Improving the sensitivity of the sequence profile method.

The sequence profile method (Gribskov M, McLachlan AD, Eisenberg D, 1987, Proc Natl Acad Sci USA 84:4355-4358) is a powerful tool to detect distant relationships between amino acid sequences. A profile is a table of position-specific scores and gap penalties, providing a generalized description of a protein motif, which can be used for sequence alignments and database searches instead of an individual sequence. A sequence profile is derived from a multiple sequence alignment. We have found 2 ways to improve the sensitivity of sequence profiles: (1) Sequence weights: Usage of individual weights for each sequence avoids bias toward closely related sequences. These weights are automatically assigned based on the distance of the sequences using a published procedure (Sibbald PR, Argos P, 1990, J Mol Biol 216:813-818). (2) Amino acid substitution table: In addition to the alignment, the construction of a profile also needs an amino acid substitution table. We have found that in some cases a new table, the BLOSUM45 table (Henikoff S, Henikoff JG, 1992, Proc Natl Acad Sci USA 89:10915-10919), is more sensitive than the original Dayhoff table or the modified Dayhoff table used in the current implementation. Profiles derived by the improved method are more sensitive and selective in a number of cases where previous methods have failed to completely separate true members from false positives.

A Headspace needle-trap method for the analysis of volatile organic compounds in whole blood

Needle trap devices (NTDs) are a relatively new and promising tool for headspace (HS) analysis. In this study, a dynamic HS sampling procedure is evaluated for the determination of volatile organic compounds (VOCs) in whole blood samples. A full factorial design was used to evaluate the influence of the number of cycles and incubation time and it is demonstrated that the controlling factor in the process is the number of cycles. A mathematical model can be used to determine the most appropriate number of cycles required to adsorb a prefixed amount of VOCs present in the HS phase whenever quantitative adsorption is reached in each cycle. Matrix effect is of great importance when complex biological samples, such as blood, are analyzed. The evaluation of the salting out effect showed a significant improvement in the volatilization of VOCs to the HS in this type of matrices. Moreover, a 1:4 (blood:water) dilution is required to obtain quantitative recoveries of the target analytes when external calibration is used. The method developed gives detection limits in the 0.020–0.080 μg L−1 range (0.1–0.4 μg L−1 range for undiluted blood samples) with appropriate repeatability values (RSD < 15% at high level and <23% at LOQ level). Figure of merits of the method can be improved by using a smaller phase ratio (i.e., an increase in the blood volume and a decrease in the HS volume), which lead to lower detection limits, better repeatability values and greater sensibility. Twenty-eight blood samples have been evaluated with the proposed method and the results agree with those indicated in other studies. Benzene was the only target compound that gave significant differences between blood levels detected in volunteer non-smokers and smokers

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

A clustering method for robust and reliable large scale functional and structural protein sequence annotation

Bioinformatics, in the last few decades, has played a fundamental role to give sense to the huge amount of data produced. Obtained the complete sequence of a genome, the major problem of knowing as much as possible of its coding regions, is crucial. Protein sequence annotation is challenging and, due to the size of the problem, only computational approaches can provide a feasible solution. As it has been recently pointed out by the Critical Assessment of Function Annotations (CAFA), most accurate methods are those based on the transfer-by-homology approach and the most incisive contribution is given by cross-genome comparisons. In the present thesis it is described a non-hierarchical sequence clustering method for protein automatic large-scale annotation, called “The Bologna Annotation Resource Plus” (BAR+). The method is based on an all-against-all alignment of more than 13 millions protein sequences characterized by a very stringent metric. BAR+ can safely transfer functional features (Gene Ontology and Pfam terms) inside clusters by means of a statistical validation, even in the case of multi-domain proteins. Within BAR+ clusters it is also possible to transfer the three dimensional structure (when a template is available). This is possible by the way of cluster-specific HMM profiles that can be used to calculate reliable template-to-target alignments even in the case of distantly related proteins (sequence identity < 30%). Other BAR+ based applications have been developed during my doctorate including the prediction of Magnesium binding sites in human proteins, the ABC transporters superfamily classification and the functional prediction (GO terms) of the CAFA targets. Remarkably, in the CAFA assessment, BAR+ placed among the ten most accurate methods. At present, as a web server for the functional and structural protein sequence annotation, BAR+ is freely available at http://bar.biocomp.unibo.it/bar2.0.

Assessing and comparing the total antioxidant capacity of commercial beverages: application to beers, wines, waters and soft drinks using TRAP, TEAC and FRAP methods.

This work measures and tries to compare the Antioxidant Capacity (AC) of 50 commercial beverages of different kinds: 6 wines, 12 beers, 18 soft drinks and 14 flavoured waters. Because there is no reference procedure established for this purpose, three different optical methods were used to analyse these samples: Total Radical trapping Antioxidant Parameter (TRAP), Trolox Equivalent Antioxidant Capacity (TEAC) and Ferric ion Reducing Antioxidant Parameter (FRAP). These methods differ on the chemical background and nature of redox system. The TRAP method involves the transfer of hydrogen atoms while TEAC and FRAP involves electron transfer reactions. The AC was also assessed against three antioxidants of reference, Ascorbic acid (AA), Gallic acid (GA) and 6-hydroxy-2,5,7,8-tetramethyl- 2-carboxylic acid (Trolox). The results obtained were analyzed statistically. Anova one-way tests were applied to all results and suggested that methods and standards exhibited significant statistical differences. The possible effect of sample features in the AC, such as gas, flavours, food colouring, sweeteners, acidity regulators, preservatives, stabilizers, vitamins, juice percentage, alcohol percentage, antioxidants and the colour was also investigated. The AC levels seemed to change with brand, kind of antioxidants added, and kind of flavour, depending on the sample. In general, higher ACs were obtained for FRAP as method, and beer for kind of sample, and the standard expressing the smaller AC values was GA.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/ lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in viva. A suitable in viva model for studying these events is the alveolar bone of young rats because it is continuously. Thus, sections of aldehyde fixed alveolar undergoing intense resorption/remodeling bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Identifying sequence regions undergoing conformational change via predicted continuum secondary structure

Motivation: Conformational flexibility is essential to the function of many proteins, e.g. catalytic activity. To assist efforts in determining and exploring the functional properties of a protein, it is desirable to automatically identify regions that are prone to undergo conformational changes. It was recently shown that a probabilistic predictor of continuum secondary structure is more accurate than categorical predictors for structurally ambivalent sequence regions, suggesting that such models are suited to characterize protein flexibility. Results: We develop a computational method for identifying regions that are prone to conformational change directly from the amino acid sequence. The method uses the entropy of the probabilistic output of an 8-class continuum secondary structure predictor. Results for 171 unique amino acid sequences with well-characterized variable structure (identified in the 'Macromolecular movements database') indicate that the method is highly sensitive at identifying flexible protein regions, but false positives remain a problem. The method can be used to explore conformational flexibility of proteins (including hypothetical or synthetic ones) whose structure is yet to be determined experimentally.

TATPred:a Bayesian method for the identification of twin arginine translocation pathway signal sequences

The twin arginine translocation (TAT) system ferries folded proteins across the bacterial membrane. Proteins are directed into this system by the TAT signal peptide present at the amino terminus of the precursor protein, which contains the twin arginine residues that give the system its name. There are currently only two computational methods for the prediction of TAT translocated proteins from sequence. Both methods have limitations that make the creation of a new algorithm for TAT-translocated protein prediction desirable. We have developed TATPred, a new sequence-model method, based on a Nave-Bayesian network, for the prediction of TAT signal peptides. In this approach, a comprehensive range of models was tested to identify the most reliable and robust predictor. The best model comprised 12 residues: three residues prior to the twin arginines and the seven residues that follow them. We found a prediction sensitivity of 0.979 and a specificity of 0.942.

Tool support for testing concurrent Java components

Concurrent programs are hard to test due to the inherent nondeterminism. This paper presents a method and tool support for testing concurrent Java components. Too[ support is offered through ConAn (Concurrency Analyser), a too] for generating drivers for unit testing Java classes that are used in a multithreaded context. To obtain adequate controllability over the interactions between Java threads, the generated driver contains threads that are synchronized by a clock. The driver automatically executes the calls in the test sequence in the prescribed order and compares the outputs against the expected outputs specified in the test sequence. The method and tool are illustrated in detail on an asymmetric producer-consumer monitor. Their application to testing over 20 concurrent components, a number of which are sourced from industry and were found to contain faults, is presented and discussed.

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays.

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl(-1) of RNA material, without prior PCR amplification and use of labels.

A study of the thermal decomposition of allyl t-butyl peroxide and 3-hydroperoxy-1-propene (allyl hydroperoxide) in toluene /|nby Krishnankutty Nair V. G. -- 260 St. Catharines [Ont. : s. n.],

Kinetics and product studies of the decompositions of allyl-t-butyl peroxide and 3-hydroperoxy- l-propene (allyl hydroperoxide ) in tolune were investigated. Decompositions of allyl-t-butyl peroxide in toluene at 130-1600 followed first order kinetics with an activation energy of 32.8 K.cals/mol and a log A factor of 13.65. The rates of decomposition were lowered in presence of the radical trap~methyl styrene. By the radical trap method, the induced decomposition at 1300 is shown to be 12.5%. From the yield of 4-phenyl-l,2- epoxy butane the major path of induced decomposition is shown to be via an addition mechanism. On the other hand, di-t-butYl peroxyoxalate induced decomposition of this peroxide at 600 proceeded by an abstraction mechanism. Induced decomposition of peroxides and hydroperoxides containing the allyl system is proposed to occur mainly through an addition mechanism at these higher temperatures. Allyl hydroperoxide in toluene at 165-1850 decomposes following 3/2 order kinetics with an Ea of 30.2 K.cals per mole and log A of 10.6. Enormous production of radicals through chain branching may explain these relatively low values of E and log A. The complexity of the reaction is indicated a by the formation of various products of the decomposition. A study of the radical attack of the hydro peroxide at lower temperatures is suggested as a further work to throw more light on the nature of decomposition of this hydroperoxide.

Genotyping Klebsiella pneumoniae isolated from hepatic abscesses in three patients from Bogota, Colombia

Pyogenic liver abscess caused by Klebsiella pneumoniae represents an ever increasing entity which has mainly been described as occurring in Asia, even though, on a smaller scale, cases are being more frequently described from the USA and Europe, 13% overall mortality being reached worldwide. Affected patients are severely sick, suffering from fever, sweating, having increased acute phase reactants and risk factors such as Diabetes Mellitus, alcoholism and the inherent characteristics of the bacteria causing the disease. Objective: in this work we used a Multilocus Sequencing Typing (MLST), a nucleotide sequence-based method in order to characterize the genetic relationships among bacterial isolates. Materials and methods: the report is focused on three cases involving patients suffering from pyogenic liver abscess caused by Klebsiella pneumoniae in two hospitals in Bogota, Colombia, where phenotyping and hypermucoviscosity studies were carried out, as well as the genotyping of cultured Klebsiella isolates. Reults: it was found that the isolated microorganism in cases I and II corresponded to the same K. pneumoniae strain, having 100% sequence identity for the 5 genes being studied while the strain in Case III was genotypically different. Conclusion: it is important to carry out multidisciplinary studies allowing all pyogenic liver abscess cases reported in Colombia to be complied to ascertain the frequency of microorganisms causing this pathology in our country, as well as a genotyping study of different K. pneumoniae strains to compare them and confirm clonal and pathogenicity relationships through housekeeping gene analysis.

Measuring bee diversity in different European habitats and biogeographical regions

Bee pollinators are currently recorded with many different sampling methods. However, the relative performances of these methods have not been systematically evaluated and compared. In response to the strong need to record ongoing shifts in pollinator diversity and abundance, global and regional pollinator initiatives must adopt standardized sampling protocols when developing large-scale and long-term monitoring schemes. We systematically evaluated the performance of six sampling methods (observation plots, pan traps, standardized and variable transect walks, trap nests with reed internodes or paper tubes) that are commonly used across a wide range of geographical regions in Europe and in two habitat types (agricultural and seminatural). We focused on bees since they represent the most important pollinator group worldwide. Several characteristics of the methods were considered in order to evaluate their performance in assessing bee diversity: sample coverage, observed species richness, species richness estimators, collector biases (identified by subunit-based rarefaction curves), species composition of the samples, and the indication of overall bee species richness (estimated from combined total samples). The most efficient method in all geographical regions, in both the agricultural and seminatural habitats, was the pan trap method. It had the highest sample coverage, collected the highest number of species, showed negligible collector bias, detected similar species as the transect methods, and was the best indicator of overall bee species richness. The transect methods were also relatively efficient, but they had a significant collector bias. The observation plots showed poor performance. As trap nests are restricted to cavity-nesting bee species, they had a naturally low sample coverage. However, both trap nest types detected additional species that were not recorded by any of the other methods. For large-scale and long-term monitoring schemes with surveyors with different experience levels, we recommend pan traps as the most efficient, unbiased, and cost-effective method for sampling bee diversity. Trap nests with reed internodes could be used as a complementary sampling method to maximize the numbers of collected species. Transect walks are the principal method for detailed studies focusing on plant-pollinator associations. Moreover, they can be used in monitoring schemes after training the surveyors to standardize their collection skills.