The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

Uncommon non-oncogenic HPV genotypes, TP53 and MDM2 genes polymorphisms in HIV-infected women in Southern Brazil

Background: It is believed that Human Papillomavirus (HPV) and Human Immunodeficiency Virus coinfection contributes to increase the risk for cervical intraepithelial injuries. Several factors may contribute to cervical cancer (CC) development, including genetic variants such as TP53 and MDM2 gene polymorphisms. Materials and methods: A hundred HIV-infected women were examined for HPV detection and its genotypes, as well as the frequencies of the SNPs Arg72Pro and SNP309 and their associations with CC risk factors. Nested Polymerase Chain Reaction (nPCR) was used for HPV detection and PCR-RFLP for TP53 and MDM2 SNP309 genotyping. Results: HPV DNA was detected in 68% of samples. A higher frequency of low-risk HPV genotypes (66.7%) was observed when compared to high-risk genotypes (33.3%). Nine different HPV genotypes were identified, with the highest prevalence of HPV-6, followed by HPV-16 and 31. p53 Arg72Arg and SNP309 TG genotype were the most prevalent. HPV genotyping was performed by sequencing. Conclusion: The data obtained suggest that HIV-infected women are more susceptible to be infected by low-risk HPV (LR-HPV) genotypes than by high-risk (HR-HPV), and Pro72Pro of TP53 gene and TG of MDM2 SNP309 genotypes apparently seem to be protective factors among HIV-infected women for HPV acquisition and HR-HPV infection, respectively, in a sample of Southern Brazilian woman. Future investigations in larger populations are necessary to better understand the potential roles of these SNPs and the behavior of non-oncogenic HPV genotypes in HIV-mediated immunosuppression cases. .

High incidence of oncogenic HPV genotypes found in women from Southern Brazil

Oncogenic HPV genotypes are strongly associated with premalignant and malignant cervical lesion. The purpose was to determine human papillomavirus (HPV) prevalence and genotypes, and to estimate cervical cancer risk factor associations. Cervical samples were obtained from 251 women seeking gynecological care at the Pelotas School of Medicine Clinic. This is a cross-sectional study. HPV-DNA was amplified by nested-PCR using MY09/11 and GP5/6 primers, and the sequencing was used for genotyping. Sociodemographic and behavioral risk factors were obtained by closed questionnaire, and its relationship to HPV infection prevalence were analyzed. Statistical analyses were performed using SPSS 16.0 software, and differences were considered significant at p < 0.05. As results, the prevalence of HPV infection was 29.9%. The most frequent genotype was HPV-16 (41.3%), followed by HPV-18 (17.3%), and HPV-33 (9.3%). Others nine HPV genotypes were also found. On this population, prevalence of oncogenic HPV genotypes was high, but does not seem to confer relationship with the risk factors investigated. Future investigations in larger populations are necessary, for the proposition of more appropriated monitoring strategies and treatment according to the Brazilian health service reality, as well as patients.

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.

Comparison between two methods for molecular characterization of human papillomavirus

A sensitive method of detection for Human Papillomavirus (HPV) is important to facilitate the early treatment of cervical cancer precursors.Objective: to analyze the spectrum of HPV infection and compare the sensibility of DNA HPV detection using polymerase chain reaction (PCR) and nestedPCR (nPCR) methods in a group of 251 women of Pelotas-RS. Methods: genomic DNA was extracted from the collected samples and was submitted toPCR methods with the primers MY09/11 and nPCR with the pair of primers MY09/MY11 and GP5+/6+. The results were applied to the softwares Epi-Info v.3.5.1* and STATA v.11 * for analyzes. Results: the prevalence of HPV infection was 6.8% with the use of primers MY09/11. When associated withprimers GP5/6, this result increased to 29.9% (p < 0.001). Conclusion: the increase founded in HPV DNA detection from 6.8 to 29.9% suggests that thetechnique of nPCR MY09/11 followed by GP5/6 is the most sensitive method to detect HPV DNA from cervical specimens.

Neuropeptide Y gene expression around meal time in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (−2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (P<0.05) and significantly reduced 2 h later (P<0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (P<0.05) at −2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.

TP53 gene polymorphism: Importance to cancer, ethnicity and birth weight in a Brazilian cohort.

Arg72Pro SNP of p53 has been associated with many types of cancer as well as with survival and longevity. We evaluated the Arg72Pro SNP frequencies of a Brazilian birth cohort and their association with current, demographic and birth epidemiological parameters available. In 1982, all hospital births of Pelotas, southern Brazil, were identified and studied prospectively. In 2004–5, blood samples were collected and DNA extracted. PCR-RFLP was used to genotype the Arg72Pro SNP in 3794 individual samples of the Brazil birth cohort and DNA sequencing was performed to confirm the genotypes. The genotype distribution, which was in Hardy–Weinberg equilibrium, showed a predominance of the arginine amino acid with a frequency of 46.9% Arg/Arg, 42.2% Arg/Pro and 10.9% Pro/Pro. The allele frequency was 0.68 of Arginine and 0.32 of Proline. The Arg72Pro SNP genotype and allelic frequency were related to skin colour where proline amino acid was observed more among black subjects, while arginine amino acid was observed more among white subjects. The individuals without family history of cancer and those with low birth weight were associated with arginine amino acid. The Arg72Pro SNP was strongly associated with important epidemiological variables confirming that genetic profiles on cohort studies can improve our understanding of the susceptibility of diseases and its risk factors.

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.

Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide.

Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA–DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.

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.

Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.

Identifi cation, tissue distribution and evaluation of brain neuropeptide Y gene expression in the Brazilian fl ounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fi sh. However, the present knowledge about feeding behaviour in fi sh is still limited and based on studies in a few species. The Brazilian fl ounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of fl ounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian fl ounder brain. NPY expression was detected in all the peripheral tissues analysed. No signifi cant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 ± 3°C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian fl ounder are affected by temperature.