Caracterização molecular de isolados de Flavobacterium columnare de tilapia do nilo e piracanjuba por meio de RAPD-PCR

Flavobacterium columnare is a cosmopolite bacteria and it is one of the main problem in Brazilian aquaculture, causing high mortalities index and economic damage. The main factors that contribute to columnaris disease are inadequate water quality, excess handling, high density of fish and temperature variations. For a successful epidemiological study and disease control, it is essential to differentiate the F. columnare from other yellow pigmentation bacteria. The present study used molecular techniques to characterize, by RAPD-PCR, two strains of F. columnare isolated from Oreochromis niloticus and Brycon orbignyanus. Data were analyzed as binary (0 and 1) and a genetic similarity matrix was generated by Jaccard's coefficient. Cluster analysis was performed by the neighbor joining method. The RAPD-PCR technique confirmed to be a usefull tool to obtain genetic profiles from F. columnare isolates based on the oligonucleotides used and to verify genetic similarity.

Identificação e análise da expressão de genes relacioanados com tolerância à seca em soja através de microarranjos de DNA e PCR em tempo real

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class (Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue (Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.

BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was

Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level

Background: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

High Prevalence of bla(CTX-M) Extended Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae Isolates from a Tertiary Care Hospital: First report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil

The aim of this study was to investigate the presence and prevalence of bla(TEM), bla(SHV), and bla(CTX-M) and bla(GES)-like genes, responsible for extended spectrum beta-lactamases (ESBLs) production in clinical isolates of Klebsiella pneumoniae collected from a Brazilian tertiary care hospital. Sixty-five ESBL producing K. pneumoniae isolates, collected between 2005 and 2007, were screened by polymerase chain reaction (PCR). Identification of bla genes was achieved by sequencing. Genotyping of ESBL producing K. pneumoniae was performed by the enterobacterial repetitive intergenic consensus-PCR with cluster analysis by the Dice coefficient. The presence of genes encoding ESBLs was confirmed in 59/65 (90.8%) isolates, comprising 20 bla(CTX-M-2), 14 bla(CTX-M-59), 12 bla(CTX-M-15), 9 bla(SHV-12), 1 bla(SHV-2), 1 bla(SHV-2a), 1 bla(SHV-5), and 1 bla(SHV-31) genes. The ESBL genes bla(SHV-12), bla(SHV-31), and bla(CTX-M-15), and the chromosome-encoded SHV-type beta-lactamase capable of hydrolyzing imipenem were detected in Brazil for the first time. The analysis of the enterobacterial repetitive intergenic consensus-PCR band patterns revealed a high rate of multiclonal bla(CTX-M) carrying K. pneumoniae isolates (70.8%), suggesting that dissemination of encoding plasmids is likely to be the major cause of the high prevalence of these genes among the K. pneumoniae isolates considered in this study.

Endophytic Colonization of Potato (Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte, Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.

Analysis of genetic diversity within Australian lucerne cultivars and implications for future genetic improvement

Lucerne (Medicago sativa L.) is autotetraploid, and predominantly allogamous. This complex breeding structure maximises the genetic diversity within lucerne populations making it difficult to genetically discriminate between populations. The objective of this study was to evaluate the level of random genetic diversity within and between a selection of Australian-grown lucerne cultivars, with tetraploid M. falcata included as a possible divergent control source. This diversity was evaluated using random amplified polymorphic DNA (RAPDs). Nineteen plants from each of 10 cultivars were analysed. Using 11 RAPD primers, 96 polymorphic bands were scored as present or absent across the 190 individuals. Genetic similarity estimates (GSEs) of all pair-wise comparisons were calculated from these data. Mean GSEs within cultivars ranged from 0.43 to 0.51. Cultivar Venus (0.43) had the highest level of intra-population genetic diversity and cultivar Sequel HR (0.51) had the lowest level of intra-population genetic diversity. Mean GSEs between cultivars ranged from 0.31 to 0.49, which overlapped with values obtained for within-cultivar GSE, thus not allowing separation of the cultivars. The high level of intra- and inter-population diversity that was detected is most likely due to the breeding of synthetic cultivars using parents derived from a number of diverse sources. Cultivar-specific polymorphisms were only identified in the M. falcata source, which like M. sativa, is outcrossing and autotetraploid. From a cluster analysis and a principal components analysis, it was clear that M. falcata was distinct from the other cultivars. The results indicate that the M. falcata accession tested has not been widely used in Australian lucerne breeding programs, and offers a means of introducing new genetic diversity into the lucerne gene pool. This provides a means of maximising heterozygosity, which is essential to maximising productivity in lucerne.

Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010.

Most of the non-B HIV-1 subtypes are predominant in Sub-Saharan Africa and India although they have been found worldwide. In the last decade, immigration from these areas has increased considerably in Spain. The objective of this study was to evaluate the prevalence of non-B subtypes circulating in a cohort of HIV-1-infected immigrants in Seville, Southern Spain and to identify drug resistance-associated mutations. METHODS: Complete protease and first 220 codons of the reverse transcriptase coding regions were amplified and sequenced by population sequencing. HIV-1 subtypes were determined using Stanford University Drug Resistance Database, and phylogenetic analysis was performed comparing multiple reported sequences. Drug resistance mutations were defined according to the International AIDS Society-USA. RESULTS: From 2000 to 2010 a total of 1,089 newly diagnosed HIV-1-infected patients were enrolled in our cohort. Of these, 121 were immigrants, of which 98 had ethical approval and informed consent to include in our study. Twenty-nine immigrants (29/98, 29.6%) were infected with non-B subtypes, of which 15/29 (51.7%) were CRF02-AG, mostly from Sub-Saharan Africa, and 2/29 (6.9%) were CRF01-AE from Eastern Europe. A, C, F, J and G subtypes from Eastern Europe, Central-South America and Sub-Saharan Africa were also present. Some others harboured recombinant forms CRF02-AG/CRF01-AE, CRF2-AG/G and F/B, B/C, and K/G, in PR and RT-coding regions. Patients infected with non-B subtypes showed a high frequency of minor protease inhibitor resistance mutations, M36I, L63P, and K20R/I. Only one patient, CRF02_AG, showed major resistance mutation L90M. Major RT inhibitor resistance mutations K70R and A98G were present in one patient with subtype G, L100I in one patient with CRF01_AE, and K103N in another patient with CRF01_AE. Three patients had other mutations such as V118I, E138A and V90I. CONCLUSIONS: The circulation of non-B subtypes has significantly increased in Southern Spain during the last decade, with 29.6% prevalence, in association with demographic changes among immigrants. This could be an issue in the treatment and management of these patients. Resistance mutations have been detected in these patients with a prevalence of 7% among treatment-naïve patients compared with the 21% detected among patients under HAART or during treatment interruption.

Diversity of the bacterial community in the surface soil of a pear orchard based on 16S rRNA gene analysis

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environments

Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice.

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.

OBJECTIVE: The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. DESIGN: We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. METHODS: Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. RESULTS: Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. CONCLUSION: As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.

We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vβ region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vβ chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.