Isolation and characterization of microsatellite loci in Colletotrichum acutatum, the causal agent of postbloom fruit drop on citrus

From a genomic enriched library, we developed 27 primer pairs from microsatellite flanking sequences for Colletotrichum acutatum, associated to postbloom fruit drop disease on citrus. Loci were characterized using 40 monosporic C. acutatum isolates. Nine primer pairs successfully amplified polymorphic microsatellite regions, with 3-6 alleles per locus, and mean heterozygosities ranging 0.093-0.590 across loci. The suitability of these primers was investigated in four Colletotrichum species as well. These microsatellite markers will be useful for genetic analyses and epidemiological studies of C. acutatum.

A new set of microsatellite markers for the genetic characterization of Ceratocystis fimbriata, an economically important plant pathogen

Ceratocystis fimbriata is a fungal pathogen which attacks several economically important plants, but occurs in host-associated, morphologically indistinguishable forms. In Brazil, this fungus seriously attacks mango trees (Mangifera indica), causing severe loss of yield. This work aimed to develop and characterize a novel set of microsatellite markers for this important pathogen, providing researchers with new molecular tools for the characterization of isolates. Twenty polymorphic primer pairs were designed from a microsatellite-enriched library. We tested the usefulness of these markers through genotyping thirteen isolates of the fungus. On average, 6.65 alleles per locus were detected, revealing the ability of this set of markers to characterize C. fimbriata isolates associated to mango and to other plant species.

Development and characterization of microsatellite loci in the marsh deer (Blastocerus dichotomus Cervidae)

Marsh deer (Blastocerus dichotomus) is one of the most exposed large mammals in South America. To aid in the conservation management of the species, nine polymorphic microsatellite loci were isolated and tested on up to 50 animals, showing 3-12 alleles and expected heterozygosity values varying from 0.69 to 0.89. These markers should be of considerable utility in future population and ecological genetics studies of this species. The marsh deer (Blastocerus dichotomus) is the biggest South American species of deer. Originally distributed across a large part of South America, stretching from the south bank of the Amazon river to northern Argentina, significant wild populations are now restricted to the Pantanal, swamplands that cover about 40% of southwest Brazil. The marsh deer is listed as Vulnerable on the Red List of the IUCN. Three populations of the species from three areas in the Parana River basin (between the states of Sao Paulo and Mato Grosso do Sul) were recently studied by observing protein polymorphism at 17 loci (Oliveira et al. 2005). Now we are presenting data about isolation of microsatellite markers to improve the results regarding population structure.

Genetic analysis of forest species Eugenia uniflora L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA)(n) and (CA)(n) microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeir (a) over tildeo Preto, Tambau and S (a) over tildeo Jose do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guacu river basins, in state of S (a) over tildeo Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H(E) = 0.83) and a low genetic differentiation (mean F(ST) = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.

A quantitative and qualitative evaluation of reports of clinical trials published in six Brazilian dental journals indexed in the Scientific Electronic Library Online (SciELO)

INTRODUCTION: Open access publishing is becoming increasingly popular within the biomedical sciences. SciELO, the Scientific Electronic Library Online, is a digital library covering a selected collection of Brazilian scientific journals many of which provide open access to full-text articles.This library includes a number of dental journals some of which may include reports of clinical trials in English, Portuguese and/or Spanish. Thus, SciELO could play an important role as a source of evidence for dental healthcare interventions especially if it yields a sizeable number of high quality reports. OBJECTIVE: The aim of this study was to identify reports of clinical trials by handsearching of dental journals that are accessible through SciELO, and to assess the overall quality of these reports. MATERIAL AND METHODS: Electronic versions of six Brazilian dental Journals indexed in SciELO were handsearched at www.scielo.br in September 2008. Reports of clinical trials were identified and classified as controlled clinical trials (CCTs - prospective, experimental studies comparing 2 or more healthcare interventions in human beings) or randomized controlled trials (RCTs - a random allocation method is clearly reported), according to Cochrane eligibility criteria. CRITERIA TO ASSESS METHODOLOGICAL QUALITY INCLUDED: method of randomization, concealment of treatment allocation, blinded outcome assessment, handling of withdrawals and losses and whether an intention-to-treat analysis had been carried out. RESULTS: The search retrieved 33 CCTs and 43 RCTs. A majority of the reports provided no description of either the method of randomization (75.3%) or concealment of the allocation sequence (84.2%). Participants and outcome assessors were reported as blinded in only 31.2% of the reports. Withdrawals and losses were only clearly described in 6.5% of the reports and none mentioned an intention-to-treat analysis or any similar procedure. CONCLUSIONS: The results of this study indicate that a substantial number of reports of trials and systematic reviews are available in the dental journals listed in SciELO, and that these could provide valuable evidence for clinical decision making. However, it is clear that the quality of a number of these reports is of some concern and that improvement in the conduct and reporting of these trials could be achieved if authors adhered to internationally accepted guidelines, e.g. the CONSORT statement.

Bone response to a Ca- and P-enriched titanium surface obtained by anodization

This study evaluated bone response to a Ca- and P- enriched titanium (Ti) surface treated by a multiphase anodic spark deposition coating (BSP-AK). Two mongrel dogs received bilateral implantation of 3 Ti cylinders (4.1 x 12 mm) in the humerus, being either BSP-AK treated or untreated (machined - control). At 8 weeks postimplantation, bone fragments containing the implants were harvested and processed for histologic and histomorphometric analyses. Bone formation was observed in cortical area and towards the medullary canal associated to approximately 1/3 of implant extension. In most cases, in the medullary area, collagen fiber bundles were detected adjacent and oriented parallel to Ti surfaces. Such connective tissue formation exhibited focal areas of mineralized matrix lined by active osteoblasts. The mean percentages of bone-to-implant contact were 2.3 (0.0-7.2 range) for BSP-AK and 0.4 (0.0-1.3 range) for control. Although the Mann-Whitney test did not detect statistically significant differences between groups, these results indicate a trend of BSP-AK treated surfaces to support contact osteogenesis in an experimental model that produces low bone-to-implant contact values.

Antidepressant-like effects of an apolar extract and chow enriched with Nepeta cataria (catnip) in mice

Nepeta cataria (catnip) is a plant used in pet toys and to treat human diseases. Catnip has also been used in the treatment of some depressive disorders. In this paper, we studied the antidepressant, anxiogenic, and motor activity effects of acute and repeated feeding of chow enriched with 10% N. cataria leaves and the acute and repeated administration of apolar and polar extracts of N. cataria leaves in male mice. The results showed that repeated feeding and acute and repeated administration with the apolar extract reduced immobility in the behavioral despair test but did not alter elevated plus maze and open-field parameters. Acute feeding and the acute and repeated administration of the polar extracts of N. cataria leaves did not alter the behavior of mice. These data suggest that N. cataria has antidepressant properties. Moreover, this antidepressant activity was present in the apolar extract.

NEW MICROSATELLITE LOCI FOR WATER YAM (DIOSCOREA ALATA, DIOSCOREACEAE) AND CROSS-AMPLIFICATION FOR OTHER DIOSCOREA SPECIES

Premise of the study: Dioscorea alata L. is one of the most widely distributed species of the genus in the humid and semihumid tropics and is associated with traditional agriculture. Only a few microsatellite markers have been developed so far for this and other Dioscorea species. Methods and Results: We isolated 14 codominant polymorphic microsatellite markers using a microsatellite-enriched genomic library technique. Ten microsatellite loci were selected, and 80 D. alata accessions from different regions in Brazil were evaluated with nine polymorphic loci. The polymorphism information content (PIC) varied from 0.39 to 0.78 and the power discrimination (PD) ranged from 0.15 to 0.91. Six of the markers showed transferability for the species D. bulbifera, D. cayenensis-D. rotundata, and D. trifida. Conclusions: The SSR markers obtained are an important tool for further studies aiming to characterize the genetic diversity in D. alata and other Dioscorea spp. accessions.

Effects of enriched colostrum supply in intestinal mucosa morphology of newborn calves

Colostrum intake in neonatal calves is essential to obtain passive immunity and to influence metabolism, endocrine systems and the nutritional state. This study compares morphologic features of small intestine of calves fed a colostrum second meal at 12 hours of life with concentrations of immunoglobulin G (IgG) higher than 100mg mL(-1) (fresh colostrum or artificially prepared with addition of lyophilized colostrum) or smaller than 30mg mL(-1). Twenty-four Holstein calves were randomly grouped according to concentration of IgG intake at 12 hours of life: low (less than 30mg mL(-1)); high (more than 100mg mL(-1)); plus lyophilized colostrum (more than 120mg mL(-1)). Intestinal tissue samples were collected at 0, 10, 24 and 72 hours after birth to evaluate morphology in segments: duodenum; proximal, middle and distal jejunum and ileum by examined with a scanning electron microscope. Villi of all segments showed more organized and uniform morphology characteristics with age. Only the animals fed colostrum with more than 100mg mL(-1) of IgG at 24 hours after birth still showed the distal jejunum villi disoriented and more united in comparison to the other ages and groups. Intake of lyophilized colostrum affected ileum morphology along experiment period. Higher concentration of immunologic and bioactive elements supplied for longer period of time could be responsible for promoting effects on the enterocytes. Use of colostrum with high IgG concentrations, with or without addition of lyophilized colostrum, influenced the precocity of the intestinal mucosa development in newborn calves, suggesting earlier maturation of the mucosa.

CHARACTERIZATION OF MICROSATELLITE LOCI IN HIMATANTHUS DRASTICUS (APOCYNACEAE), A MEDICINAL PLANT FROM THE BRAZILIAN SAVANNA

Premise of the study: We developed a new set of microsatellite markers for studying the genome of the janaguba tree, Himatanthus drasticus (Mart.) Plumel, which is used in folk medicine in northeastern Brazil. These novel markers are being used to evaluate the effect of harvesting on the genetic structure and diversity of natural populations of this species. Methods and Results: Microsatellite loci were isolated from an enriched H. drasticus genomic library. Nine primer pairs successfully amplified polymorphic microsatellite regions, with an average of 8.5 alleles per locus. The average values of observed and expected heterozygosity were 0.456 and 0.601, respectively. Conclusions: The microsatellite markers described here are valuable tools for population genetics studies of H. drasticus. The majority of the primers also amplified sequences in the genome of another species of the same genus. This new set of markers may be useful in designing a genetic conservation strategy and a sustainable management plan for the species.

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility ( het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer ( HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated ""gene dumps'' and, perhaps, simultaneously, as "" gene factories''.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

Background: Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results: The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions: In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.

Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive ""Lid""

Background: The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Methodology: Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 angstrom resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. Conclusion: The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six alpha-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this ""lid"" may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4(+)CD25(+) T Cells by Phage Display

Thymic CD4(+)CD25(+) cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4(+)CD25(+) T regulatory cells we targeted thymic CD4(+)CD25(+) cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4(+)CD25(+) T cells. After four rounds of selection on CD4(+)CD25(+) enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4(+)CD25(+) cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.