In vitro Approach to Bacterial Chemotaxis

An in vitro approach to study bacterial motility and chemotaxis is described. The approach is based on a preparation of flagellated cell envelopes. The envelopes are prepared from bacteria by a penicillin treatment and subsequent osmotic lysis. When the envelopes are energized, their flagella rotate. The direction of rotation in wild type envelopes is counterclockwise. Inclusion of the CheY protein within the envelopes may restore clockwise rotation. The advantages and disadvantages of this approach are pointed out.     

A Double-Pulse Approach For Electrotransfection

Gene transfer and expression can be obtained by delivering calibrated electric pulses on cells in the presence of plasmids coding for the activity of interest. The electric treatment affects the plasma membrane and induces the formation of a transient complex between nucleic acids and the plasma membrane. It results in a delivery of the plasmid in the cytoplasm. Expression is only obtained if the plasmid is translocated inside the nucleus. This is a key limit in the process. We previously showed that delivery of a high-field short-duration electric pulse was inducing a structural alteration of the nuclear envelope. This study investigates if the double-pulse approach (first pulse to transfer the plasmid to the cytoplasm, and second pulse to induce the structural alteration of the envelope) was a way to enhance the protein expression using the green fluorescent protein as a reporter. We observed that not only the double-pulse approach induced the transfection of a lower number of cells but moreover, these transfected cells were less fluorescent than the cells treated only with the first pulse.     

A Methodological Framework for the Reconstruction of Contiguous Regions of Ancestral Genomes and Its Application to Mammalian Genomes

The reconstruction of ancestral genome architectures and gene orders from homologies between extant species is a long-standing problem, considered by both cytogeneticists and bioinformaticians. A comparison of the two approaches was recently investigated and discussed in a series of papers, sometimes with diverging points of view regarding the performance of these two approaches. We describe a general methodological framework for reconstructing ancestral genome segments from conserved syntenies in extant genomes. We show that this problem, from a computational point of view, is naturally related to physical mapping of chromosomes and benefits from using combinatorial tools developed in this scope. We develop this framework into a new reconstruction method considering conserved gene clusters with similar gene content, mimicking principles used in most cytogenetic studies, although on a different kind of data. We implement and apply it to datasets of mammalian genomes. We perform intensive theoretical and experimental comparisons with other bioinformatics methods for ancestral genome segments reconstruction. We show that the method that we propose is stable and reliable: it gives convergent results using several kinds of data at different levels of resolution, and all predicted ancestral regions are well supported. The results come eventually very close to cytogenetics studies. It suggests that the comparison of methods for ancestral genome reconstruction should include the algorithmic aspects of the methods as well as the disciplinary differences in data aquisition.     

A Cross-Lingual Similarity Measure for Detecting Biomedical Term Translations

Bilingual dictionaries for technical terms such as biomedical terms are an important resource for machine translation systems as well as for humans who would like to understand a concept described in a foreign language. Often a biomedical term is first proposed in English and later it is manually translated to other languages. Despite the fact that there are large monolingual lexicons of biomedical terms, only a fraction of those term lexicons are translated to other languages. Manually compiling large-scale bilingual dictionaries for technical domains is a challenging task because it is difficult to find a sufficiently large number of bilingual experts. We propose a cross-lingual similarity measure for detecting most similar translation candidates for a biomedical term specified in one language (source) from another language (target). Specifically, a biomedical term in a language is represented using two types of features: (a) intrinsic features that consist of character n-grams extracted from the term under consideration, and (b) extrinsic features that consist of unigrams and bigrams extracted from the contextual windows surrounding the term under consideration. We propose a cross-lingual similarity measure using each of those feature types. First, to reduce the dimensionality of the feature space in each language, we propose prototype vector projection (PVP)—a non-negative lower-dimensional vector projection method. Second, we propose a method to learn a mapping between the feature spaces in the source and target language using partial least squares regression (PLSR). The proposed method requires only a small number of training instances to learn a cross-lingual similarity measure. The proposed PVP method outperforms popular dimensionality reduction methods such as the singular value decomposition (SVD) and non-negative matrix factorization (NMF) in a nearest neighbor prediction task. Moreover, our experimental results covering several language pairs such as English–French, English–Spanish, English–Greek, and English–Japanese show that the proposed method outperforms several other feature projection methods in biomedical term translation prediction tasks.     

Waveform Similarity Analysis: A Simple Template Comparing Approach for Detecting and Quantifying Noisy Evoked Compound Action Potentials

Experimental electrophysiological assessment of evoked responses from regenerating nerves is challenging due to the typical complex response of events dispersed over various latencies and poor signal-to-noise ratio. Our objective was to automate the detection of compound action potential events and derive their latencies and magnitudes using a simple cross-correlation template comparison approach. For this, we developed an algorithm called Waveform Similarity Analysis. To test the algorithm, challenging signals were generated in vivo by stimulating sural and sciatic nerves, whilst recording evoked potentials at the sciatic nerve and tibialis anterior muscle, respectively, in animals recovering from sciatic nerve transection. Our template for the algorithm was generated based on responses evoked from the intact side. We also simulated noisy signals and examined the output of the Waveform Similarity Analysis algorithm with imperfect templates. Signals were detected and quantified using Waveform Similarity Analysis, which was compared to event detection, latency and magnitude measurements of the same signals performed by a trained observer, a process we called Trained Eye Analysis. The Waveform Similarity Analysis algorithm could successfully detect and quantify simple or complex responses from nerve and muscle compound action potentials of intact or regenerated nerves. Incorrectly specifying the template outperformed Trained Eye Analysis for predicting signal amplitude, but produced consistent latency errors for the simulated signals examined. Compared to the trained eye, Waveform Similarity Analysis is automatic, objective, does not rely on the observer to identify and/or measure peaks, and can detect small clustered events even when signal-to-noise ratio is poor. Waveform Similarity Analysis provides a simple, reliable and convenient approach to quantify latencies and magnitudes of complex waveforms and therefore serves as a useful tool for studying evoked compound action potentials in neural regeneration studies.     

Detecting Protein Candidate Fragments Using a Structural Alphabet Profile Comparison Approach

Predicting accurate fragments from sequence has recently become a critical step for protein structure modeling, as protein fragment assembly techniques are presently among the most efficient approaches for de novo prediction. A key step in these approaches is, given the sequence of a protein to model, the identification of relevant fragments - candidate fragments - from a collection of the available 3D structures. These fragments can then be assembled to produce a model of the complete structure of the protein of interest. The search for candidate fragments is classically achieved by considering local sequence similarity using profile comparison, or threading approaches. In the present study, we introduce a new profile comparison approach that, instead of using amino acid profiles, is based on the use of predicted structural alphabet profiles, where structural alphabet profiles contain information related to the 3D local shapes associated with the sequences. We show that structural alphabet profile-profile comparison can be used efficiently to retrieve accurate structural fragments, and we introduce a fully new protocol for the detection of candidate fragments. It identifies fragments specific of each position of the sequence and of size varying between 6 and 27 amino-acids. We find it outperforms present state of the art approaches in terms (i) of the accuracy of the fragments identified, (ii) the rate of true positives identified, while having a high coverage score. We illustrate the relevance of the approach on complete target sets of the two previous Critical Assessment of Techniques for Protein Structure Prediction (CASP) rounds 9 and 10. A web server for the approach is freely available at http://bioserv.rpbs.univ-paris-diderot.fr/SAFrag.     

A New Approach to Bacterial Vaccines

Immunizing antigens against only 10 bacterial diseases—cholera, diphtheria, paratyphoid, pertussis, plague, scarlet fever, staphylococcal disease, tetanus, tuberculosis and typhoid—have been licensed for sale in Canada and the United States. Convincing evidence of efficacy is available for only four of these—diphtheria and tetanus toxoids, and pertussis and typhoid vaccines.The principles which determine the efficacy of different immunizing antigens are not always the same. Toxoids, for example, stimulate the formation of antitoxin-producing mechanisms which can neutralize toxins produced by invading organisms, thereby rendering them harmless. Conversely, vaccines stimulate the formation of antibacterial mechanisms which stop the growth of organisms before they can produce disease.Use of enzyme-lysed vaccines for prevention of staphylococcal disease represents a new approach in vaccine research. Animal tests have shown lysed vaccines to be 10 to 100 times less toxic, and about eight times more effective, than whole bacterial vaccines. Studies with lysed vaccines for other diseases are now in progress.     

Trypanothione Reductase: A Target Protein for a Combined In Vitro and In Silico Screening Approach

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC₅₀ values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC₅₀ values down to 2 μM.     

Selection for Unequal Densities of σ70 Promoter-Like Signals in Different Regions of Large Bacterial Genomes

The evolutionary processes operating in the DNA regions that participate in the regulation of gene expression are poorly understood. In Escherichia coli, we have established a sequence pattern that distinguishes regulatory from nonregulatory regions. The density of promoter-like sequences, that could be recognizable by RNA polymerase and may function as potential promoters, is high within regulatory regions, in contrast to coding regions and regions located between convergently transcribed genes. Moreover, functional promoter sites identified experimentally are often found in the subregions of highest density of promoter-like signals, even when individual sites with higher binding affinity for RNA polymerase exist elsewhere within the regulatory region. In order to see the generality of this pattern, we have analyzed 43 additional genomes belonging to most established bacterial phyla. Differential densities between regulatory and nonregulatory regions are detectable in most of the analyzed genomes, with the exception of those that have evolved toward extreme genome reduction. Thus, presence of this pattern follows that of genes and other genomic features that require weak selection to be effective in order to persist. On this basis, we suggest that the loss of differential densities in the reduced genomes of host-restricted pathogens and symbionts is an outcome of the process of genome degradation resulting from the decreased efficiency of purifying selection in highly structured small populations. This implies that the differential distribution of promoter-like signals between regulatory and nonregulatory regions detected in large bacterial genomes confers a significant, although small, fitness advantage. This study paves the way for further identification of the specific types of selective constraints that affect the organization of regulatory regions and the overall distribution of promoter-like signals through more detailed comparative analyses among closely related bacterial genomes.     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp fight end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to fight fragment, obtaining a “rescue” plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstⅠ digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

以犬2型腺病毒为载体进行疫苗和基因治疗的研究,是近年的研究热点.目前的焦点问题是新型载体的构建,经典的方法是体外连接,费时、费力.     

A Statistical Model of Protein Sequence Similarity and Function Similarity Reveals Overly-Specific Function Predictions

Background

Predicting protein function from primary sequence is an important open problem in modern biology. Not only are there many thousands of proteins of unknown function, current approaches for predicting function must be improved upon. One problem in particular is overly-specific function predictions which we address here with a new statistical model of the relationship between protein sequence similarity and protein function similarity.

Methodology

Our statistical model is based on sets of proteins with experimentally validated functions and numeric measures of function specificity and function similarity derived from the Gene Ontology. The model predicts the similarity of function between two proteins given their amino acid sequence similarity measured by statistics from the BLAST sequence alignment algorithm. A novel aspect of our model is that it predicts the degree of function similarity shared between two proteins over a continuous range of sequence similarity, facilitating prediction of function with an appropriate level of specificity.

Significance

Our model shows nearly exact function similarity for proteins with high sequence similarity (bit score >244.7, e-value >1e⁻⁶², non-redundant NCBI protein database (NRDB)) and only small likelihood of specific function match for proteins with low sequence similarity (bit score <54.6, e-value <1e⁻⁰⁵, NRDB). For sequence similarity ranges in between our annotation model shows an increasing relationship between function similarity and sequence similarity, but with considerable variability. We applied the model to a large set of proteins of unknown function, and predicted functions for thousands of these proteins ranging from general to very specific. We also applied the model to a data set of proteins with previously assigned, specific functions that were electronically based. We show that, on average, these prior function predictions are more specific (quite possibly overly-specific) compared to predictions from our model that is based on proteins with experimentally determined function.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Crumbs Affects Protein Dynamics In Anterior Regions Of The Developing Drosophila Embryo

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.     

Evidence For the Presence of A Cdc2-Like Protein Kinase In the Dinoflagellate Crypthecodinium Cohnii

ABSTRACT. The unusual nature of mitosis and ancestral organization of the dinoflagellate nucleus prompted the question of whether the cdc 2-like histone H1 kinase, a presumed ubiquitous cell cycle regulator in eukaryotes, is present in these primitive organisms. Western blotting of Crypthecodinium cohnii protein extracts using antibody against the Pro-Ser-Thr-Ala-Ile-Arg-Glu (=PSTAIRE) amino acid sequence motif, conserved in all cdc 2 homologues known, revealed one prominent band corresponding to a protein with an apparent relative molecular weight ≈ 34,000, identical in mobility to that from HeLa cells and Physarum polycephalum , higher and lower eukaryotic controls, respectively. Incubation of C. cohnii cell lysates with p13 ^{suc 1}-sepharose beads, which preferentially, though not exclusively, bind p34 ^{cdc 2}, resulted in precipitation of a 34-kDa protein which was reactive with anti-PSTAIRE antibody, selectively competed for by the PSTAIRE peptide and able to phosphorylate histone H1 in vitro. We conclude that the dinoflagellate C. cohnii contains a protein very similar to the cdc 2 gene product from fission yeast and its homologues in all eukaryotes studied thus far.     

A Streamlined Method for Detecting Structural Variants in Cancer Genomes by Short Read Paired-End Sequencing

Defining the architecture of a specific cancer genome, including its structural variants, is essential for understanding tumor biology, mechanisms of oncogenesis, and for designing effective personalized therapies. Short read paired-end sequencing is currently the most sensitive method for detecting somatic mutations that arise during tumor development. However, mapping structural variants using this method leads to a large number of false positive calls, mostly due to the repetitive nature of the genome and the difficulty of assigning correct mapping positions to short reads. This study describes a method to efficiently identify large tumor-specific deletions, inversions, duplications and translocations from low coverage data using SVDetect or BreakDancer software and a set of novel filtering procedures designed to reduce false positive calls. Applying our method to a spontaneous T cell lymphoma arising in a core RAG2/p53-deficient mouse, we identified 40 validated tumor-specific structural rearrangements supported by as few as 2 independent read pairs.     

A Device For Static Elimination In Ultramicrotomy

The device described here was originally developed in an attempt to reduce the effects of static electricity during cryo-ultramicrotomy with a dry knife. It has, however, found considerable application in conventional ultramicrotomy.     

A Multidimensional Approach to Protein Characterization

When mass spectrometry (MS) is used to study protein primary structure, it is used in a static mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS. If a series of mass spectra is collected as something else in the experiment is changing, we increase the dimensionality of the MS data. For example, measuring mass spectra as a function of time after exposure of a protein to deuterated solvents can provide information about protein structure. Likewise, by measuring mass spectra of a protein as the concentration of a binding ligand is changed, one can infer the stoichiometry of the complex. Another important, but fundamentally different way of increasing the dimensionality of mass spectral data is by coupling the mass spectrometer to a one- or two-dimensional separation technique.