Distinct carbapenems susceptibility profiles in isogenic isolates of Klebsiella pneumoniae presenting Ompk36 disruption and expression of down-regulated blaKPC-2 / Perfis distintos de suscetibilidade a carbapenêmicos em isolados isogênicos de Klebsiella pneumoniae apresentando interrupção de Ompk36 e expressão de blaKPC-2 negativamente regulada

The isolation of multidrug-resistant Klebsiella pneumoniae in hospitals is a major public health threat, increasing patient hospitalization costs, morbidity and mortality. Therefore, this work investigated the resistance mechanisms that produced different carbapenems susceptibility profiles in two isogenic strains of K. pneumoniae isolated from the same patient in a public hospital in Recife, Pernambuco. The genes that encode the main porins in K. pneumoniae, ompK35 and ompK36, and several beta-lactamase genes were analyzed. The expression of these genes was evaluated by quantitative real time PCR (polymerase chain reaction) with reverse transcriptase (RT-qPCR). SDS-PAGE (sodium dodecyl sulphate­polyacrylamide gel electrophoresis) was performed to analyze the outer membrane proteins. The analysis of the ompK36 genetic environment disclosed an IS903 insertion sequence disrupting this gene in the ertapenem resistant isolate (KPN133). The blaKPC-2 gene showed down-regulated expression in both isolates. Our findings show that changes in porins, especially OmpK36, are more determinant to carbapenems susceptibility profile of bacterial isolates than variations in blaKPC gene expression.

O isolamento de Klebsiella pneumoniae multirresistente em hospitais é uma grande ameaça à saúde pública, aumentando os custos de internação, morbidade e mortalidade dos pacientes. Portanto, este trabalho investigou os mecanismos de resistência que produziram diferentes perfis de suscetibilidade aos carbapenêmicos em duas cepas isogênicas de K. pneumoniae isoladas do mesmo paciente em um hospital público em Recife, Pernambuco. Foram analisados ​​os genes que codificam as principais porinas em K. pneumoniae, ompK35 e ompK36, e diversos genes de beta-lactamases. A expressão desses genes foi avaliada por PCR (reação em cadeia da polimerase quantitativa em tempo real) com transcriptase reversa (RT-qPCR). SDS-PAGE (dodecil sulfato de sódio-poliacrilamida gel eletroforese) foi realizada para analisar as proteínas da membrana externa. A análise do ambiente genético ompK36 revelou uma sequência de inserção IS903 interrompendo este gene no isolado resistente ao ertapenem (KPN133). O gene blaKPC-2 apresentou expressão negativamente regulada em ambos os isolados. Nossos achados mostram que alterações nas porinas, especialmente OmpK36, são mais determinantes no perfil de suscetibilidade aos carbapenêmicos de isolados bacterianos do que variações na expressão do gene blaKPC.

Maternal obesity and ovarian failure: is leptin the culprit?

At the time of its discovery and characterization in 1994, leptin was mostly considered a metabolic hormone able to regulate body weight and energy homeostasis. However, in recent years, a great deal of literature has revealed leptin's pleiotropic nature, through its involvement in numerous physiological contexts including the regulation of the female reproductive tract and ovarian function. Obesity has been largely associated with infertility, and leptin signalling is known to be dysregulated in the ovaries of obese females. Hence, the disruption of ovarian leptin signalling was shown to contribute to the pathophysiology of ovarian failure in obese females, affecting transcriptional programmes in the gamete and somatic cells. This review attempts to uncover the underlying mechanisms contributing to female infertility associated with obesity, as well as to shed light on the role of leptin in the metabolic dysregulation within the follicle, the effects on the oocyte epigenome, and the potential long-term consequence to embryo programming.(AU)

Reproductive physiology of the heat-stressed dairy cow: implications for fertility and assisted reproduction

Heat stress causes a large decline in pregnancy success per insemination during warm times of the year. Improvements in fertility are possible by exploiting knowledge about how heat stress affects the reproductive process. The oocyte can be damaged by heat stress at the earliest stages of folliculogenesis and remains sensitive to heat stress in the peri-ovulatory period. Changes in oocyte quality due to heat stress are the result of altered patterns of folliculogenesis and, possibly, direct effects of elevated body temperature on the oocyte. While adverse effects of elevated temperature on the oocyte have been observed in vitro, local cooling of the ovary and protective effects of follicular fluid may limit these actions in vivo. Heat stress can also compromise fertilization rate. The first seven days of embryonic development are very susceptible to disruption by heat stress. During these seven days, the embryo undergoes a rapid change in sensitivity to heat stress from being very sensitive (2- to 4-cell stage) to largely resistant (by the morulae stage). Direct actions of elevated temperature on the embryo are likely to be an important mechanism for reduction in embryonic survival caused by heat stress. An effective way to avoid effects of heat stress on the oocyte, fertilization, and early embryo is to bypass the effects through embryo transfer because embryos are typically transferred into females after acquisition of thermal resistance. There may be some opportunity to mitigate effects of heat stress by feeding antioxidants or regulating the endocrine environment of the cow but neither approach has been reduced to practice. The best long-term solution to the problem of heat stress may be to increase genetic resistance of cows to heat stress. Thermotolerance genes exist within dairy breeds and additional genes can be introgressed from other breeds by traditional means or gene editing.

Reproductive physiology of the heat-stressed dairy cow: implications for fertility and assisted reproduction

Heat stress causes a large decline in pregnancy success per insemination during warm times of the year. Improvements in fertility are possible by exploiting knowledge about how heat stress affects the reproductive process. The oocyte can be damaged by heat stress at the earliest stages of folliculogenesis and remains sensitive to heat stress in the peri-ovulatory period. Changes in oocyte quality due to heat stress are the result of altered patterns of folliculogenesis and, possibly, direct effects of elevated body temperature on the oocyte. While adverse effects of elevated temperature on the oocyte have been observed in vitro, local cooling of the ovary and protective effects of follicular fluid may limit these actions in vivo. Heat stress can also compromise fertilization rate. The first seven days of embryonic development are very susceptible to disruption by heat stress. During these seven days, the embryo undergoes a rapid change in sensitivity to heat stress from being very sensitive (2- to 4-cell stage) to largely resistant (by the morulae stage). Direct actions of elevated temperature on the embryo are likely to be an important mechanism for reduction in embryonic survival caused by heat stress. An effective way to avoid effects of heat stress on the oocyte, fertilization, and early embryo is to bypass the effects through embryo transfer because embryos are typically transferred into females after acquisition of thermal resistance. There may be some opportunity to mitigate effects of heat stress by feeding antioxidants or regulating the endocrine environment of the cow but neither approach has been reduced to practice. The best long-term solution to the problem of heat stress may be to increase genetic resistance of cows to heat stress. Thermotolerance genes exist within dairy breeds and additional genes can be introgressed from other breeds by traditional means or gene editing.(AU)

Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis

Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.(AU)

Jaburetox: update on a urease-derived peptide

Abstract Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jburell gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 g it provoked death of Triatoma infestans bugs. JBTX (105-106 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.

Jaburetox: update on a urease-derived peptide

Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jburell gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 μg it provoked death of Triatoma infestans bugs. JBTX (10−5-10−6 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.(AU)

Jaburetox: update on a urease-derived peptide

Urease from Canavalia ensiformis seeds was the first enzyme ever to be crystallized, in 1926. These proteins, found in plants, bacteria and fungi, present different biological properties including catalytic hydrolysis of urea, and also enzyme-independent activities, such as induction of exocytosis, pro-inflammatory effects, neurotoxicity, antifungal and insecticidal properties. Urease is toxic to insects and fungi per se but part of this toxicity relies on an internal peptide (~11 kDa), which is released upon digestion of the protein by insect enzymes. A recombinant form of this peptide, called jaburetox (JBTX), was constructed using jburell gene as a template. The peptide exhibits liposome disruption properties, and insecticidal and fungicidal activities. Here we review the known biological properties activities of JBTX, and comment on new ones not yet fully characterized. JBTX was able to cause mortality of Aedes aegypti larvae in a feeding assay whereas in a dose as low as of 0.1 μg it provoked death of Triatoma infestans bugs. JBTX (10−5-10−6 M) inhibits the growth of E. coli, P. aeruginosa and B. cereus after 24 h incubation. Multilamellar liposomes interacting with JBTX undergo reorganization of the membrane's lipids as detected by small angle X-ray scattering (SAXS) studies. Encapsulating JBTX into lipid nanoparticles led to an increase of the peptide's antifungal activity. Transgenic tobacco and sugarcane plants expressing the insecticidal peptide JBTX, showed increased resistance to attack of the insect pests Spodoptera frugiperda, Diatraea saccharalis and Telchin licus licus. Many questions remain unanswered; however, so far, JBTX has shown to be a versatile peptide that can be used against various insect and fungus species, and in new bacterial control strategies.(AU)

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthaseacyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.(AU)

Characteristics of dr1790 disruptant and its functional analysis in Deinococcus radiodurans

Deinococcus radiodurans (DR) is an extremophile that is well known for its resistance to radiation, oxidants and desiccation. The gene dr1790 of D. radiodurans was predicted to encode a yellow-related protein. The primary objective of the present study was to characterize the biological function of the DR1790 protein, which is a member of the ancient yellow/major royal jelly (MRJ) protein family, in prokaryotes. Fluorescence labeling demonstrated that the yellow-related protein encoded by dr1790 is a membrane protein. The deletion of the dr1790 gene decreased the cell growth rate and sensitivity to hydrogen peroxide and radiation and increased the membrane permeability of D. radiodurans. Transcript profiling by microarray and RT-PCR analyses of the dr1790 deletion mutant suggested that some genes that are involved in protein secretion and transport were strongly suppressed, while other genes that are involved in protein quality control, such as chaperones and proteases, were induced. In addition, the expression of genes with predicted functions that are involved in antioxidant systems, electron transport, and energy metabolism was significantly altered through the disruption of dr1790. Moreover, the results of proteomic analyses using 2-DE and MS also demonstrated that DR1790 contributed to D. radiodurans survival. Taken together, these results indicate that the DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses.(AU)

Genetic structuring of Salminus hilarii Valenciennes, 1850 (Teleostei: Characiformes) in the rio Paraná basin as revealed by microsatellite and mitochondrial DNA markers

Genetic variation of Salminus hilarii was assessed by screening microsatellite loci and mitochondrial D-loop DNA across four sampling in the upper rio Paraná basin of Brazil. Genetic diversity - measured as mean expected heterozygosity (0.904) and mean number of alleles across populations (13.7) - was reasonably high. Differentiation of microsatellite allele frequencies among populations was shown to be low but significant by AMOVA Φ ST (0.0192), and high by D EST (0.185). D-loop variation was high, with haplotypic diversity of 0.950 and nucleotide diversity of 0.011. Mitochondrial DNA-based estimates for population differentiation were high, with an overall Φ ST of 0.173. The results of tests of nuclear and mitochondrial variation yielded no unequivocal inference of historical demographic bottleneck or expansion. Genetic differentiation observed among S. hilarii populations in the rio Grande may be caused by a combination of historical differentiation and recent gene-flow disruption caused by the dams followed by reproduction of isolated spawning assemblages in mid-sized tributaries of the respective reservoirs. We present spatially more intensive sampling of S. hilariipopulations across the rio Paraná basin in order to more effectively distinguish between historical and contemporary differentiation.(AU)

A variabilidade genética de Salminus hilarii foi avaliada por lócus microssatélites e sequências D-Loop do DNA mitocondrial em quatro populações da região da bacia do Alto Paraná. A diversidade genética - medida pela heterozigosidade média (0,904) e número de alelos médios das populações (13,7) - foi razoavelmente alta. A diferenciação das frequências alélicas entre as populações foi baixa, mas significativa pela AMOVA Φ ST (0,0192), e alta pelo D EST (0,185). A variação mitocondrial foi alta com uma diversidade haplotípica de 0,950 e uma diversidade nucleotídica de 0,011. Estimativas de diferenciação populacional baseadas no DNA mitocondrial foram altas, com um valor global de Φ ST de 0,173. Os resultados dos testes da variação nuclear e mitocondrial demonstram nenhuma inequívoca inferência histórica de contração e expansão demográfica. A diferenciação genética observada entre as populações de S. hilarii no rio Grande pode ter sido causada pela combinação de diferenciação histórica e interrupção recente do fluxo gênico causada pela construção de barragens seguida por um isolamento reprodutivo de populações em tributários de médio porte dos respectivos reservatórios. Nós apresentamos uma amostragem mais ampla e intensiva de populações de S. hilarii ao longo da bacia do alto rio Paraná para se efetivamente distinguir se a diferenciação genética das populações encontrada é histórica ou contemporânea.(AU)

Ovarian aging in growth hormone receptor knockout mice supplemented with 17-Estradiol

Camundongos knockout para o gene do receptor do hormônio de crescimento (GH) (GHRKO) têm atividade de GH ausente. Estes camundongos são o principal modelo para acessar os efeitos do GH e são conhecidos por terem maior longevidade e as fêmeas apresentarem vida reprodutiva prolongada. O 17-estradiol (17-E2) é um hormônio capaz de prolongar a longevidade dos camundongos. Assim, o objetivo deste estudo foi avaliar a reserva ovariana, em ovários de camundongos normais e GHRKO suplementados ou não com 17-estradiol. Nossa hipótese era que o knok out do GHR e o tratamento com 17-estradiol diminuiriam a ativação de folículos primordiais de forma complementar em camundongos.Seis camundongos GHRKO e seis camundongos wild-type (WT) foram divididos em quatro grupos: camundongos normais GHRKO (GHRKO; n = 3), camundongos GHRKO suplementados com 17-E2 (GHRKO-E2; n = 3), wild-type (WT) camundongos com dieta normal (WT; n = 3) e camundongos WT suplementados com 17-E2 (WT-E2; n = 3). E2 foi fornecido na comida durante quatro meses. Lâminas histológicas foram preparadas a partir dos ovários dos camundongos. Imagens das seções ovarianas foram capturadas por uma câmera acoplada a um microscópio, e os folículos foram contados e medidos. A análise estatística considerou o efeito do genótipo (GHRKO vs WT), tratamento (E2 vs CONT) e sua interação. Tanto o genótipo (p = 0,0117) quanto a dieta (p = 0,0136) tiveram efeito no número de folículos primordiais e na interação entre eles (p = 0,004). O número de folículos primordiais foi maior no grupo GHRKO-CONT (2632 ± 181) do que nos outros grupos. O tratamento com 17a-E2 reduziu o n�ero de fol�ulos primordiais nos ratinhos GHRKO para um n�el semelhante ao WT, mas n� teve efeito nos ratinhos WT. Camundongos WT tiveram um número maior de folículos primários do que ratos GHRKO (p = 0,006). A dieta 17-E2 não exerceu efeito sobre o número de folículos primários (p = 0,785). Em síntese, camundongos GHRKO não tratados tiveram um aumento na reserva ovariana do que os camundongos WT. No entanto, os genótipos tratados com 17-E2 responderam diferentemente. Quando tratado, grupo GHRKO diminuiu a reserva de folículos primordiais enquanto os camundongos WT mantiveram o número de folículos primordiais, o que indica que o 17-E2 tem um papel na ativação folicular que não atua da mesma forma quando os níveis de GH/IGF são baixos.

Mice with growth hormone (GH) receptor gene disruption (GHRKO) have absent GH activity. These mice are the main model to access the effects of GH and are known for having extended lifespan and female reproductive longevity. 17-estradiol (17-E2) is a molecule reported to extend mice lifespan. Hence, the aim of this study was to evaluate the ovarian reserve, in ovaries of normal and GHRKO mice supplemented or not with 17-estradiol. Our hypothesis was that GHR disruption and 17-Estradiol treatment would decrease the activation of primordial follicles in a complementary way in mice. Six GHRKO mice and six wild-type (WT) mice were divided into four groups: GHRKO mice normal diet (GHRKO; n=3), GHRKO mice supplemented with 17-E2 (GHRKO-E2; n=3), WT mice normal diet (WT; n=3) and WT mice supplemented with 17-E2 (WT-E2; n=3). E2 was provided in the food for four months. Histological slides were prepared from the mice ovaries. Images of the ovarian sections were captured by a camera coupled to a microscope, and the follicles were counted and measured. The statistical analysis considered the effect of the genotype (GHRKO vs WT), treatment (E2 vs CONT) and its interaction. Both genotype (p=0.0117) and diet (p=0.0136) had effects on the number of primordial follicles count as well as the interaction between them (p=0.004). The number of primordial follicles was higher on the GHRKO-CONT group (2632±181) than in the other groups. 17-E2 treatment reduced the number of primordial follicles in the GHRKO mice to a level similar to WT, but had no effect on WT mice. WT mice had a higher number of primary follicles than GHRKO mice (p=0.006). 17-E2 diet exerted no effect on the number of primary follicles (p=0.785). In summary, non-treated GHRKO mice had an increased primordial follicle reserve than WT mice. However, the genotypes treated with 17-E2 responded differently, GHRKO decreased the primordial follicle reserve and WT mice maintained the primordial follicles number, which indicates that 17-E2 has a differential role on follicle activation when there is a lack of GH /IGF-I.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Cellular and molecular basis of therapies to ameliorate effects of heat stress on embryonic development in cattle

Much of the effect of heat stress on establishment and maintenance of pregnancy involves changes in ovarian function and embryonic development that reduce the competence of the oocyte to be fertilized and the resultant embryo to develop. There are three possible therapeutic approaches to manipulate the connection between hyperthermia and cellular responses to elevated temperature to improve fertility during heat stress. Embryo transfer is based on the idea that 1) most effects of heat stress on fertility involve actions during folliculogenesis or on cleavagestage embryos and 2) the embryo has acquired resistance to elevated temperature by the time it is transferred at t he morula or blastocyst stage. The mechanisms for acquis ition of thermotolerance involve changes in production of reactive oxygen species in response to heat shock as well as accumulation of antioxidants in the embryo. Synthesis of heat shock proteins may not be the controlling factor for acq uisition of thermotolerance because transcript abundance for HSPA1A and HSP90AA1 is higher for the two-cell embryo than morula. Involvement of reactive oxygen species in actions of elevated temperature on embryo survival is indicative that provision of ant ioxidants to heat-stressed cows could improve fertility. More work is needed but there are indications that pregnancy rates can be improved by feeding supplemental β-carotene or administration of melatonin implants. It is also evident that there are genes that control thermoto lerance at the cellular level. Brahman, Nelore and Romosinuano embryos have increased resistance to heat shock as compared to Holstein or Angus embryos. Mutations in the gene for heat shock protein 70 that control resistance of cells t o heat shock have been identified in Holsteins. Selection for the desirable alleles of genes conferring cellular thermotolerance could lead to development of strains of cattle whose fertility is resistant to disruption by heat stress. Pursuing these and other therapeutic approaches for reducing consequences of heat stress for livestock species should be a priority because of the prospects for continuing global climate change.

Cellular and molecular basis of therapies to ameliorate effects of heat stress on embryonic development in cattle

Much of the effect of heat stress on establishment and maintenance of pregnancy involves changes in ovarian function and embryonic development that reduce the competence of the oocyte to be fertilized and the resultant embryo to develop. There are three possible therapeutic approaches to manipulate the connection between hyperthermia and cellular responses to elevated temperature to improve fertility during heat stress. Embryo transfer is based on the idea that 1) most effects of heat stress on fertility involve actions during folliculogenesis or on cleavagestage embryos and 2) the embryo has acquired resistance to elevated temperature by the time it is transferred at t he morula or blastocyst stage. The mechanisms for acquis ition of thermotolerance involve changes in production of reactive oxygen species in response to heat shock as well as accumulation of antioxidants in the embryo. Synthesis of heat shock proteins may not be the controlling factor for acq uisition of thermotolerance because transcript abundance for HSPA1A and HSP90AA1 is higher for the two-cell embryo than morula. Involvement of reactive oxygen species in actions of elevated temperature on embryo survival is indicative that provision of ant ioxidants to heat-stressed cows could improve fertility. More work is needed but there are indications that pregnancy rates can be improved by feeding supplemental β-carotene or administration of melatonin implants. It is also evident that there are genes that control thermoto lerance at the cellular level. Brahman, Nelore and Romosinuano embryos have increased resistance to heat shock as compared to Holstein or Angus embryos. Mutations in the gene for heat shock protein 70 that control resistance of cells t o heat shock have been identified in Holsteins. Selection for the desirable alleles of genes conferring cellular thermotolerance could lead to development of strains of cattle whose fertility is resistant to disruption by heat stress. Pursuing these and other therapeutic approaches for reducing consequences of heat stress for livestock species should be a priority because of the prospects for continuing global climate change.(AU)

P53 gene: major mutations in neoplasias and anticancer gene therapy

The p53 gene encodes a protein that has molecular weight of 53kD and is also called p53 protein, being constantly studied for its classic concept of "genome guardian". This gene plays a range of essential functions to ensure the cell cycle control, in addition to playing a central role in carcinogenesis. With respect to neoplasias, it prevents the neoplastic transformation through three intricate mechanisms. Depending on the extent of the mutation, different responses may be sent by p53 and those range since the disruption of the cell cycle, the correction of the mutation through the activation of repair proteins or still, the induction of senescence or cell death by apoptosis. This review aims to address the structural and functional aspects of the p53 gene and protein, and also reaffirm their participation in the carcinogenesis control, approaching their major mutations and the anticancer gene therapy involving this gene.

O gene p53 codifica uma proteína que tem peso molecular de 53kD e é também chamada de proteína p53, sendo constantemente estudado por seu conceito clássico de "guardião do genoma". Esse gene desempenha uma série de funções essenciais para garantir o controle do ciclo celular, além de desempenhar um papel central na carcinogênese. Com relação a neoplasias, impede a transformação neoplásica através de três mecanismos intrincados. Dependendo da extensão da mutação, diferentes respostas podem ser enviadas por p53 desde a ruptura do ciclo celular, a correção da mutação através da ativação de proteínas de reparo ou, ainda, a indução de senescência ou morte celular por apoptose. Esta revisão visa a abordar os aspectos estruturais e funcionais do gene p53 e proteína, e também reafirmar a sua participação no controle da carcinogênese, abordando suas principais mutações e a contribuição para a terapia gênica anticâncer.

P53 gene: major mutations in neoplasias and anticancer gene therapy

The p53 gene encodes a protein that has molecular weight of 53kD and is also called p53 protein, being constantly studied for its classic concept of "genome guardian". This gene plays a range of essential functions to ensure the cell cycle control, in addition to playing a central role in carcinogenesis. With respect to neoplasias, it prevents the neoplastic transformation through three intricate mechanisms. Depending on the extent of the mutation, different responses may be sent by p53 and those range since the disruption of the cell cycle, the correction of the mutation through the activation of repair proteins or still, the induction of senescence or cell death by apoptosis. This review aims to address the structural and functional aspects of the p53 gene and protein, and also reaffirm their participation in the carcinogenesis control, approaching their major mutations and the anticancer gene therapy involving this gene.

O gene p53 codifica uma proteína que tem peso molecular de 53kD e é também chamada de proteína p53, sendo constantemente estudado por seu conceito clássico de "guardião do genoma". Esse gene desempenha uma série de funções essenciais para garantir o controle do ciclo celular, além de desempenhar um papel central na carcinogênese. Com relação a neoplasias, impede a transformação neoplásica através de três mecanismos intrincados. Dependendo da extensão da mutação, diferentes respostas podem ser enviadas por p53 desde a ruptura do ciclo celular, a correção da mutação através da ativação de proteínas de reparo ou, ainda, a indução de senescência ou morte celular por apoptose. Esta revisão visa a abordar os aspectos estruturais e funcionais do gene p53 e proteína, e também reafirmar a sua participação no controle da carcinogênese, abordando suas principais mutações e a contribuição para a terapia gênica anticâncer.

Correlation between ebv co-infection and HPV16 genome integrity in Tunisian cervical cancer patients

Infection with high risk Human papillomavirus (HR-HPV) is necessary but not sufficient to cause cervical carcinoma. This study explored whether multiple HR-HPV or coinfection with Epstein-Barr virus (EBV) influence the integration status of HPV16 genome. The presence and typing of HPV in a series of 125 cervical specimens were assessed by polymerase chain reaction (PCR) using the specific primers for the HPV L1 region. As for EBV infection, the viral EBNA1 gene was used for its detection through PCR amplification. Disruption of the HPV E2 gene was assessed by amplification of the entire E2 gene with single set of primers, while E2 transcripts were evaluated by a reverse transcription PCR method (RT-PCR). The overall prevalence of HPVDNA was of 81.8% in cervical cancers versus 26.9% in benign lesions. In HPV positive cases, HPV16 and HPV18 were the most prevalent types, followed by HPV types 33, 31. EBV EBNA1 prevalence was statistically more frequent in cervical carcinomas than in benign lesions (29.5%, vs 9.6%; P=0.01). No viral infection was detected in healthy control women. The uninterrupted E2 gene was correlated with the presence of E2 transcripts originating from the HPV episomal forms. It was observed that integration was more common in HPV18 and EBV coinfection. The presence of EBV caused a five-fold [OR= 5; CI= 1.15-21.8; P = 0.04] increase in the risk of HPV16 genome integration in the host genome. This study indicates that EBV infection is acting as a cofactor for induction of cervical cancer by favoring HPVDNA integration.

CLONAGEM DO GENE DA PROTEÍNA DE CHOQUE TÉRMICO DE 70 kDa (HSP70) DE Trypanosoma evansi

BOAVENTURA, Bruna Larissa. CLONAGEM DO GENE DA PROTEÍNA DE CHOQUE TÉRMICO DE 70 kDa (HSP70) de Trypanosoma evansi.66f. Tripanossomoses são doenças causadas por protozoários pertencentes ao gênero Trypanosoma que podem infectar uma ampla gama de animais, inclusiveo homem. OTrypanosoma evansi é um hemoparasito que acomete várias espécies de animais, principalmente equinos, sendo responsável pela enfermidade conhecida como tripanossomose equina, tendo relato também de sua ocorrência em humanos,sendo, por tanto uma zoonose. Possui ampla distribuição geográfica, e é importante causa de prejuízos econômicos em criações de equinos. O controle do agente é feito por meio da utilização de tripanocidas, porém, ao longo dos anos têm sido descritas cepas resistentes às drogas atualmente disponíveis. As proteínas de choque térmico da família HSP 70 são componentes centrais da rede celular, auxiliando em diversas funções como dobramento de proteínas e se destacando como importantes alvos para terapias gênicas e biomarcadores por serem proteínas altamente conservadas em diversos parasitos. A realização deste trabalho teve como objetivo clonar o gene codificante para a proteína HSP70 de T. evansi (TEHSP70) e realizar estudos in silico com a proteína TEHSP70. A clonagem do gene TEHSP70 em pGEM- T easy®, foi bem sucedida e a partir do seu sequenciamento obteve-se a sequencia de nucleotídeos que, traduzida, forneceu a sequencia primária da proteína alvo TEHSP70. A sequência de TEHSP70 foi comparada com de ortólogos de genes de Trypanosomas disponíveis em banco de dados públicos e os resultados mostraram que TEHSP70 possui alta similaridade filogenética com as espécies de Trypanosomas de maior ocorrência. A análise de bioinformática realizada na HSP70 e sua co-chaperona HSP40 demonstrou que a região C-terminal da HSP70 pode atuar como um potente alvo para interrupção do ciclo biológico do parasito.O estudo de proteínas funcionais, envolvidas no metabolismo do parasito, pode servir de base para o desenvolvimento de novas drogas.

BOAVENTURA, Bruna Larissa. CLONING OF HEAT SHOCK PROTEIN HSP70 GENE IN Trypanosoma evansi. 2016. 66p. Trypanosomes are diseases caused by protozoa belonging to the genus Trypanosoma that can infect a wide range of animals, including man.Trypanosoma evansi is a hemoparasite that affects several species of animals, mainly equines, being responsible for the disease known as equine trypanosomiasis, and also reports of its occurrence in humans, being, therefore, a zoonosis.It has a wide geographical distribution, and is an important cause of economic losses in equine breeding. Control of the agent is done through the use oftrypanocides, however, over the years, resistant strains of drugs currently available have been described.The heat shock proteins of the HSP 70 family are central components of the cellular network, assisting in several functions such as protein folding and stand out as important targets for gene therapies and biomarkers because they are proteins highly conserved in several parasites.The objective of this work was to clone the gene coding for the HSP70 protein of T. evansi (TEHSP70) and to perform in silico studies with the TEHSP70 protein.Cloning of the TEHSP70 gene in pGEM-T easy® was successful and from its sequencing the nucleotide sequence was obtained which translated gave the primary sequence of the target protein TEHSP70. The sequence of TEHSP70 was compared with that of orthologs of Trypanosome genes available in public database and the results showed that TEHSP70 has high phylogenetic similarity with the most frequent species of Trypanosomes.The bioinformatics analysis performed on HSP70 and its HSP40 co-chaperone demonstrated that the C-terminal region of HSP70 may act as a potent target for disruption of the parasite's life cycle.The study of functional proteins, involved in the metabolism of the parasite, can serve as the basis for the development of new drugs.

RITMOS DIARIOS DAS ATIVIDADES DE ENZIMAS DIGESTIVAS, DOS PARAMETROS IMUNOLOGICOS E DE ATIVIDADE LOCOMOTORA DA TILAPIA DO NILO

O ciclo claro-escuro e a alimentacao sao fatores sincronizadores dos ritmos biologicos em peixes, fato que demandou a pesquisa ora desenvolvida, cujo objetivo foi avaliar os ritmos das funcoes fisiologicas, metabolicas e locomotora da tilapia do Nilo. Experimento 1: avaliar a sincronizacao dos ritmos diarios da atividade locomotora, da amilase, da protease alcalina e dos niveis da glicose ao horario de alimentacao. Juvenis (n=120) (65,04±0,58g) foram divididos em 2 grupos. Um grupo foi alimentado uma vez ao dia, as 11h (ZT6) (grupo ML) e o outro, alimentado as 23h (ZT18) (grupo MD). A atividade locomotora dos animais foi mensurada por meio de fotocelula fixada nos tanques. A atividade locomotora, bem como analise do Cosinor para os parametros de glicose, protease alcalina e amilase foram analisados. Todos os resultados foram expressos como media ± S.E.M. e avaliados por ANOVA one-way, teste de Duncan. Os peixes alimentados em ML demonstraram uma atividade locomotora diurna (74,01% da atividade total) e sincronizacao ao horario de alimentacao. Esse grupo demonstrou antecipacao da atividade locomotora ao horario da alimentacao. Nos peixes em MD houve um rompimento no padrao diario de atividade locomotora (58,94% periodo de luz e 41,06%, escuro). A atividade de protease alcalina no intestino medio dos dois grupos (ML e MD) demostrou padrao ritmico diario (COSINOR, P<0,05). A amilase nao demostrou padrao ritmico de atividades (COSINOR, P>0,05), nem diferenca significativa (P>0,05). Os resultados demonstraram uma variacao ritmica diaria para a glicose plasmatica, Cosinor, P<0,05 e diferenca significativa (ANOVA, P<0,05). O horario da alimentacao exerceu influencia sobre os ritmos diarios dos parametros comportamentais, fisiologicos e metabolicos da tilapia do Nilo, que demonstrou plasticidade do sistema circadiano com capacidade de controle do padrao de atividade locomotora. Experimento 2: avaliou as variacoes diarias nas respostas imunologicas e hematologicas de tilapia. Juvenis (n=144) (38.06±0.42g) foram distribuidos em 2 grupos e mantidos em temperatura de 28°C e fotoperiodo 12:12LD. Um grupo foi vacinado contra S. agalactiae (T1) e outro grupo recebeu solucao salina como controle (T2). Apos trinta dias da imunizacao todos os peixes foram desafiados com a bacteria S. agalactiae). A atividade de peroxidase demonstrou um ritmo diario (COSINOR, p<0,05), no s peixes do tratamento controle. Os niveis de IgM no soro dos peixes vacinados, demonstrou um padrao ritmico diurno, Cosinor (p<0,05) e diferenca significativa entre os pontos de coleta (p<0,05). O grupo controle (T2) apresentou expressoes ritmicas genicas de TGF1, TNF3 e IL12 (COSINOR, p<0,05), todos com acrofase diurna. No grupo controle, o VG e trombocitos demostraram variacao ritmica (COSINOR, p<0,05), como tambem o VG, o VCM e o CHCM demonstraram diferenca estatistica (p<0,05). Nos peixes vacinados houve diferenca significativa para o VG e CHCM (p<0,05). Ha evidencias de variacao diaria das funcoes imunologicas e hematologica da tilapias do Nilo, sincronizadas com o ciclo claro-escuro do dia. Isso reflete a importancia da dimensao temporal na gestao do cultivo dessa especie.

The light-dark cycle and food are factors synchronizers of biological rhythms in fish, a fact which required research now developed, whose objective was to evaluate the rhythms of physiological functions, and locomotor metabolics of Nile tilapia. Experiment 1: assess the synchronization of daily rhythms of locomotor activity, amylase, alkaline protease and glucose levels at feeding time. Juveniles (n = 120) (65.04 ± 0.58g) were divided into 2 groups. One group was fed once a day, at 11am (ZT6) (ML group) and the other fed to 23h (ZT18) (MD group). The locomotor activity of the animals was measured by photocell fixed in the tanks. The locomotor activity, as well as Cosinor analysis for glucose parameters, alkaline protease and amylase were analyzed. All results were expressed as mean ± S.E.M. and evaluated by one-way ANOVA, Duncan test. Fish fed on ML showed a diurnal locomotor activity (74.01% of the total activity) and synchronizing the feeding schedule. This group showed anticipation of locomotor activity at the time of feeding. In fish in MD there was a disruption in the daily pattern of locomotor activity (58.94% light period and 41.06%, dark). The alkaline protease activity in the midgut of the two groups (ML and MD) demonstrated daily rhythmic pattern (COSINOR, p<0.05). The amylase did not show rhythmic pattern of activities (COSINOR, p>0.05), no significant difference (p>0.05). The results show a daily rhythm variation for plasma glucose, Cosinor, p<0.05 and significant differences (ANOVA, p<0.05). The power of time exerted influence on the daily rhythms of behavioral, physiological and metabolic parameters of Nile tilapia, which demonstrated the plasticity of the circadian system with standard control capability of locomotor activity. Experiment 2: evaluated the daily changes in immunological and hematological responses of tilapia. Juveniles (n = 144) (38.06 ± 0.42g) were divided into 2 groups and kept at 28°C, photoperiod 12:12LD. One group was vaccinated against S. agalactiae (T1) and another group received saline as control (T2). Thirty days after immunization all fishes were challenged with the bacterium S. agalactiae). Peroxidase activity showed a daily rhythm (COSINOR, p<0.05) in the control treatment of fish. IgM levels in fish serum vaccinated showed a diurnal rhythm pattern, Cosinor (p<0.05) and significant difference between the collection points (p<0.05). The control group (T2) showed rhythmic gene expressions of TGF1, TNF3 and IL12 (COSINOR, p<0.05), all with daytime acrophase. In the control group, GV and thrombocytes demonstrated rhythmic variation (COSINOR, p<0.05), as well as GV, MCV and MCHC showed statistical difference (p<0.05). In fish vaccinated significant difference to the GV and MCHC (p<0.05). There is evidence of daily variation of immunologic and hematologic functions of the Nile tilapia, synchronized with the light-dark cycle of the day. This reflects the importance of the temporal dimension in this kind cultivation management.