Complete mitochondrial genomes of three species of fresh flies of forensic entomology interest from the genus Sarcophaga (Sarcophagidae) from Portugal and Brazil

The Sarcophagidae family of fresh flies bears strong importance in the context of forensic entomology due to their application in the estimation of the Post Mortem Interval (PMI). Sarcophaga is the major genus in the Sarcophagidae family and includes cosmopolitan species, which are distributed worldwide. In this communication, we present the analysis of the complete mitochondrial genome (mtDNA) of two species from Portugal – S. melanura and S. dux – and one from Brazil – S. ruficornis. The mtDNA of these species range from 14,882 bp to 15,190 bp and have 22 tRNA genes, 13 protein-coding genes (PCG), and two rRNAs distributed along both strands. Our data include the first record of complete Sarcophaga mtDNA sequences from species collected in Portugal and in Brazil. These genomes represent an advance in the understanding about this group, expand the database, and can be used for the development of new markers for species identification41237239CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES25/2014; 23038.006839 / 2014-33; 0092/ 17-7This work was funded by Coordenac¸~ao de Aperfeic¸oamento de Pessoal de N ıvel Superior (CAPES) (Edital Ci^ encias Forenses no. 25/2014, Process 23038.006839 / 2014-33), International postdoc fellowship (CAPES 0092/ 17-7) and Conselho Nacional de Desenvolvimento Cient ıfico e Tecnologico (CNPq). Financial support was also provided by CESAM (UID/ AMB/50017 - POCI-01-0145-FEDER-007638), and by FCT/MCTES through national funds (PIDDAC). Co-funding was provided by the FEDER, within the PT2020 Partnership Agreement and Compete 202

Vascular Endothelial Growth Factor Increases During Blood-brain Barrier-enhanced Permeability Caused By Phoneutria Nigriventer Spider Venom.

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na(+), K(+) and Ca(2+) channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.201472196

Complete mitochondrial genomes from four species of the genus Oxysarcodexia (Sarcophagidae) with forensic entomology interest

The Sarcophagidae family is one of the most important in forensic entomology. Its richest genus, Oxysarcodexia, is well-distributed in Brazil and Latin America and includes species that can be used in forensic investigations to estimate the Post Mortem Interval (PMI). In this communication, we present the mitochondrial genome (mtDNA) of four Oxysarcodexia species: O. avuncula (one specimen), O. terminalis (one specimen), O. thornax (three specimens), and O. varia (one specimen). These mitochondrial genomes (mtDNA) range from 14,998 to 15,613 bp and have 22 tRNA genes, 13 protein-coding genes (PCG), and 2 rRNAs distributed along both strands. The mitogenomes from Oxysarcodexia showed in the present work represents an important contribution to the knowledge regarding the Sarcophagidae phylogenetic structure and is an important source of information for the development of novel DNA markers for forensic identification4143043

Characterization of a new Autographa californica multiple nucleopolyhedrovirus (AcMNPV) polyhedra mutant

In the very late phase of baculovirus infection, virions are occluded in a crystalline matrix called polyhedra, which is mainly composed of polyhedrin. This protein is highly conserved among baculoviruses and changes in its amino acid sequence may lead to mutant polyhedra. During the purification of an AcMNPV recombinant virus, a mutant virus was isolated. Structural and ultrastrutural analysis by light and transmission electron microscopy (TEM) of insect cells infected with this mutant virus did not show polyhedra formation and differed from the wild-type infection by the presence of a proteinaceous mass dispersed in the cytoplasm and nucleus of the infected cells, which was confirmed by immunogold labelling to be polyhedrin. The polyhedrin gene was amplified by PCR and sequenced. The only change observed was the substitution of a G to a T at the nucleotide +352, which resulted in a Val to Phe change. A recombinant virus was constructed by transferring the mutant gene into a polyhedrin negative virus. The phenotype of this recombinant virus was the same as the mutant one, confirming that this single mutation alone was responsible for the mutant phenotype

Complete mitochondrial genomes from three species of the genus Peckia (Sarcophagidae) with forensic entomology interest

Peckia is one of the most important genera in the Sarcophagidae family of flesh flies. This genus is distributed in Brazil and Latin America, and its species can be used to estimate the Post Mortem Interval (PMI) in forensic investigations. In this communication, we present four mitochondrial genomes (mtDNA) from three Peckia species: P. australis, P. collusor, and P. resona. These mtDNA range from 15,116 bp to 15,234 bp in length and have 22 tRNA genes, 13 protein-coding genes (PCG), and two rRNAs distributed along both the strands. These data expand the knowledge about the Sarcophagidae genomes and present, for the first time, four complete mtDNA sequences of the Peckia genus. We show novel complete mtDNA sequences of flesh fly species of forensic importance. Our data expand the knowledge on the molecular database for the identification of these species, and is an important step towards increasing the databases and can help on the identification of new species, particularly in the forensic context41220221CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESNão tem25/2014; 23038.006839/2014-33; 0092/ 17-7This work was funded by Coordenac¸~ao de Aperfeic¸oamento de Pessoal de N ıvel Superior (CAPES) (Edital Ciências Forenses no. 25/2014, Process 23038.006839/2014-33), International postdoc fellowship (CAPES 0092/ 17-7) and Conselho Nacional de Desenvolvimento Cient ıfico e Tecnologico (CNPq). Financial support was also provided by CESAM (UID/ AMB/50017 - POCI-01-0145-FEDER-007638), and by FCT/MCTES through national funds (PIDDAC). Co-funding provided by the FEDER, within the PT2020 Partnership Agreement and Compete 202

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

BACKGROUND: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite. METHODS: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay. RESULTS: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay. CONCLUSION: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid

Expression Of Vegf And Flk-1 And Flt-1 Receptors During Blood-brain Barrier (bbb) Impairment Following Phoneutria Nigriventer Spider Venom Exposure.

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, β-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats.52572-8

Expressed sequence tags in venomous tissue of Scorpaena plumieri (Scorpaeniformes: Scorpaenidae)

Species of the family Scorpaenidae are responsible for accidents and sporadic casualties by the shore they inhabit. The species Scorpaena plumieri from this family populate the Northeastern and Eastern coast of Brazil causing human envenomation characterized by local and systemic symptoms. In experimental animals the venom induces cardiotoxic, hypotensive, and airway respiratory effects. As first step to identify the venom components we isolated gland mRNA to produce a cDNA library from the fish gland. This report describes the partial sequencing of 356 gland transcripts from S. plumieri. BLAST analysis of transcripts showed that 30% were unknown sequences, 17% hypothetical proteins, 17% related to metabolic enzymes, 14% belonged to signal transducing functions and the remaining groups (7-8%) composed by gene related with expressing proteins, regulatory proteins and structural proteins. A considerable number of these EST were not found in available databases suggesting the existence of new proteins and/or functions yet to be discovered. By screening the library with antibodies against a lectin fraction from S. plumieri venom we identified several clones whose DNA sequence showed similarities with lectins found in fish. In silico analysis of these clones confirm the identity of these molecules in the venom gland of S. plumieri. Espécies da família Scorpaenidae são responsáveis por acidentes e mortes esporádicas ao longo da costa que habitam. A espécie Scorpaena plumieri desta família povoam a costa Leste e Nordeste do Brasil, causando envenenamento humano caracterizado por sintomas locais e sistêmicos. Em modelos experimentais animais a peçonha induz cardiotoxicidade, efeitos hipotensivos e alterações nas vias aéreas respiratórias. Como primeiro passo para identificar os componentes da peçonha foram isolados os mRNA das glândulas do peixe para produzir uma biblioteca de cDNAs. Esse artigo descreve o sequenciamento parcial de 356 transcritos das glândulas de S. plumieri. Análises em bancos de dados (BLAST) dos transcritos demonstraram que 30% eram sequências desconhecidas, 17% proteínas hipotéticas, 17% relacionadas às enzimas do metabolismo, 14% pertenciam a funções de transdução de sinais e os demais grupos (7-8%) formados por genes relacionados com a expressão de proteínas, proteínas regulatórias e estruturais. Um número considerável destes EST não foi encontrado em bases de dados disponíveis, sugerindo a existência de novas proteínas e/ou funções ainda a serem descobertas. Ao fazer um barrido da biblioteca com anticorpos produzidos contra uma fração das lectinas do veneno de S. plumieri, identificamos vários clones, cuja sequência de DNA mostram semelhanças com lectinas encontradas em peixes. A análise in silico destes clones confirmam a identidade destas moléculas na glândula de peçonha de S. plumieri

Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes

Fish germ cell transplantation presents several important potential applications for aquaculture, including the preservation of germplasm from endangered fish species with high genetic and commercial values. Using this technique in studies developed in our laboratory with adult male Nile tilapias (Oreochromis niloticus), all the necessary procedures were successfully established, allowing the production of functional sperm and healthy progeny approximately 2 months after allogeneic transplantation. In the present study, we evaluated the viability of the adult Nile tilapia testis to generate sperm after xenogeneic transplant of germ cells from sexually mature Jundia catfish (Rhamdia quelen) that belong to a different taxonomic order. Therefore, in order to investigate at different time-periods post-transplantation, the presence and development of donor PKH26 labeled catfish germ cells were followed in the tilapia seminiferous tubules. From 7 to 20 days post-transplantation, only PKH26 labeled spermatogonia were observed, whereas spermatocytes at different stages of development were found at 70 days. Germ cell transplantation success and progression of spermatogenesis were indicated by the presence of labeled PKH26 spermatids and sperm on days 90 and 120 post-transplantation, respectively. Confirming the presence of the catfish genetic material in the tilapia testis, all recipient tilapias evaluated (n = 8) showed the genetic markers evaluated. Therefore, we demonstrated for the first time that the adult Nile tilapia testis offers the functional conditions for development of spermatogenesis with sperm production from a fish species belonging to a different order, which provides an important new venue for aquaculture advancement. © 2016 Elsevier Inc