Analysis of expressed sequence tags from the agarophyte Gracilaria tenuistipitata (Rhodophyta)

A total of 3,631 expressed sequence tags (ESTs) were established from two size-selected cDNA libraries made from the tetrasporophytic phase of the agarophytic red alga Gracilaria tenuistipitata. The average sizes of the inserts in the two libraries were 1,600 bp and 600 bp, with an average length of the edited sequences of 850 bp. Clustering gave 2,387 assembled sequences with a redundancy of 53%. Of the ESTs, 65% had significant matches to sequences deposited in public databases, 11% to proteins without known function, and 35% were novel. The most represented ESTs were a Na/K-transporting ATPase, a hedgehog-like protein, a glycine dehydrogenase and an actin. Most of the identified genes were involved in primary metabolism and housekeeping. The largest functional group was thus genes involved in metabolism with 14% of the ESTs; other large functional categories included energy, transcription, and protein synthesis and destination. The codon usage was examined using a subset of the data, and the codon bias was found to be limited with all codon combinations used.

Genome Sequence of Exiguobacterium antarcticum B7, Isolated from a Biofilm in Ginger Lake, King George Island, Antarctica

Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e. g., the capacity to adapt to cold, which make this genus a target for discovering new enzymes, such as lipases and proteases, in addition to improving our understanding of the mechanisms of adaptation and survival at low temperatures. This study presents the genome of E. antarcticum B7, isolated from a biofilm sample of Ginger Lake on King George Island, Antarctic peninsula.

Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in Sao Paulo, Brazil

This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coil (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coil strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type. (C) 2010 Elsevier Ltd. All rights reserved.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

SIS: a program to generate draft genome sequence scaffolds for prokaryotes

Background: Decreasing costs of DNA sequencing have made prokaryotic draft genome sequences increasingly common. A contig scaffold is an ordering of contigs in the correct orientation. A scaffold can help genome comparisons and guide gap closure efforts. One popular technique for obtaining contig scaffolds is to map contigs onto a reference genome. However, rearrangements that may exist between the query and reference genomes may result in incorrect scaffolds, if these rearrangements are not taken into account. Large-scale inversions are common rearrangement events in prokaryotic genomes. Even in draft genomes it is possible to detect the presence of inversions given sufficient sequencing coverage and a sufficiently close reference genome. Results: We present a linear-time algorithm that can generate a set of contig scaffolds for a draft genome sequence represented in contigs given a reference genome. The algorithm is aimed at prokaryotic genomes and relies on the presence of matching sequence patterns between the query and reference genomes that can be interpreted as the result of large-scale inversions; we call these patterns inversion signatures. Our algorithm is capable of correctly generating a scaffold if at least one member of every inversion signature pair is present in contigs and no inversion signatures have been overwritten in evolution. The algorithm is also capable of generating scaffolds in the presence of any kind of inversion, even though in this general case there is no guarantee that all scaffolds in the scaffold set will be correct. We compare the performance of SIS, the program that implements the algorithm, to seven other scaffold-generating programs. The results of our tests show that SIS has overall better performance. Conclusions: SIS is a new easy-to-use tool to generate contig scaffolds, available both as stand-alone and as a web server. The good performance of SIS in our tests adds evidence that large-scale inversions are widespread in prokaryotic genomes.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Probing the anomalous extinction of four young star clusters: the use of colour-excess, main-sequence fitting and fractal analysis

Aims. We studied four young star clusters to characterise their anomalous extinction or variable reddening and asses whether they could be due to contamination by either dense clouds or circumstellar effects. Methods. We evaluated the extinction law (R-V) by adopting two methods: (i) the use of theoretical expressions based on the colour-excess of stars with known spectral type; and (ii) the analysis of two-colour diagrams, where the slope of the observed colour distribution was compared to the normal distribution. An algorithm to reproduce the zero-age main-sequence (ZAMS) reddened colours was developed to derive the average visual extinction (A(V)) that provides the closest fit to the observational data. The structure of the clouds was evaluated by means of a statistical fractal analysis, designed to compare their geometric structure with the spatial distribution of the cluster members. Results. The cluster NGC 6530 is the only object of our sample affected by anomalous extinction. On average, the other clusters suffer normal extinction, but several of their members, mainly in NGC 2264, seem to have high R-V, probably because of circumstellar effects. The ZAMS fitting provides A(V) values that are in good agreement with those found in the literature. The fractal analysis shows that NGC 6530 has a centrally concentrated distribution of stars that differs from the substructures found in the density distribution of the cloud projected in the A(V) map, suggesting that the original cloud was changed by the cluster formation. However, the fractal dimension and statistical parameters of Berkeley 86, NGC 2244, and NGC 2264 indicate that there is a good cloud-cluster correlation, when compared to other works based on an artificial distribution of points.

Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from Sao Paulo State, Brazil

Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes. using polymerase chain reaction (PCR) and comparing strains by means of Pulsed field gel electrophoresis (PFGE). Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only, alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.

Time sequence of the intensification of the liver glucose production induced by high-fat diet in mice

It is well established that the development of insulin resistance shows a temporal sequence in different organs and tissues. Moreover, considering that the main aspect of insulin resistance in liver is a process of glucose overproduction from gluconeogenesis, we investigated if this metabolic change also shows temporal sequence. For this purpose, a well-established experimental model of insulin resistance induced by high-fat diet (HFD) was used. The mice received HFD (HFD group) or standard diet (COG group) for 1, 7, 14 or 56?days. The HFD group showed increased (P?<?0.05 versus COG) epididymal, retroperitoneal and inguinal fat weight from days 1 to 56. In agreement with these results, the HFD group also showed higher body weight (P?<?0.05 versus COG) from days 7 to 56. Moreover, the changes induced by HFD on liver gluconeogenesis were progressive because the increment (P?<?0.05 versus COG) in glucose production from l-lactate, glycerol, l-alanine and l-glutamine occurred 7, 14, 56 and 56 days after the introduction of the HFD schedule, respectively. Furthermore, glycaemia and cholesterolemia increased (P?<?0.05 versus COG) 14?days after starting the HFD schedule. Taken together, the results suggest that the intensification of liver gluconeogenesis induced by an HFD is not a synchronous all-or-nothing process but is specific for each gluconeogenic substrate and is integrated in a temporal manner with the progressive augmentation of fasting glycaemia. Copyright (c) 2012 John Wiley & Sons, Ltd.

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Lithium in M 67: From the main sequence to the red giant branch

Context. Lithium abundances in open clusters are a very effective probe of mixing processes, and their study can help us to understand the large depletion of lithium that occurs in the Sun. Owing to its age and metallicity, the open cluster M 67 is especially interesting on this respect. Many studies of lithium abundances in M 67 have been performed, but a homogeneous global analysis of lithium in stars from subsolar masses and extending to the most massive members, has yet to be accomplished for a large sample based on high-quality spectra. Aims. We test our non-standard models, which were calibrated using the Sun with observational data. Methods. We collect literature data to analyze, for the first time in a homogeneous way, the non-local thermal equilibrium lithium abundances of all observed single stars in M 67 more massive than similar to 0.9 M-circle dot. Our grid of evolutionary models is computed assuming a non-standard mixing at metallicity [Fe/H] = 0.01, using the Toulouse-Geneva evolution code. Our analysis starts from the entrance into the zero-age main-sequence. Results. Lithium in M 67 is a tight function of mass for stars more massive than the Sun, apart from a few outliers. A plateau in lithium abundances is observed for turn-off stars. Both less massive (M >= 1.10 M-circle dot) and more massive (M >= 1.28 M-circle dot) stars are more depleted than those in the plateau. There is a significant scatter in lithium abundances for any given mass M <= 1.1 M-circle dot. Conclusions. Our models qualitatively reproduce most of the features described above, although the predicted depletion of lithium is 0.45 dex smaller than observed for masses in the plateau region, i.e. between 1.1 and 1.28 solar masses. More work is clearly needed to accurately reproduce the observations. Despite hints that chromospheric activity and rotation play a role in lithium depletion, no firm conclusion can be drawn with the presently available data.

Facial Profile Evaluation of Isolated Pierre Robin Sequence

Objective: To evaluate numerically the facial profile of children with isolated Pierre Robin sequence (PRS) and to compare them with a control group that has no pathologies and exhibits regular and balanced facial growth, with no skeletal alterations. Patients: Eighty-three children aged 5 to 10 years (PRS group, n = 60; control group, n = 23) were selected. Setting: Hospital for Rehabilitation of Craniofacial Anomalies, University of Sao Paulo (HRAC-USP). Children from the control group were taken from the program of Interceptive Orthodontics at HRAC-USP. Design: Angular and ratio analyses of the facial profiles in both groups were realized through digital photographs. The PRS group was subdivided into two groups-complete and incomplete-according to the sagittal extension of the cleft palate, to investigate the possible influence of cleft extension on the face. Results: The facial convexity angle and the facial inferior third angle were considerably higher in the PRS groups than in the control group and were not significantly different between PRS groups. Nasolabial angle did not differ between groups. Conclusion: The facial profile was more convex in individuals with PRS than in those with regular facial growth and with no pathology. The mandible was responsible for the convexity of the profile in PRS because of its lack off anterior projection. An important relationship between the extension of the cleft palate and alterations in facial profile in PRS was not observed.

Bacillus cereus infection outbreak in captive psittacines

This study reports an uncommon epizootic outbreak of Bacillus cereus that caused the sudden death of 12 psittacines belonging to the species Anodorhynchus hyacinthinus (1 individual), Diopsittaca nobilis (1 individual), Ara severe (1 individual) and Ara ararauna (9 individuals) in a Brazilian zoo. Post-mortem examination of the animals reveled extensive areas of lung hemorrhage, hepatic congestion, hemorrhagic enteritis and cardiac congestion. Histopathological examination of the organs showed the presence of multiple foci of vegetative cells of Gram-positive bacilli associated with discrete and moderate mononuclear inflammatory cell infiltrate. Seventeen B. cereus strains isolated from blood and sterile organs of nine A. ararauna were analyzed in order to investigate the genetic diversity (assessed by Rep-PCR) and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. Amplification of genomic DNA by Rep-PCR of B. cereus strains generated two closely related profiles (Rep-PCR types A and B) with three bands of difference. All strains were classified as belonging to the toxigenic profile I which contained HBL and NHE gene complexes, entFM and cytK genes. Altogether, microbiological and histopathological findings and the evidence provided by the success of the antibiotic prophylaxis, corroborate that B. cereus was the causative agent of the infection that killed the birds. (C) 2012 Elsevier B.V. All rights reserved.