Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Effect of porcine somatotropin on metabolism and testicular characteristics of prepubertal pigs / Efeito da somatotrofina suína sobre o metabolismo e características testiculares de suínos pré-púberes

The effect of pST on the testicular characteristics and metabolic parameters of prepubertal pigs was evaluated. Experiment 1 aimed to determine the interval between applications of pST based on the concentrations of circulating IGF-I. Experiment 2 aimed to evaluate the effect of pST on metabolic parameters, testicular characteristics, and expression of GHR, IGF-I and PCNA. In Experiment 1 twelve piglets with 30 days of age were used. The pST Group (n = 6) was submitted to one i.m. injection of pST and the Control Group (n = 6) to one placebo injection. Blood collections were performed until day 7 post pST application to determine IGF-I concentration and metabolic profile. In Experiment 2 twelve piglets with 22 days of age were used. The pST Group was submitted to pST injections every three days, and the Control Group received placebo doses during 30 days. Blood collections were performed every 3 days. Samples of liver and testicular tissue were collected to determine gene expression and testicular characteristics. In Experiment 1 IGF-I concentration was higher for the pST Group (P = 0.02). In Experiment 2 the pST Group had higher body and testicular weight (P=0.06) and increased gene expression of PCNA in testes (P < 0.05). However, a reduction in the number of seminiferous tubules, and Sertoli cells, and in GHR expression (P < 0.05) was observed. Thus, pST administration increased body and testis development in prepubertal pigs, however it reduced the density of seminiferous tubules and Sertoli cells.

Foi investigado o efeito da pST sobre características testiculares e metabolismo de suínos pré-púberes. O Experimento 1 determinou o intervalo entre aplicações de pST, baseado nas concentrações de IGF-I. O Experimento 2 avaliou o efeito da pST sobre o metabolismo, características testiculares e expressão gênica de GHR, IGF-I e PCNA. No Experimento 1, foram usados 12 leitões com 30 dias de idade. O grupo pST (n = 6) foi submetido a uma injeção IM de pST e o grupo Controle (n = 6) a uma injeção de placebo. Coletas de sangue foram realizadas até o dia sete após a aplicação de pST para determinação dos níveis de IGF-I e parâmetros metabólicos. No Experimento 2, foram usados 12 leitões com 22 dias de idade. O grupo pST foi submetido às aplicações de pST a cada 3 dias, e o grupo Controle, às doses de placebo, durante 30 dias. Coletas de sangue foram realizadas a cada três dias. Amostras de fígado e testículo foram coletadas para determinar a expressão gênica e características testiculares. No Experimento 1, a concentração de IGF-I foi maior no grupo pST (P = 0,02). No Experimento 2, o grupo pST teve maior peso corporal e testicular (P = 0,06) e aumento na expressão de PCNA no testículo (P < 0,05). Contudo, foi observada uma redução no número de túbulos seminíferos, células de Sertoli e GHR (P < 0,05). Assim, a administração de pST aumentou o desenvolvimento testicular e corporal de suínos pré-púberes, porém reduziu a densidade de túbulos seminíferos e células de Sertoli.

NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus / Expressão gênica do NPY e do sbGnRH em machos juvenis e adultos de linguado Paralichthys orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

O objetivo deste estudo foi avaliar a expressão gênica do neuropeptídeo Y (NPY) e da variante sea bream do hormônio liberador de gonadotrofinas (sbGnRH) em linguados machos juvenis e adultos. O hipotálamo foi isolado para a extração de RNA total. Após a síntese de cDNA, a PCR em tempo real foi usada para avaliar a expressão gênica. Foi observado um aumento de aproximadamente duas vezes nos níveis de NPY e de aproximadamente três vezes nos níveis de sbGnRH nos peixes adultos. Esses resultados demonstram que estes peptídeos podem estar envolvidos na regulação, via hipotálamo, da maturação sexual no linguado.

MicroRNAs e seu papel no desenvolvimento embrionário / MicroRNAs and its role in embryonic development

MicroRNAs (miRNAs) são pequenas moléculas de RNA com aproximadamente 22 nucleotídeos incapazes de codificar proteínas e que apresentam função na regulação pós-transcricional da expressão gênica. Vários estudos vêm demonstrando o importante papel dos miRNAs na regulação do desenvolvimento embrionário de diferentes espécies, desde o controle da expressão de RNAs mensageiros durante o desenvolvimento inicial embrionário até a determinação de linhagens celulares durante a organogênese. Esta revisão irá abordar os principais miRNAs e seu papel na biologia reprodutiva, com ênfase no desenvolvimento embrionário de mamíferos.

MicroRNAs (miRNAs) are small RNA molecules with around 22 nucleotides that are unable to encode proteins and play a key role on post-transcription regulation process. Several studies have demonstrated the relevant role of miRNAs on the regulation of embryonic development on different species, from the control of gene expression during the early embryo development to determination of cellular lineages over the organogenesis. This review will present the miRNAs and its role on reproductive biology focusing on the mammalian embryo development.

Transgene transmission in South American catfi sh (Rhamdia quelen) larvae by sperm-mediated gene transfer.

The silver catfi sh (Rhamdia quelen) is an endemic American fi sh species. The sperm of each species has its own peculiarities and biological characteristics, which infl uence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfi sh. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/ rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E, INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups (P>0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fl uorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the fi rst report on transgene transmission of exogenous DNA into silver catfi sh larvae through SMGT technology.