Alteraciones en el material genético de pintores de carros expuestos a solventes orgánicos en una localidad de Bogotá

Los solventes orgánicos son sustancias químicas que por sus propiedades físico-químicas son fácilmente inhalados o absorbidos por la piel, pueden causar daños de diversa índole en la salud. En Colombia existen normas que contemplan las medidas de protección, sin embargo persiste la informalidad en el sector de pintores de autos, por lo cual los trabajadores expuestos, a largo plazo pueden ver afectada su salud. En este estudio se analizó la relación entre individuos expuestos laboralmente a los solventes orgánicos versus no expuestos con respecto a la longitud de sus telómeros y formación de fragilidades. Se emplearon muestras de sangre extraídas por venopunción, recolectada en dos tubos: uno con Heparina, destinado al cultivo de linfocitos, para obtener cromosomas metafásicos y evaluar en ellos la presencia de fragilidades; el otro tubo con EDTA, fue empleado para la extracción de ADN y se utilizó para obtener los valores de longitud telomérica mediante la técnica de PCR cuantitativa. Los análisis estadísticos se realizaron aplicando la prueba de rangos de Wilcoxon, en el caso de la presencia de fragilidades se analizó la razón No.Fragilidades/No.Metafases, aplicando el método de Wilcoxon se encontró que existe diferencia estadísticamente significativa entre expuestos y no expuestos (p = 0,036), en donde los expuestos presentan mayor frecuencia de fragilidades. Por otra parte el valor relativo de longitud telomérica del grupo de expuestos fue mayor que el observado en el grupo de no expuestos, esta diferencia fue estadísticamente significativa (Wilcoxon, p = 0.002).

Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or beta-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10-7 g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.

Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 102 to 1.4 × 103 per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of “Candidatus Cardinium hertigii” and as a separate novel cluster.

High-resolution melt and morphological analyses of mealybugs (Hemiptera: Pseudococcidae) from cacao: tools for the control of Cacao Swollen Shoot Virus spread

BACKGROUND: Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are key vectors of badnaviruses, including Cacao Swollen Shoot Virus (CSSV) the most damaging virus affecting cacao (Theobroma cacao L.). The effectiveness of mealybugs as virus vectors is species dependent and it is therefore vital that CSSV resistance breeding programmes in cacao incorporate accurate mealybug identification. In this work the efficacy of a CO1-based DNA barcoding approach to species identification was evaluated by screening a range of mealybugs collected from cacao in seven countries. RESULTS: Morphologically similar adult females were characterised by scanning electron microscopy and then, following DNA extraction, were screened with CO1 barcoding markers. A high degree of CO1 sequence homology was observed for all 11 individual haplotypes including those accessions from distinct geographical regions. This has allowed for the design of a High Resolution Melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. CONCLUSIONS: HRM Analysis (HRMA) readily differentiated between mealybug pests of cacao that can not necessarily be identified by conventional morphological analysis. This new approach, therefore, has potential to facilitate breeding for resistance to CSSV and other mealybug transmitted diseases.

Prevalence of Human Papillomaviruses in Urine Samples of Male Patients Infected With HIV-1 in Sao Paulo, Brazil

Human papillomavirus is a DNA virus that includes 118 genotypes. HPV16 is responsible for 80% of cervical cancer in women. Men are important reservoirs and major transmitters of HPV to their partners. The aim of this study was to detect HPV DNA and to determine the prevalence of HPV types 6, 11, 16, and 18 in urine samples of men infected with HIV-1. This study included 223 patients infected with HIV-1 from the Center of Reference on HIV/AIDS (CRT-SP) and an outpatient clinic of HIV. Urine samples were collected and after DNA extraction real-time PCR was performed for detection of HPV DNA. Positive samples were then tested by conventional PCR using type-specific primers for the four HPV types. A total of 223 men infected with HIV-1 were tested, 81% of whom were on HAART. Four (5.8%) were positive for HPV6, 18 (26.1%) were positive for HPV11, 22 (31.9%) were positive for HPV16 and five (7.2%) were positive for HPV18 by conventional PCR. Twenty (29%) patients had other HPV types and five patients (1.5%) had multiple types. The mean T CD4+cells count was 517 and 441 cells/mm(3) (P=0.30), in HPV negative and positive men, respectively. The HIV viral load was higher in the HPV negative group than for in the men with HPV (P=0.0002). A 30.9% prevalence of HPV was found in asymptomatic urine samples of men infected with HIV-1. This study suggests that urine may be a useful specimen for HPV screening. J. Med. Virol 81:2007-2011, 2009. (C) 2009 Wiley-Liss, Inc.

Prevalência da infecção por HPV e perfil das mulheres frente ao exame de papanicolau no município de São José de Mipibú/RN.

The Human Papillomavirus (HPV) infection is the major sexually transmitted disease all over the world. There are many factors associated to infection and the virus persistency in the organism. This study aims to evaluate the women's knowledge, attitudes and practice about the Papanicolaou test (Pap), as well as analyze the HPV and Chlamydia trachomatis infections prevalences in sexually active women from the city of São José do Mipibu/RN/Brazil. This research was divided in two steps (step I and step II), using different methodologies and samples each. The samples collected in each step, even socio-demographic or from uterus cervix, are from different patients e were analyzed separated. In step I was evaluated 267 rural and urban zone women s knowledge, attitudes and practices about the Pap by home interview. In the step II were included 605 women with age ranged from 15 to 71 years old, with mean of 33,5 years old and from each one were collected two cervical samples, one for Pap and other for molecular biology, beside the epidemiological interview to investigate the correlation between prevalence of HPV infection and risk factors. To molecular analyses, the samples were processed using a mammal rapid DNA extraction technique protocol. For C. trachomatis DNA detection were used the CP24/27 primers, and GP5+/GP6+ to HPV. PCR products were analyzed by electrophoresis on 8% polyacrylamide gels, followed by silver staining. The results of the step I showed that, in spite of only 46,1% of the interviewed women they have demonstrated to possess appropriate knowledge on the Pap test, the attitude and practice proportions were significantly larger, 63,3% and 64,4% respectively. The largest education degree presented association with adaptation of the knowledge, attitudes and practice, while neglect, lack of solicitation of the exam for the doctor and shame, came as main barriers for the accomplishment of the exam. In the stage II the HPV general prevalence was 28,9%, being 26,7% in the women with normal cytology or benign alterations, 26,7% in the ones that had atypical squamous cells of undetermined significance (ASC-US) and 80% in those with Low grade squamous intraepithelial lesion (LSIL). the HPV infection prevalence was larger in the patients with up to 30 years of age and in the unmarried women, and those that had more than one sexual partner presented larger infection risk. The results show that the sexual relationship with multiple partners increased the infection risk for HPV and consequently the possibility of the occurrence of lesions uterine cervix

Prevalência da infecção por papilomavírus humano, herpes simplex tipo 1 e 2 e Chlamydia trachomatis em um segmento da população feminina da grande Natal/RN

Sexually transmitted diseases (STDs) are among the largest public health problems, especially in developing countries. The acquisition of these infections during early sexual activity is common and many infections have a benign course. However, in some pathogens remain in the state of latency can be reactivated and cause productive infection that may progress to severe forms. In addition, some of them are transmitted vertically resulting in congenital infection, causing immediate damage or long-term child. The classic risk factors for sexually transmitted agents are: early onset of sexual and reproductive health, multiple sexual partners throughout life, use of oral contraceptives and co-infections with different pathogens. We present the results of a cross-sectional study aimed to estimate the prevalence of genital infection by human papillomavirus (HPV), Herpes simplex virus (HSV) and Chlamydia trachomatis (CT) in a segment of the female population of the metropolitan area Christmas, among those who enrolled voluntarily sought, Basic Health Units for the examination of cancer screening cervix in the period 2008 to 2010. All participants, a total of 261 women answered a standard questionnaire by which identified the socio-demographic characteristics, classical risk factors for STDs, reproductive and sexual activity and smoking. Of each patient were obtained two samples, one for the completion of the Pap test for detection of cellular changes and the other processed for DNA extraction and analyzed by PCR (polymerase chain reaction) to detect the three pathogens studied. The population of the study was composed of sexually active women aged between 13 and 79 years, mean 38.7 years, most of them being married, low education levels and low incomes. The majority (87%) had normal results on cytology and only 2.7% had low-grade cytological abnormalities. Prevalence rates were 37.9% for HPV, 4.6% for CT and 26% for HSV. HPV prevalence was higher in women under 25, unmarried and in those who had multiple sexual partners. Women with simultaneous infection by HSV-1 and 2 had higher prevalence of HPV infection. The prevalence of HSV infection showed no association whatsoever with the risk factors analyzed and HSV-1 was the predominant type among the cases of genital HSV infection. The overall prevalence of C. Trachomatis was relatively low, thus providing greater value in younger women aged less than or equal to 20 years

Prevalência da infecção pelo Papilomavírus humano, Chlamydia Trachomatis e Herpes Simples do tipo 2 em adolescentes atendidas em unidades de saúde pública de Natal

This study assessed the level of knowledge, attitude and practice of Pap smear and human papillomavirus (HPV), in addition to analyzing the prevalence of genital HPV infection, Herpes Simplex Type 2 (HSV-2) and Chlamydia trachomatis in teenagers. The study consisted of two approaches, one based only on interviews conducted with adolescents enrolled in public schools or in public health facilities in the city of Natal. The other approach involved only a group of 132 adolescents enrolled among those admitted to two health units in Natal-RN. This second group of participants two specimens were collected for laboratory analysis: one was directed to prepare the blade for the Pap test, and other processed for DNA extraction for molecular analysis, focusing on the detection of HPV, HSV-2 and C . trachomatis. The presence of DNA of the three pathogens was investigated by the technique of polymerase chain reaction (PCR). The presence of each of the three pathogens was analyzed in terms of socio-demographic characteristics, as well as sexual and reproductive activity to identify risk factors for infection and development of lesions of the uterine cervix. The results show that the adolescents in this study had levels of knowledge and attitude very low, both in relation to cytology to HPV as though they have made a reasonable percentage of adequate practice exam and prevention of HPV infection. The overall prevalence of HPV infection was 54.5% and 48.2% in adolescents with normal cytology and 86.4% in those with abnormal cytology. We observed a higher proportion of cases of infection in the age group of 18 to 21. The prevalence of HPV infection was slightly higher among pregnant teenagers. The overall prevalence of HSV-2 infection was 13.6% and 11.8% in women with normal cytology and 22.7% in those with abnormal cytology. A higher proportion of cases of infection was found in the age group from 14 to 17, with a slightly higher prevalence among pregnant women. The C. trachomatis was found with an overall prevalence of 19.7% and 21.8% in adolescents with normal cytology and 9.1% in those with abnormal cytology. The prevailing rate was highest in the age group 18 to 21 years and in nonpregnant

Prevalência da infecção genital pelo vírus herpes simples tipo 1 e 2 em mulheres grávidas e não grávidas de Natal/RN

Herpes simplex is a virus that can be transmitted sexually and is potentially associated with vertical transmission. This study evaluated the prevalence of genital infection by herpes simplex virus (HSV) types 1 and 2 in pregnant and nonpregnant care in the city of Natal / RN, including a total of 222 women, 92 pregnant and 130 nonpregnant. The participants answered a questionnaire to obtain data and socio-demographic characteristics, as well as potential risk factors for sexually transmitted diseases. After the interview, we collected two cervical specimens, one for the Pap test and the other for DNA extraction and analyzed by polymerase chain reaction (PCR) to detect both virus serotypes. Then the women underwent a clinical examination by colposcopy. For statistical analysis, we used the chi-square and logistic regression by SSPS 17.0 Statistic. Most women were up to 30 years of age, nonwhite ethnicity, married, elementary education, family income below the poverty level; initiated sexual activity with age up to 18 years; had more than one sexual partner lifelong and was not pregnant, but has had at least one child. The HSV-1 showed a prevalence of 26.1% among pregnant women and 30.0% in non-pregnant women. While HSV-2 prevalence was found with 10.9% and 19.2% in pregnant and nonpregnant women, respectively. The largest proportion of morphological changes of the uterine cervix was detected among nonpregnant women, both in cytology and in colposcopy. The women were nonwhite ethnicity, married, became pregnant aged less than or equal to 18 years and who had one to two pregnancies had a lower risk of acquiring genital HSV infection. There was a high prevalence of genital HSV infection, HSV-1 is more prevalent than HSV-2. No association was found between morphological changes of the uterine cervix and the presence of the virus in pregnant and nonpregnant women, nor between genital HSV infection and the classic risk factors for sexually transmitted diseases

RAPD marker use for improving resistance to Helicoverpa zea in corn

Helicoverpa zea is responsible for great losses to the corn, Zen mays L., crops final productivity, and the best way to control it is by improving genetic resistance. In collaboration with corn improvement and increasing resistance to insects through molecular marker assisted selection, this work had as an objective the selection of resistant (RP) and susceptible progenies (SP) to H. zea based on the RAPD technique. Molecular markers were Found, among the resistant progenies and it is suggested that linkage of these within the Zapalote Chico corn race, be used to extract resistance genes from this race as a donor. The progenies were selected from a population of half-sibs exhibiting a broader genetic base (FCAVJ-VF14). After DNA extraction, two sample bulks were formed; one made up of the six most resistant plants, the other of the six least resistant plants. Eighty-six primers were tested for PCR reactions with the resistant and susceptible bulks and analyzed on agarose electrophoresis for the detection of RAPD band polymorphism. The results of the banding patterns and similarity values indicated a nucleotide sequence amplified by the primer OPC-2 as a possible molecular marker for the identification of resistant progenies and a homology region between them and the Zapalote Chico corn race.

Estudo de polimorfismos em genes relacionados ao metabolismo do ácido fólico e sua associação com o desenvolvimento de fendas orais não-sindrômicas

The congenital facial clefts are characterized by incomplete formation of the structures that separate the oral and nasal cavity. It is known that several environmental and genetic factors are involved in its development, among these, polymorphisms associated with folic acid metabolism have been investigated. In this sense, the objective was to observe the frequency of polymorphisms C677T and A1298C methylenetetrahydrofolate reductase gene (MTHFR), methionine synthase A2756G of (MTR), A66G of methionine synthase reductase (MTRR) A80G and the reduced folate carrier (RFC1) in patients with non-syndromic oral clefts, trying to match them with their development. Methods: We studied 140 patients with non-syndromic oral clefts and their mothers and 175 control subjects with their mothers, who underwent a questionnaire to obtain family information. Were collecting blood for DNA extraction from patients and their mothers to identify the genotypes of both by PCRRFLP, in addition to carrying out the determination of glucose, AST, ALT and serum creatinine, folic acid and vitamin B12 Serum and plasma homocysteine, and the hemogram. Results: Most patients have cleft lip and palate (55.8%), followed by isolated cleft palate (24.2%) and cleft lip (20%). Regarding gender, 62% of patients were male and 48% female and, after subdivision of the type of screwdriver according to sex was found a prevalence of males in the cracks of the type lip and palate (69 %) and lip (69.2%) and in the case of cleft palate was a female predominance (59%). The average concentration of serum folate in the group of mothers of cleft patients was significantly lower (13.8 ± 2.4 ng / mL) compared with the group of mothers of control subjects (18.8 ± 3.4 ng / mL) This was also observed for the group of cleft children as compared to controls, the dosage of folic acid had a significant difference with values of 15.6 ± 0.6 (ng / mL) and 17.9 ± 0.6 (ng / mL), respectively. For the biochemical measurements of glucose, AST, ALT and creatinine were not statistically different, nor was observed for haematological parameters performed. In assessing the frequency of polymorphisms C677T and A1298C MTHFR, A2756G MTR, MTRR A66G and A80G of the RFC1 there was no statistically significant difference in genotype distribution between cases and controls both for mothers and in the cleft. Conclusion: Although not observed association of polymorphisms with the development of cracks, the decrease in serum folate in the group of cleft patients and their mothers may reflect a disturbance in the metabolism of this metabolite, necessitating further studies such as studies methylation and expression to further elucidate the involvement of folate in the development of oral clefts

Characterization of the genetic variability among Caesalpinia pulcherrima (L.) Sw. (Fabaceae) plants using RAPD molecular markers

O flamboyanzinho (Caesalpinia pulcherrima, Fabaceae) é muito utilizado como cerca-viva e na arborização de ruas, parques e jardins. de florescimento exuberante, suas flores podem apresentar coloração rosa, laranja ou amarela, conforme a variedade. Como características florais são essenciais para a definição do valor comercial de espécies ornamentais, o objetivo principal do trabalho foi verificar a existência de polimorfismo entre plantas de C. pulcherrima, que produzem flores de diferentes colorações, por marcadores moleculares RAPD. Foram escolhidas aleatoriamente 30 plantas adultas, cultivadas no município de Jaboticabal, Estado de São Paulo, para a retirada de amostras foliares para a extração de DNA. Dentre essas matrizes, 20 possuem flores alaranjadas, oito, flores amarelas, e apenas duas produzem flores de coloração rosa. Dos 140 primers de RAPD avaliados, 94 foram capazes de ampliar fragmentos definidos, gerando 246 bandas, das quais se observou 100% de bandas monomórficas, indicando que nenhum dos primers utilizados detectou polimorfismo entre os tratamentos. Concluiu-se que a técnica para detecção de polimorfismo por marcadores RAPD não foi eficiente, e que existe a necessidade de se testarem marcadores moleculares mais específicos. Além disso, a característica morfológica cor de flor é, possivelmente, controlada por vários genes ou pela combinação deles.

Análise da variabilidade genética de acessos dePsidium spp. (Myrtaceae) avaliados quanto à reação a Meloidogyne enterolobii

This study aimed to characterize molecular of 13 accessions of Psidium spp. (Myrtaceae) that was been identified for the reaction to rootknot guava nematode. The DNA extraction of the samples was carried according to the protocol of Shillito & Saul (1988). The molecular markers type fAFLP, were obtained from fAFLP Regular Plant Genomes Fingerprinting Kit' (Applied Biosystems from Brasil Ltda.) and were tested 24 selectives combinations of primers, of which 18 showed amplification that produced 272 polymorphic markers. To the analysis of the markers were employed the softwares GeneScan (ABI Prism versao 1.0) and Genotyper (ABI Prism version 1.03), and the data collected were transformed into a binary matrix that was analyzed in the software PAUP (Phylogenetic Analysis Using Parcimony - version 3.01). Were calculated genetic distance index intra and interespecific between the genotipes. It was found that the AFLP markers were efficient in the discrimination between accessions, as well as in showing genetic similarity among accessions identified as resistant to the nematode Meloidogyne enterolobii, which could be discussed in the future.

Diversidade genética entre progênies e matrizes de rambutan

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

Aims: Detection of Xylella fastidiosa in citrus plants and insect vectors.Methods and Results: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay.Conclusions: the use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively.Significance and Impact of the study: the employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.