interpreted in search for congruence between genetic breaks and historic geomorphologic events documented for the region stretching northeastern to central-western of the Brazilian shield. The Rio São Francisco and the Serra Geral de Goiás were found to represent relevant geographic barriers to gene flow for G. agilis populations as well as for populations of several other widespread taxa.

Cryptic Diversity of the European Blind Mole Rat Nannospalax leucodon Species Complex: Implications for Conservation.

We explored the cryptic speciation of the Nannospalax leucodon species complex, characterised by intense karyotype evolution and reduced phenotypic variability that has produced different lineages, out of which 25 are described as chromosomal forms (CFs), so many cryptic species remain unnoticed. Although some of them should be classified as threatened, they lack the official nomenclature necessary to be involved in conservation strategies. Reproductive isolation between seven CFs has previously been demonstrated. To investigate the amount and dynamics of genetic discrepancy that follows chromosomal changes, infer speciation levels, and obtain phylogenetic patterns, we analysed mitochondrial 16S rRNA and MT-CYTB nucleotide polymorphism among 17 CFs-the highest number studied so far. Phylogenetic trees delineated 11 CFs as separate clades. Evolutionary divergence values overlapped with acknowledged higher taxonomic categories, or sometimes exceeded them. The fact that CFs with higher 2n are evolutionary older corresponds to the fusion hypothesis of Nannospalax karyotype evolution. To participate in conservation strategies, N. leucodon classification should follow the biological species concept, and proposed cryptic species should be formally named, despite a lack of classical morphometric discrepancy. We draw attention towards the syrmiensis and montanosyrmiensis CFs, estimated to be endangered/critically endangered, and emphasise the need for detailed monitoring and population survey for other cryptic species.

Population genetic structure of the biological control agent Macrolophus pygmaeus in Mediterranean agroecosystems.

Biological control of agricultural pests relies on knowledge of agroecosystem functionality, particularly when affected by the use of mass-produced biological agents. Incorporating pre- and/or post-release information such as genetic diversity and structure on these agents using molecular-based approaches could advance our knowledge of how they perform in agroecosystems. We evaluated the population genetics of Macrolophus pygmaeus, the most widely used predatory mirid against many arthropod pests of greenhouse crops in the Mediterranean region, using the mitochondrial Cytb sequence and microsatellite data, and population genetics and phylogeny approaches. We investigated commercially mass-produced insects (i.e., commercial insects either mass-reared in the laboratory for many generations, or purchased by farmers and released in the greenhouses) and "wild" insects (i.e., that occur naturally outside or are collected in nature for release in the greenhouses). The mirids were mainly collected in agroecosystems in which solanaceous plants are grown in northern Spain, southern France and Greece. Both molecular markers and approaches distinguished 2 genetically differentiated populations. The less genetically diverse population, hereafter named the "commercial" strain included all individuals from laboratory mass-rearings and most releases of commercially bred individuals. The most genetically diverse population mainly comprised individuals originating from noncultivated environments, or from releases of "wild" individuals. Rare examples of hybridization between M. pygmaeus from the 2 populations were observed and asymmetric gene flow was revealed. These findings provide new insights into what happens to M. pygmaeus released in the agroecosystems we studied, and show that it is possible to monitor some commercial strains.

Glycogen synthase kinase-3 (GSK3) controls deoxyglucose-induced mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells.

Mitochondrial biogenesis, a mitochondrial growth and division process, is crucial for adaptation to metabolic stress. The present study demonstrated that treatment with a specific inhibitor of GSK3, SB216763, attenuated induction of mitochondrial biogenesis by a glycolysis inhibitor, 2-deoxyglucose (2-DG), without affecting this biogenesis at basal condition. Additionally, overexpression of WT-GSK3ß promoted whereas GSK3ß-KD attenuated 2-DG-induced mitochondrial protein expression. The mitochondrial biogenesis attenuation by GSK3 inhibitor was not due to inhibition of protein degradation. Furthermore, GSK3 inhibition further reduced transcription of mitochondrial (COXII), but not nuclear (VDAC) gene by 2-DG suggesting its participation in 2-DG-induced mitochondrial transcription. Together, our results show that GSK3 regulates mitochondrial biogenesis induced by glycolysis inhibition.

Monitoramento da suscetibilidade ao piretróide Deltametrina em populações de Triatoma sordida Stal, 1859 (Hemiptera: Reduviidae) / Monitoring susceptibility to pyrethroid deltamethrin in populations of Triatoma sordida Stal, 1859 (Hemiptera: Reduviidae)

A resistência de triatomíneos a inseticidas já foi relatada na Venezuela, Argentina e Bolívia, sendo possível a existência de outros focos em regiões ainda mais amplas. Deste modo, torna-se necessário desenvolver estudos que investiguem a dispersão geográfica desses vetores resistentes, bem como os mecanismos envolvidos, com vistas a elaborar estratégias adequadas às necessidades locais, ou até mesmo que impeçam o aparecimento deste fenômeno. O objetivo deste estudo foi padronizar ensaios biológicos para monitoramento de resistência de triatomíneos a inseticidas em laboratório e caracterizar a suscetibilidade de quatorze populações de Triatoma sordida Stal, 1859 de Minas Gerais (Sudeste do Brasil) ao piretróide Deltametrina. A área em que os triatomíneos foram coletados foi submetida ao controle químico por cerca de 30 anos. A linhagem referência de suscetibilidade é oriunda de Uberaba. A metodologia foi orientada pelas recomendações da WHO (1994) e OPS (2005), tendo sido redefinidos alguns detalhes técnicos (dose diagnóstica adequada para triagem de resistência em triatomíneos, local ideal de aplicação do inseticida nas ninfas I, idade e geração dos insetos utilizados). A linha base de suscetibilidade da linhagem referência foi inicialmente determinada. A Deltametrina, solubilizada em acetona, foi aplicada topicamente no dorso do abdômen de ninfas I da geração F2 (cinco dias de idade, jejum, ± 1,2 mg. de peso) com auxílio de uma microseringa.

Os resultados dos ensaios dose-resposta foram analisados com auxílio do programa PROBIT (Finney, 1971). Em seguida, as populações de campo foram submetidas a dose diagnóstica de 1XDL99 da linhagem referência de suscetibilidade e 2XDD99 da linhagem referência de suscetibilidade. Todas as populações apresentaram-se menos suscetíveis ao inseticida quando comparadas com a linhagem referência de suscetibilidade. As razões de resistência encontradas variaram de 2,6 a 6,8. A comparação de um fragmento de DNA de 317 pb do gene mitocondrial do citocromo b (mtCytB) foi usada para inferir sobre a variabilidade genética das quatorze populações de T. sordida estudadas. Inferências filogenéticas, usando P. megistus como grupo externo, não revelaram a formação de clados. Os 150 espécimes analisados apresentaram 50 haplótipos gerando uma diversidade (Hd) de 0,831. Foi possível distinguir seis populações geneticamente diferentes. Análise do Network revelou a presença de vários loops, demonstrando homoplasia e a ocorrência de mutações reversas/paralelas. Com o marcador genético utilizado não foi possível estabelecer correlação entre a variabilidade genética das populações estudadas e a resistência a Deltametrina. Giordano et al. (2005) sugerem que os microssatélites podem ser uma ferramenta mais sensível e eficiente para este objetivo. A possível natureza bioquímica da resistência encontrada está sendo verificada

Monitoramento da suscetibilidade ao piretróide Deltametrina em populações de Triatoma sordida Stål, 1859 (Hemiptera: Reduviidae)

A resistência de triatomíneos a inseticidas já foi relatada na Venezuela, Argentina e Bolívia, sendo possível a existência de outros focos em regiões ainda mais amplas. Deste modo, torna-se necessário desenvolver estudos que investiguem a dispersão geográfica desses vetores resistentes, bem como os mecanismos envolvidos, com vistas a elaborar estratégias adequadas às necessidades locais, ou até mesmo que impeçam o aparecimento deste fenômeno. O objetivo deste estudo foi padronizar ensaios biológicos para monitoramento de resistência de triatomíneos a inseticidas em laboratório e caracterizar a suscetibilidade de quatorze populações de Triatoma sordida Stal, 1859 de Minas Gerais (Sudeste do Brasil) ao piretróide Deltametrina. A área em que os triatomíneos foram coletados foi submetida ao controle químico por cerca de 30 anos. A linhagem referência de suscetibilidade é oriunda de Uberaba. A metodologia foi orientada pelas recomendações da WHO (1994) e OPS (2005), tendo sido redefinidos alguns detalhes técnicos (dose diagnóstica adequada para triagem de resistência em triatomíneos, local ideal de aplicação do inseticida nas ninfas I, idade e geração dos insetos utilizados). A linha base de suscetibilidade da linhagem referência foi inicialmente determinada. A Deltametrina, solubilizada em acetona, foi aplicada topicamente no dorso do abdômen de ninfas I da geração F2 (cinco dias de idade, jejum, ± 1,2 mg. de peso) com auxílio de uma microseringa.

Os resultados dos ensaios dose-resposta foram analisados com auxílio do programa PROBIT (Finney, 1971). Em seguida, as populações de campo foram submetidas a dose diagnóstica de 1XDL99 da linhagem referência de suscetibilidade e 2XDD99 da linhagem referência de suscetibilidade. Todas as populações apresentaram-se menos suscetíveis ao inseticida quando comparadas com a linhagem referência de suscetibilidade. As razões de resistência encontradas variaram de 2,6 a 6,8. A comparação de um fragmento de DNA de 317 pb do gene mitocondrial do citocromo b (mtCytB) foi usada para inferir sobre a variabilidade genética das quatorze populações de T. sordida estudadas. Inferências filogenéticas, usando P. megistus como grupo externo, não revelaram a formação de clados. Os 150 espécimes analisados apresentaram 50 haplótipos gerando uma diversidade (Hd) de 0,831. Foi possível distinguir seis populações geneticamente diferentes. Análise do Network revelou a presença de vários loops, demonstrando homoplasia e a ocorrência de mutações reversas/paralelas. Com o marcador genético utilizado não foi possível estabelecer correlação entre a variabilidade genética das populações estudadas e a resistência a Deltametrina. Giordano et al. (2005) sugerem que os microssatélites podem ser uma ferramenta mais sensível e eficiente para este objetivo. A possível natureza bioquímica da resistência encontrada está sendo verificada