CLP-based protein fragment assembly

The paper investigates a novel approach, based on Constraint Logic Programming (CLP), to predict the 3D conformation of a protein via fragments assembly. The fragments are extracted by a preprocessor-also developed for this work- from a database of known protein structures that clusters and classifies the fragments according to similarity and frequency. The problem of assembling fragments into a complete conformation is mapped to a constraint solving problem and solved using CLP. The constraint-based model uses a medium discretization degree Ca-side chain centroid protein model that offers efficiency and a good approximation for space filling. The approach adapts existing energy models to the protein representation used and applies a large neighboring search strategy. The results shows the feasibility and efficiency of the method. The declarative nature of the solution allows to include future extensions, e.g., different size fragments for better accuracy.Comment: special issue dedicated to ICLP 201

A Constraint Solver for Flexible Protein Models

This paper proposes the formalization and implementation of a novel class of constraints aimed at modeling problems related to placement of multi-body systems in the 3-dimensional space. Each multi-body is a system composed of body elements, connected by joint relationships and constrained by geometric properties. The emphasis of this investigation is the use of multi-body systems to model native conformations of protein structures---where each body represents an entity of the protein (e.g., an amino acid, a small peptide) and the geometric constraints are related to the spatial properties of the composing atoms. The paper explores the use of the proposed class of constraints to support a variety of different structural analysis of proteins, such as loop modeling and structure prediction. The declarative nature of a constraint-based encoding provides elaboration tolerance and the ability to make use of any additional knowledge in the analysis studies. The filtering capabilities of the proposed constraints also allow to control the number of representative solutions that are withdrawn from the conformational space of the protein, by means of criteria driven by uniform distribution sampling principles. In this scenario it is possible to select the desired degree of precision and/or number of solutions. The filtering component automatically excludes configurations that violate the spatial and geometric properties of the composing multi-body system. The paper illustrates the implementation of a constraint solver based on the multi-body perspective and its empirical evaluation on protein structure analysis problems

Novel design and controls for focused DNA microarrays: applications in quality assurance/control and normalization for the Health Canada ToxArray™

BACKGROUND: Microarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes. These normalization approaches are not appropriate for focused arrays with small sets of genes where a large portion may be expected to change. Furthermore, many microarrays lack control features that can be used for quality assurance (QA). Here, we describe a novel external control series integrated with a design feature that addresses the above issues. RESULTS: An EC dilution series that involves spike-in of a single concentration of the A. thaliana chlorophyll synthase gene to hybridize against spotted dilutions (0.000015 to 100 μM) of a single complimentary oligonucleotide representing the gene was developed. The EC series is printed in duplicate within each subgrid of the microarray and covers the full range of signal intensities from background to saturation. The design and placement of the series allows for QA examination of frequently encountered problems in hybridization (e.g., uneven hybridizations) and printing (e.g., cross-spot contamination). Additionally, we demonstrate that the series can be integrated with a LOWESS normalization to improve the detection of differential gene expression (improved sensitivity and predictivity) over LOWESS normalization on its own. CONCLUSION: The quality of microarray experiments and the normalization methods used affect the ability to measure accurate changes in gene expression. Novel methods are required for normalization of small focused microarrays, and for incorporating measures of performance and quality. We demonstrate that dilution of oligonucleotides on the microarray itself provides an innovative approach allowing the full dynamic range of the scanner to be covered with a single gene spike-in. The dilution series can be used in a composite normalization to improve detection of differential gene expression and to provide quality control measures

Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

Background: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion: While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.

LOGIC AND CONSTRAINT PROGRAMMING FOR COMPUTATIONAL SUSTAINABILITY

Computational Sustainability is an interdisciplinary field that aims to develop computational and mathematical models and methods for decision making concerning the management and allocation of resources in order to help solve environmental problems. This thesis deals with a broad spectrum of such problems (energy efficiency, water management, limiting greenhouse gas emissions and fuel consumption) giving a contribution towards their solution by means of Logic Programming (LP) and Constraint Programming (CP), declarative paradigms from Artificial Intelligence of proven solidity. The problems described in this thesis were proposed by experts of the respective domains and tested on the real data instances they provided. The results are encouraging and show the aptness of the chosen methodologies and approaches. The overall aim of this work is twofold: both to address real world problems in order to achieve practical results and to get, from the application of LP and CP technologies to complex scenarios, feedback and directions useful for their improvement

High throughput prediction of inter-protein coevolution

Inter-protein co-evolution analysis can reveal in/direct functional or physical protein interactions. Inter-protein co-evolutionary analysis compares the correlation of evolutionary changes between residues on aligned orthologous sequences. On the other hand, modern methods used in experimental cell biological research to screen for protein-protein interaction, often based on mass spectrometry, often lead to identification of large amount of possible interacting proteins. If automatized, inter-protein co-evolution analysis can serve as a valuable step in refining the results, typically containing hundreds of hits, for further experiments. Manual retrieval of tens of orthologous sequences, alignment and phylogenetic tree preparations of such amounts of data is insufficient. The aim of this thesis is to create an assembly of scripts that automatize high-throughput inter-protein co-evolution analysis. Scripts were written in Python language. Scripts are using API client interface to access online databases with sequences of input protein identifiers. Through matched identifiers, over 85 representative orthologous sequences from vertebrate species are retrieved from OrthoDB orthologues database. Scripts align these sequences with PRANK MSA algorithm and create corresponding phylogenetic tree. All protein pairs are structured for multicore computation with CAPS programme on CSC supercomputer. Multiple CAPS outputs are abstracted into comprehensive form for comparison of relative co-adaptive co-evolution between proposed protein pairs. In this work, I have developed automatization for a protein-interactome screen done by proximity labelling of B cell receptor and plasma membrane associated proteins under activating or non-activating conditions. Applying high-throughput co-evolutionary analysis to this data provides a completely new approach to identify new players in B cell activation, critical for autoimmunity, hypo-immunity or cancer. Results showed unsatisfying performance of CAPS, explanation and alternatives were given

Discovery and characterization of heterogeneous and multipotent fibroblast populations isolated from excised cleft lip tissue.

BACKGROUND Regularly discarded lip tissue obtained from corrective surgeries to close the cleft lip represents an easily accessible and rich source for the isolation of primary fibroblasts. Primary fibroblasts have been described to show compelling similarities to mesenchymal stem cells (MSCs). Hence, cleft lip and palate (CLP) lip-derived fibroblasts could be thought as an intriguing cell source for personalized regenerative therapies in CLP-affected patients. METHODS Initially, we thoroughly characterized the fibroblastic nature of the lip-derived mesenchymal outgrowths by molecular and functional assays. Next, we compared their phenotype and genotype to that of bone marrow-mesenchymal stem cells (BM-MSCs) and of human lung-derived fibroblasts WI38, by assessing their morphology, surface marker expression, trilineage differentiation potential, colony-forming (CFU) capacity, and immunomodulation property. Finally, to better decipher the heterogeneity of our CLP cultures, we performed a single cell clonal analysis and tested expanded clones for surface marker expression, as well as osteogenic and CFU potential. RESULTS We identified intriguingly similar phenotypic and genotypic properties between CLP lip fibroblasts and BM-MSCs, which makes them distinct from WI38. Furthermore, our own data in combination with the complex anatomy of the lip tissue indicated heterogeneity in our CLP cultures. Using a clonal analysis, we discovered single cell-derived clones with increased levels of the MSC markers CD106 and CD146 and clones with variabilities in their commitment to differentiate into bone-forming cells and in their potential to form single cell-derived colonies. However, we were not able to gain clones possessing superior MSC-like capacities when compared to the heterogeneous parental CLP population. Additionally, all clones could still generate contractile forces and retained robust levels of the fibroblast specific marker FSP1, which was not detectable in BM-MSCs. CONCLUSIONS Our results suggest that we isolate heterogeneous populations of fibroblasts from discarded CLP lip tissue, which show a prominently multipotent character in their entirety avoiding the need for elaborate subpopulation selections in vitro. These findings suggest that CLP lip fibroblasts might be a novel potential cell source for personalized regenerative medicine of clinical benefit for CLP patients

Cost Function Networks to Solve Large Computational Protein Design Problems

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Bioinformatic and Proteomic Investigation of Chloroplast Transit Peptide Motifs and Genesis

The eukaryotic mitochondrion was formed by the endosymbiotic association of an - proteobacterium and a primordial phagocytic eukaryote. A second, and later, endosymbiosis between the eukaryote and a cyanobacterium gave rise to the chloroplast of plants. Following each of these events most of the organellar DNA was exported to the nucleus. A system evolved wherein proteins produced on cytosolic ribosomes are targeted to organelle protein translocators by N-terminal targeting sequences. Protein sorting between the chloroplast and the mitochondrion in the plant cell by the general import pathways shows remarkable fidelity despite a lack of sequence conservation among transit peptides and pre-sequences and despite very little sequence difference between these two targeting peptides. There is evidence for a hydrophobic recognition motif in mitochondrial presequences, and a similar motif has been proposed for the chloroplast transit peptide. We have developed novel motif-finding methods and applied them to our own chloroplast proteome data and to literature mitochondrial data. We fail to find a hydrophobic motif that discriminates the chloroplast and the mitochondrion. Another little understood phenomenon of organelle protein trafficking is how the targeting sequence is acquired after transfer of organelle DNA to the nucleus. It has been hypothesized that the transit peptide is acquired by exon shuffling. We find no correlation of transit peptide lengths with exon boundaries. Furthermore, using highly expressed cyanobacterial proteins conserved in plants, we find that the transit peptide appears as likely to be attached within the primordial sequence as without, indicating a more stochastic process for the origin of the transit peptide

The Identification of RCY339 as a K.lactic cdc14 Mutant

In budding yeast there exist regulators that control the steps in the process of forming the new daughter cell. Some of these regulators are called the mitotic exit network (MEN), which help the cell go from mitosis to G1. We have been studying cell cycle control in the budding yeast K.lactis. Our K.lactis temperature sensitive mutant, RCY339, arrests after anaphase, which is suggested by its large bud, segregated DNA, and elongated spindle at the non-permissive temperature. A candidate gene altered in our mutant is CDC14, the last gene in the MEN pathway. A wild type copy of the K.lactis CDC14 gene complements the temperature sensitive defect in RCY339. However, it remained to be determined whether suppression was occurring, or if CDC14 was indeed mutated. In this study, classical genetic tests were used to confirm whether or not CDC14 contains the mutation that has caused RCY339 to arrest after anaphase. Results from these tests verified that the ts mutation in RCY339 is linked to the CDC 14 locus. The cdc14 allele of RCY339 was sequenced, to show that a mutation indeed existed, causing an amino acid change from a conserved proline to a serine in the encoded protein. Due to similarities observed in the cdc 14 mutant in K.lactis and S.cerevisiae, a S. cerevisiae cdc14 mutant was transformed with a wild type copy of CDC14 from K.lactis. Our transformation was no longer temperature sensitive, leading us to believe that CDC14 proteins from S.cerevisiae and K.lactis are functionally equivalent