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

Genome characterization and population genetic structure of the zoonotic pathogen, streptococcus canis

Background - Streptococcus canis is an important opportunistic pathogen of dogs and cats that can also infect a wide range of additional mammals including cows where it can cause mastitis. It is also an emerging human pathogen. Results - Here we provide characterization of the first genome sequence for this species, strain FSL S3-227 (milk isolate from a cow with an intra-mammary infection). A diverse array of putative virulence factors was encoded by the S. canis FSL S3-227 genome. Approximately 75% of these gene sequences were homologous to known Streptococcal virulence factors involved in invasion, evasion, and colonization. Present in the genome are multiple potentially mobile genetic elements (MGEs) [plasmid, phage, integrative conjugative element (ICE)] and comparison to other species provided convincing evidence for lateral gene transfer (LGT) between S. canis and two additional bovine mastitis causing pathogens (Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. dysgalactiae), with this transfer possibly contributing to host adaptation. Population structure among isolates obtained from Europe and USA [bovine = 56, canine = 26, and feline = 1] was explored. Ribotyping of all isolates and multi locus sequence typing (MLST) of a subset of the isolates (n = 45) detected significant differentiation between bovine and canine isolates (Fisher exact test: P = 0.0000 [ribotypes], P = 0.0030 [sequence types]), suggesting possible host adaptation of some genotypes. Concurrently, the ancestral clonal complex (54% of isolates) occurred in many tissue types, all hosts, and all geographic locations suggesting the possibility of a wide and diverse niche. Conclusion - This study provides evidence highlighting the importance of LGT in the evolution of the bacteria S. canis, specifically, its possible role in host adaptation and acquisition of virulence factors. Furthermore, recent LGT detected between S. canis and human bacteria (Streptococcus urinalis) is cause for concern, as it highlights the possibility for continued acquisition of human virulence factors for this emerging zoonotic pathogen

Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Sharing Mitochondrial Clients

The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.Comment: This is a reformatted version from the recent official publication in PLoS ONE (2015). This version differs substantially from first three arXiV versions. This version uses a fixed-width font for DNA sequences as was done in the earlier arXiv versions but which is missing in the official PLoS ONE publication. The title has also been shortened slightly from the official publicatio

Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality

Innate lymphoid cells (ILCs) play key roles in host defense, barrier integrity, and homeostasis and mirror adaptive CD4(+) T helper (Th) cell subtypes in both usage of effector molecules and transcription factors. To better understand the relationship between ILC subsets and their Th cell counterparts, we measured genome-wide chromatin accessibility. We find that chromatin in proximity to effector genes is selectively accessible in ILCs prior to high-level transcription upon activation. Accessibility of these regions is acquired in a stepwise manner during development and changes little after in vitro or in vivo activation. Conversely, dramatic chromatin remodeling occurs in naive CD4(+) T cells during Th cell differentiation using a type-2-infection model. This alteration results in a substantial convergence of Th2 cells toward ILC2 regulomes. Our data indicate extensive sharing of regulatory circuitry across the innate and adaptive compartments of the immune system, in spite of their divergent developing pathways

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

Nonribosomal peptide synthetases : quaternary structure and chemoenzymatic synthesis of macrocyclic peptides

Quaternary structure and chemoenzymatic synthesis of macrocyclic peptides

Multicellular Phenotypic Studies of Single Gene Variants in Myxococcus xanthus

There are several systematic methods designed to link genes to cellular processes. These methods are derived from different hypotheses and are largely complementary to each other. This dissertation presents a systematic study of functional genetics and related phenotypes using quantitative methods. The first part of this dissertation will report the successful identification and characterization of 28 genes in the multicellular bacterium Myxococcus xanthus using three different methods: sequence homology, transcription activation and protemoics. The results from this research extended the list of M. xanthus genes involved in multicellularity, and expanded our knowledge regarding the possible molecular pathways underlying physiological and morphological changes. Although the cellular function of some of the genes in the genome of an organism can be deduced from effects of mutation on phenotype, the disruption or deletion of most genes produces little or no discernible phenotypic impact. The reason for this may be redundancy or complementation, or it may be due to the limitations inherent in available assays. The second part of this dissertation will focus on a population genetics approach to the characterization of phenotype for a collection of mutant strains containing insertion mutations in each of the ~200 ABC transporter component genes in M. xanthus. More than 50% of those mutant strains exhibit at least one phenotypic characteristic that is different from the wild type, and an average of 6% of mutant strains have a gain-of-function phenotype. We also demonstrated that the morphological features used to measure phenotype are not entirely independent variables. These results indicate that a rigorous and quantitative phenotypic characterization will provide significantly more data to understand the phenotypic space of M. xanthus, and that a more rigorous definition of phenotype may help us establish a more accurate connection between genotype and phenotype

Silencing parasitism effectors of the root lesion nematode, Pratylenchus thornei

The root lesion nematode (RLN), Pratylenchus thornei, is a biotrophic migratory pest of plant roots and its infestation causes losses in many economically important crops. RNA interference (RNAi) is a naturally occurring eukaryotic phenomenon and can be used to silence parasitism effector genes of P. thornei using host-mediated RNAi. This may be developed as an environmentally friendly and a cost-effective control strategy. The overall aims of this research were to investigate the effects of in vitro and in planta RNAi silencing of putative P. thornei parasitism effector genes, and their nematicidal effects in two host plants. Five putative target parasitism genes vital for nematode entry into roots (Pt-Eng-1, Pt-PL), feeding (Pt-CLP) and suppressing host defence responses (Pt-UEP, Pt-GST) were identified, validated in silico using comparative bioinformatics, cloned into suitable in vitro transcription and binary vectors, and advanced to RNAi studies. Partial sequences for four of these target effector genes (Pt-Eng-1, Pt-PL, Pt-CLP, Pt-GST) were identified using Rapid Amplification of cDNA (RACE) PCRs and annotated in silico. Protein families, conserved domains, taxonomic and phylogenetic relationships for all four effectors were studied. This sequence information will help inform future investigations involving gene expression and proteomics of the selected putative effectors. In vitro RNAi was used for functional characterisation of the five effector sequences. Effects on nematode phenotype, behaviour, gene expression, and longer-term effects on reproduction were assessed after soaking nematodes in dsRNA through infection of healthy wild type soybean and alfalfa roots. Soaking of mixed stage P. thornei in 1mg/mL dsRNA of target genes for 16 h did not cause phenotypic changes except for Pt-PL, which exhibited straight or slightly curved phenotypes after soaking compared to the normal sigmoid body movement, also evident for green fluorescent protein (gfp) and no dsRNA treated controls. Semi-quantitative PCRs and densitometry analysis revealed a significant reduction of transcript accumulation for all five putative parasitism effector genes. Longer-term effects assessed at 21 dpi reduced nematode reproduction by 40 to 70% for all target genes compared to respective control treatments suggesting that the effectors studied were required for nematode infectivity, survival or reproduction. In planta RNAi involved Agrobacterium-mediated plant transformations to develop axenic transgenic hairy root events of soybean (Glycine max var. Williams 82) and alfalfa (Medicago sativa), and non-axenic hairy roots (composite plants) of soybean. Both hosts were amenable to Agrobacterium-mediated transformation, but hairy root induction was faster in alfalfa than soybean. However, more events were generated for soybean than alfalfa. Transgenic hairy roots confirmed by molecular analyses were challenged with P. thornei and their presence confirmed after 14 dpi. After 21 dpi, nematode numbers and transcript abundance was assessed using semi-quantitative PCRs and densitometry analysis. Host-mediated silencing of the five putative parasitism effector genes using transgenic soybean and alfalfa hairy roots showed a significant reduction in target transcript accumulation and approximately 38 to 75% reduction in P. thornei numbers compared to untransformed wild-type controls. For some events, there was a positive correlation between reduced transcripts and nematode numbers. Based on percent reduction in transcript accumulation of the target genes relative to 18S rRNA as assessed by densitometry, the extent of gene knockdown measured (from most to least) was: Pt-Eng-1, Pt-PL, Pt-CLP, Pt-UEP, and Pt-GST. Similarly, Pt-Eng-1, Pt-PL and Pt-CLP were ranked in the same order, from the lowest to highest reproduction on soybean and alfalfa, indicating a positive correlation between the level of knockdown and reduced reproduction. In soybean, these genes were followed by Pt-GST and Pt-UEP for the percentage of reproduction recorded, whereas, in alfalfa, reduction in reproduction for these two target genes did not differ significantly. Composite soybean with wild-type shoots and transgenic hairy roots expressing Pt-Eng-1 and Pt-PL genes were developed and provided an opportunity to test the effectiveness of silencing target genes in planta and on nematode numbers in conditions that mimicked natural host infections. For both Pt-Eng-1 and Pt-PL genes, there was a significant reduction in percentage of transcript accumulation relative to 18S rRNA, which correlated with a reduction in nematode numbers by 53.4% and 48.5% for Pt-Eng-1 and Pt-PL, respectively. The amenability of P. thornei to host-mediated RNAi using effector gene sequences, and the overall results of this study, point towards the potential use of this technology to control P. thornei and related RLN species effectively in different host crops

YeATS - a tool suite for analyzing RNA-seq derived transcriptome identifies a highly transcribed putative extensin in heartwood/sapwood transition zone in black walnut [version 2; referees: 3 approved]

The transcriptome provides a functional footprint of the genome by enumerating the molecular components of cells and tissues. The field of transcript discovery has been revolutionized through high-throughput mRNA sequencing (RNA-seq). Here, we present a methodology that replicates and improves existing methodologies, and implements a workflow for error estimation and correction followed by genome annotation and transcript abundance estimation for RNA-seq derived transcriptome sequences (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). A unique feature of YeATS is the upfront determination of the errors in the sequencing or transcript assembly process by analyzing open reading frames of transcripts. YeATS identifies transcripts that have not been merged, result in broken open reading frames or contain long repeats as erroneous transcripts. We present the YeATS workflow using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut. A novel feature of the transcriptome that emerged from our analysis was the identification of a highly abundant transcript that had no known homologous genes (GenBank accession: KT023102). The amino acid composition of the longest open reading frame of this gene classifies this as a putative extensin. Also, we corroborated the transcriptional abundance of proline-rich proteins, dehydrins, senescence-associated proteins, and the DNAJ family of chaperone proteins. Thus, YeATS presents a workflow for analyzing RNA-seq data with several innovative features that differentiate it from existing software