Avaliação do envolvimento das bombas de efluxo na fármaco-resistência ao Praziquantel em Schistosoma Mansonii

O Praziquantel (PZQ) é o fármaco de primeira linha no tratamento da schistosomose, com alta taxa de cura e sem efeitos secundários significativos. Têm sido reportados cada vez mais casos de resistência ou de aumento de tolerância a este fármaco, aumentando as preocupações de emergência de estirpes resistentes ao PZQ. O Verapamil, um bloqueador de canais de cálcio, inibe o fluxo de fármaco activo e tem sido descrito como um bom inibidor das glicoproteínas-P (P-gp), sendo, por isso, utilizado em diversos estudos de fármaco-resistência. No laboratório de Helmintologia da Unidade de Ensino e Investigação em Parasitologia Médica do Instituto de Higiene e Medicina Tropical, foi seleccionada uma linha de S. mansoni, resistente a 800 mg/Kg de PZQ, após pressão de fármaco constante e crescente ao longo de vários ciclos. Para confirmar a existência de diferenças polimórficas entre a estirpes sensível e resistente de S. mansoni, extraiu-se o DNA de parasitas adultos de ambas as estirpes de S. mansoni e analisou-se por Random Amplified Polymorphic DNAPolymerase Chain Reaction (RAPD-PCR). As diferenças polimórficas entre a estirpe sensível e a resistente foram observadas e calculou-se o coeficiente de similaridade (Dice’s coefficient). Após confirmar a existência de polimorfismos entre as duas estirpes, a atividade das bombas de efluxo foi avaliada em ambas as estirpes. A avaliação foi realizada num ensaio de acumulação usando o composto Brometo de Etídio na presença e ausência de Verapamil. O papel das bombas de efluxo na resistência ao PZQ, foi ainda investigado comparando a resposta dos parasitas da estirpe sensível e resistente ao fármaco na ausência e na presença de diferentes doses de Verapamil, em cultura in vitro. Os resultados obtidos foram reforçados comparando os níveis e expressão do gene SmMDR2 em ambas as estirpes isogénicas por Real-Time PCR (qPCR). A estirpe resistente de S. mansoni, necessitou de concentrações mais elevadas de inibidor quando comparada com a estirpe sensível para obter níveis significativos de fluorescência de Brometo de Etídio. A cultura in vitro mostrou uma dose letal de PZQ mais elevada na estirpe resistente do que na estirpe sensível na ausência de Verapamil. Na presença de Verapamil houve uma redução na dose letal de PZQ nos machos de ambas as estirpes, sendo esta redução mais acentuada nos machos da estirpe resistente. As fêmeas não mostraram alterações significativas na dose letal de PZQ na presença e ausência e inibidor. Os resultados foram reforçados pela observação dos níveis do gene SmMDR2, onde os machos da estirpe resistente mostraram ter os maiores níveis de expressão e pelo aumento de expressão nos machos de ambas as estirpes após exposição ao PZQ. As fêmeas de ambas as estirpes não tiveram diferenças significativas na expressão do gene após exposição ao PZQ e as fêmeas da estirpe resistente tiveram mostraram ter os níveis de expressão do gene SmMDR2 mais baixos entre os machos e fêmeas de ambas as estirpes. Os resultados obtidos neste trabalho mostraram que os machos da estirpe resistente têm maior atividade de bombas de efluxo do que os machos da estirpe sensível e que as bombas P-gp estão envolvidas na resposta e no aumento de tolerância dos machos de S. mansoni ao PZQ.

RAPD genomic fingerprinting differentiates Thiobacillus ferrooxidans strains

The PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was optimized and used for assessing genomic variability among eight Thiobacillus ferrooxidans strains. RAPD fingerprints presented variation for the thirty primers used, giving a total of 269 polymorphic bands. Similarity coefficients between the strains were calculated, and UPGMA cluster analysis was used to generate a dendrogram showing relationships among them. Most primers divided T. ferrooxidans strains in two distinct groups - Group 1: S, SSP, V3, AMF and Group 2: CMV, FG-460, I-35, LR. We observed that the T. ferrooxidans strains used in this work have a high degree of genomic diversity and that RAPD is a powerful method to differentiate them.

Análise de genes diferencialmente expressos por Trichophyton rubrum na presença de queratina e tipagem molecular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Clinical relevance of polymorphic DNA copy number variation (CNV) in African American women with stage I-II breast cancer

Objectives. The chief goal of this study was to analyze copy number variation (CNV) in breast cancer tumors from 25 African American women with early stage breast cancer (BC) using molecular inversion probes (MIP) in order to: (1) compare the degree of CNV in tumors compared to normal lymph nodes, and (2) determine whether gains and/or losses of genes in specific chromosomes differ between pathologic subtypes of breast cancer defined by known prognostic markers, (3) determine whether gains/losses in CN are associated with known oncogenes or tumor suppressor genes, and (4) determine whether increased gains/losses in CN for specific chromosomes were associated with differences in breast cancer recurrence. ^ Methods. Twenty to 37 nanograms of DNA extracted from 25 formalin-fixed paraffin embedded (FFPE) tumor samples and matched normal lymph nodes were added to individual tubes. Oligonucleotide probes with recognition sequences at each terminus were hybridized with a genomic target sequence to form a circular structure. Probes are released from genomic DNA obtained from FFPE samples, and those which have been correctly "circularized" in the proper allele/nucleotide reaction combination are amplified using polymerase chain reaction (PCR) primers. Amplicons were fluorescently labeled and the tag sequences released from the genome homology regions by treatment with uracil-N-glycosylase to cleave the probe at the site where uracils are present, and detected using a complementary tag array developed by Affymetrix. ^ Results. Analysis of CN gains and losses from tumors and normal tissues showed marked differences in tumors with numerous chromosomes affected. Similar changes were not observed in normal lymph nodes. When tumors were stratified into four groups based on expression or lack of expression of the estrogen receptor and HER2/neu, distinct patterns of CNV for different chromosomes were observed. Gains or losses in CN for specific chromosomes correlated with amplifications/deletions of particular oncogenes or tumor suppressor genes (i.e. such as found on chromosome 17) known to be associated with aggressive tumor phenotype and poor prognosis. There was a trend for increases in CN observed for chromosome 17 to correlate inversely with time to recurrence of BC (p=0.14 for trend). CNV was also observed for chromosomes 5, 8, 10, 11, and 16, which are known sites for several breast cancer susceptibility alleles. ^ Conclusions. This study is the first to validate the MIP technique, to correlate differences in gene expression with known prognostic tumor markers, and to correlate significant increases/decreases in CN with known tumor markers associated with prognosis. The results of this study may have far reaching public health implications towards identifying new high-risk groups based on genomic differences in CNP, both with respect to prognosis and response to therapy, and to eventually identify new therapeutic targets for prevention and treatment of this disease. ^

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpson's diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection

Objectives: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). Methods: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. Results: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. Conclusion: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4 h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections. © 2005 Published by Elsevier Ltd on behalf of the British Infection Society.

葡萄属植物系统学研究

葡萄属（Vitis.L.）植物隶属于葡萄科(Vitaceae)，主要分布于北温带，最南可以分布到南美洲的委内瑞拉和亚洲的越南以及印度北部。本文通过对该属分类研究历史的回顾，认为该属存在的问题主要表现在如下几个方面： 1）葡萄属自1753 年由Carl Linne创立以来，虽经planchon于1887年做了修订，但属的范围仍需进一步界定;2）在Planchon之后的100多年中未见有一全面的分类学修订工作，出现在该属的800多个名称需要考证;3）对一些广布种的变异认识不足，导致了大量可疑种。针对这些问题本文进行了如下几个方面的工作： l、形态学：通过大量的野外工作和标本观察，对该属植物的主要性状做了分析，讨论了这些性状状态在葡萄属中的变异规律及演化趋势，将灌木状习性、退化的卷须以及不裂的叶片视为进化的性状。 2、细胞学：利用前人对葡萄属(Vitis.L.)染色体数目的统计及一些杂交实验分析的结果，结合形态学等方面的特征分析，认为在葡萄科，染色体基数X=10为原始的，而x=19则为衍生的。葡萄属的染色体基数xl9(2n=38)，多倍体较少见;麝香葡萄属[Muscadinia (Planch.) Sma11]的染色体基数为x=10 (2n=20)．与蛇葡萄属、酸蔹藤属和爬山虎属的一致。葡萄属和麝香葡萄属间的杂种是不育的。 3、孢粉学：对葡萄属32种5变种及麝香葡萄属[Muscadinia (Planch.) Sma11]1种的花粉外壁做扫描电镜观察，结果发现花粉外壁雕纹在这两属间和葡萄属内变异较小，对区分属以及属下种上类群意义不大，但对种的鉴别有重要的价值。 4、植物化学：前人对植物化学的工作表明，植物的一些次生代谢产物如类黄酮化合物在葡萄科各类群中的分布规律较好地反映了各类群间的关系。这些结果较好地支持了Planchon对葡萄属范围的界定。 5、山葡萄复合体(V.amurensis complex)包括山葡萄(V.amurensis Rupr.)、燕山葡萄(V.amuresis Rupr. var. dissecta Skvorts.=var.yanshanensisD．Z．Lu et H．P．Liang)、百花山葡萄(V.baihuashanensis M．S．Kang et D．Z．Lu)、复叶葡萄(V.piasezkii Maxim.)、少毛复叶葡萄[V. piasezkii Maxux1．var. pagnuccii (Planch.) Rehd.]共3个种和2个变种，广泛分布于中国北方，形态变异较大。本文对该复合体做了形态分析，并用RAPD (Random Amplified Polymorphic DNAs)分子标记方法分析了这几个类群的关系。综合这些结果，归并了燕山葡萄和百花山葡萄。 在上述工作的基础上，我们得出了如下的结论： l、葡萄属在葡萄科中是一个进化的类群。整理后的葡萄属包括8系62种、l亚种和15变种，其中有2个新系、1个新组合系、2个新变种、1个新组合种和l3个新异名。 2、本文赞同SmaU在1903年作出的分类学处理，把麝香葡萄作为一个独立的属，比葡萄属原始但与葡萄属有着最近的亲缘关系。 3、依据形态特征和APD分析结果把山葡萄复合体的3个种2变种归并为2种l变种，即山葡萄、复叶葡萄和少毛复叶葡萄，认为分子标记技术在分析属内近缘种闻关系上很有价值。 4、葡萄属具有东亚和北美2个现代分布中心，该属可能起源于北美的东部，在晚白垩纪经白令陆桥散布至欧亚大陆。

云南4个马品种的随机扩增多态DNA(RAPD)分析

腾冲马和新丽江马品种内的遗传变异显著高于文山马和乌蒙马,遗传变异68.06%存在于群体内。系统聚类图中品种内大多数个体聚在一起,文山马和乌蒙马聚成一个大类,腾冲马和新丽江马又聚成一个大类,说明文山马和乌蒙马以及腾冲马和新丽江马有较近的亲缘关系。

ACHT操作引起黑麦(Secale cereale)染色体的畸变和重建

用ACHT处理黑麦萌动种子，对修复前后材料的观察和分析结果表明：1. ACHT操作引起染色体数目变化和染色体断裂损失。在一定 条件和范围内，不同处理引起的这种变化具有显著差异，条件越剧烈，染色体数目变化的范围和频率愈大，断片发生的数量和频率 也愈高，同时修复前后染色体数目的变化范围和频率与断片发生的数量和频率以及它们的修复频率均表现明显的相关性。2. ACHT 操作引起染色体畸变的多样性。经ACHT处理后，胚根细胞染色体有4种断裂方式，包括着丝粒断裂、次溢痕断裂、长臂断裂和短臂 断裂等，其中着丝断裂频率最高；产生6种断片类型，包括有着丝粒和端粒的、有着丝粒而无端粒的、有部分着丝粒和端粒的、有 部分着丝粒而无端粒的、只有端粒的、既无着丝粒也无端粒的断片等。3. ACHT操作引起遗传结构重建的多样性。经ACHT处理后， 对修复24-72小时材料进行核型比较(按Stebbins 和 Levan 标准)和随体分析，处理细胞在染色体数目、大小、形态、位置等方面 均发生显著变化，说明ACHT处理使这些细胞的染色体结构和染色体组型发生了深刻变化。进一步通过Giemsa C— 带分析，观察到 多种重建染色体类型，包括易位型染色体、附加型染色体、无着丝粒染色体、化染色体、增加的m染色体以及某些带型特异的染色 体等。4. RAPD 分析从分子水平上验证了ACHT能有效地引起遗传结构的改变。所用10种引物对处理和对照材料基因组DNA的扩增产 物在条带数目、条带位置及带型特征等方面均有明显差异，其中4种引物出现条带减少，6种引物出现条带增加，后者还包括一个带 位移动。这说明两种材料的基因组DNA具有明显的RAPD反应多态性差异。This paper descripes some results draw on the basis of the observation and analysis on the rye before and after repaired through treating its budding seeds by ACHT in contrast to without ACHT: 1. ACHT manipulation caused the number variation and breakage damage of rye chromosome. Within certain conditions and timits, this phenomenon caused by different treats had signifcant difference: the more the treatment condition is drastie, the more the chageable range and frequence of rye chromosomae number, and so is the produced fragments. Meanwhile, there existed striking relationship among the changeable range and frequence of rye chromosome, the produced number and frequence of fragments and repairing frequence. 2. ACHT manipulafion engendered the diversify of rye chromosomal aberration. Four breakage patterns and six sorts of fragment were observed by watching the chromosome of the rye radicle treated by ACHT, including centric breakage (occuring in the highest frequence), secondary constriction breakage, long arm breakage and short arm breakage to the former, Comprising that with both centromere and telomere, that with centromere and without telomere, that with partial centromere and with telomere, that with partfial ceetromere and without telomere, that only with telomere and that neither with centromere nor with telomere, etc. 3. ACHT manipulation engendered the diversify or rye genetic structs reconstruction. Karureotype analysis(according to Stebbins and Levan) and satellite anaeysis were carried out to rye radicle through 24-72-hour-long recoverage after ACHT manipulation, which showed remarkable change happened on the rye chromosomal number、shape、arm ration and pattern, etc. and also on the satellite number、size、shape and location etc. Those indicated that ACHT manipulation engendered violent changes to rye chromatin structure and chromosome type. Further Giemsa C-banding analysis showed many types of reconstructed chromosome, such as translocation、addition、without centromere、st and other chromosome. 4. RAPD analysis checked the validity of ACHT on changing genetic structure of rye on the level of molecular biology. The treated and recovered rye has different amplifying band pattern by using IO valid arbitary primers selected from 40 comparing with the control.

Genetic variation and phylogeographic history of Picea likiangensis revealed by RAPD markers

Repeated cycles of retreat and recolonization during the Quaternary ice ages are thought to have greatly influenced current species distributions and their genetic diversity. It remains unclear how this climatic oscillation has affected the distribution of genetic diversity between populations of wind-pollinated conifers in the Qinghai-Tibetan region. In this study, we investigated the within-species genetic diversity and phylogenetic relationships of Picea likiangensis, a dominant forest species in this region using polymorphic DNA (RAPD) markers. Our results suggest that this species has high overall genetic diversity, with 85.42% of loci being polymorphic and an average expected heterozygosity (H (E)) of 0.239. However, there were relatively low levels of polymorphism at population levels and the differences between populations were not significant, with percentages of polymorphic bands (PPB) ranging from 46.88 to 69.76%, Nei's gene diversity (H (E)) from 0.179 to 0.289 and Shannon's indices (Hpop) from 0.267 to 0.421. In accordance with our proposed hypothesis, a high level of genetic differentiation among populations was detected based on Nei's genetic diversity (G (ST) = 0.256) and AMOVA analysis (Phi (st) = 0.236). Gene flow between populations was found to be limited (Nm = 1.4532) and far lower than reported for other conifer species with wide distribution ranges from other regions. No clusters corresponding to three morphological varieties found in the south, north and west, respectively, were detected in either UPGMA or PCO analyses. Our results suggest that this species may have had different refugia during the glacial stages in the southern region and that the northern variety may have multiple origins from these different refugia.

A biotecnologia nos programas de melhoramento de forrageiras tropicais da Embrapa Gado de Corte.

Neste trabalho procurou-se apresentar e discutir, de forma ampla, o uso da biotecnologia e seu potencial para os programas de melhoramento de forrageiras tropicais realizados na Embrapa Gado de Corte. O uso da biotecnologia nesses programas é uma atividade recente, mas importantes resultados vêm sendo gerados a fim de auxiliar o processo de obtenção de novas cultivares forrageiras. A maioria dos trabalhos apresentados utiliza marcadores Random Amplification of Polymorphic DNA (RAPD) para aplicações em curto prazo: estudos de diversidade em acessos de bancos de germoplasma, identificação de híbridos e estimação da taxa de cruzamento. Aplicações em médio e longo prazos do uso de marcadores, como mapeamento genético e seleção auxiliada por marcadores moleculares (SAMM), ainda necessitam de maiores investimentos, tanto na busca de novos marcadores, quanto no desenvolvimento de populações adequadas para esses estudos. Em 2007, teve início uma nova linha de pesquisa nessa unidade, a prospecção de genes com características econômicas. Genes para a tolerância ao alumínio são o foco dessa pesquisa que pretende explorar a sintenia entre os genomas de gramíneas, visando ao desenvolvimento de cultivares de braquiária mais tolerantes ao alumínio. A Embrapa Gado de Corte vem investindo em pessoal e aquisição de equipamentos para avançar não só na produção de cultivares de forrageiras mais adaptadas às necessidades de um mercado cada vez mais exigente, como também no crescimento institucional do setor de biotecnologia.

A Novel Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Propionibacterium acnes and Characterisation of Type I Cell Surface-Associated Antigens.

We have developed a novel Multilocus Sequence Typing Scheme (MLST) and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and Random Amplification of Polymorphic DNA (RAPD) typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (ST). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 65% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants (SLV), which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore supports an association between acne and P. acnes strains from the type IA cluster and highlights the role of a widely disseminated clonal genotype in this condition. Characterisation of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.