Polyglutamine Induced Misfolding of Huntingtin Exon1 is Modulated by the Flanking Sequences

Polyglutamine (polyQ) expansion in exon1 (XN1) of the huntingtin protein is linked to Huntington's disease. When the number of glutamines exceeds a threshold of approximately 36–40 repeats, XN1 can readily form amyloid aggregates similar to those associated with disease. Many experiments suggest that misfolding of monomeric XN1 plays an important role in the length-dependent aggregation. Elucidating the misfolding of a XN1 monomer can help determine the molecular mechanism of XN1 aggregation and potentially help develop strategies to inhibit XN1 aggregation. The flanking sequences surrounding the polyQ region can play a critical role in determining the structural rearrangement and aggregation mechanism of XN1. Few experiments have studied XN1 in its entirety, with all flanking regions. To obtain structural insights into the misfolding of XN1 toward amyloid aggregation, we perform molecular dynamics simulations on monomeric XN1 with full flanking regions, a variant missing the polyproline regions, which are hypothesized to prevent aggregation, and an isolated polyQ peptide (Qn). For each of these three constructs, we study glutamine repeat lengths of 23, 36, 40 and 47. We find that polyQ peptides have a positive correlation between their probability to form a β-rich misfolded state and their expansion length. We also find that the flanking regions of XN1 affect its probability to^x_page_count=28 form a β-rich state compared to the isolated polyQ. Particularly, the polyproline regions form polyproline type II helices and decrease the probability of the polyQ region to form a β-rich state. Additionally, by lengthening polyQ, the first N-terminal 17 residues are more likely to adopt a β-sheet conformation rather than an α-helix conformation. Therefore, our molecular dynamics study provides a structural insight of XN1 misfolding and elucidates the possible role of the flanking sequences in XN1 aggregation

BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.)

Background: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with 250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.Daniela Schulte, Ruvini Ariyadasa, Bujun Shi, Delphine Fleury, Chris Saski, Michael Atkins, Pieter deJong, Cheng-Cang Wu, Andreas Graner, Peter Langridge and Nils Stei

A Naturally Occurring Polymorphism at Drosophila melanogaster Lim3 Locus, a Homolog of Human LHX3/4, Affects Lim3 Transcription and Fly Lifespan

Lim3 encodes an RNA polymerase II transcription factor with a key role in neuron specification. It was also identified as a candidate gene that affects lifespan. These pleiotropic effects indicate the fundamental significance of the potential interplay between neural development and lifespan control. The goal of this study was to analyze the causal relationships between Lim3 structural variations, and gene expression and lifespan changes, and to provide insights into regulatory pathways controlling lifespan. Fifty substitution lines containing second chromosomes from a Drosophila natural population were used to analyze the association between lifespan and sequence variation in the 5′-regulatory region, and first exon and intron of Lim3A, in which we discovered multiple transcription start sites (TSS). The core and proximal promoter organization for Lim3A and a previously unknown mRNA named Lim3C were described. A haplotype of two markers in the Lim3A regulatory region was significantly associated with variation in lifespan. We propose that polymorphisms in the regulatory region affect gene transcription, and consequently lifespan. Indeed, five polymorphic markers located within 380 to 680 bp of the Lim3A major TSS, including two markers associated with lifespan variation, were significantly associated with the level of Lim3A transcript, as evaluated by real time RT-PCR in embryos, adult heads, and testes. A naturally occurring polymorphism caused a six-fold change in gene transcription and a 25% change in lifespan. Markers associated with long lifespan and intermediate Lim3A transcription were present in the population at high frequencies. We hypothesize that polymorphic markers associated with Lim3A expression are located within the binding sites for proteins that regulate gene function, and provide general rather than tissue-specific regulation of transcription, and that intermediate levels of Lim3A expression confer a selective advantage and longer lifespan

Supported housing programs for persons with serious mental illness in rural northern communities: A mixed method evaluation

Background: During the past two decades, consumers, providers and policy makers have recognized the role of supported housing intervention for persons diagnosed with serious mental illness (SMI) to be able to live independently in the community. Much of supported housing research to date, however, has been conducted in large urban centers rather than northern and rural communities. Northern conditional and contextual issues such as rural poverty, lack of accessible mental health services, small or non-existing housing markets, lack of a continuum of support or housing services, and in some communities, a poor quality of housing challenge the viability of effective supported housing services. The current research proposal aims to describe and evaluate the processes and outcomes of supported housing programs for persons living with SMI in northern and rural communities from the perspective of clients, their families, and community providers. Methods: This research will use a mixed method design guided by participatory action research. The study will be conducted over two years, in four stages. Stage I will involve setting up the research in each of the four northern sites. In Stage II a descriptive cross-sectional survey will be used to obtain information about the three client outcomes: housing history, quality of life and housing preference. In Stage III two participatory action strategies, focus groups and photo-voice, will be used to explore perceptions of supported housing services. In the last stage findings from the study will be re-presented to the participants, as well as other key community individuals in order to translate them into policy. Conclusion: Supported housing intervention is a core feature of mental health care, and it requires evaluation. The lack of research in northern and rural SMI populations heightens the relevance of research findings for health service planning. The inclusion of multiple stakeholder groups, using a variety of data collection approaches, contributes to a comprehensive, systems-level examination of supported housing in smaller communities. It is anticipated that the study\u27s findings will not only have utility across Ontario, but also Canada

Defects in tRNA Modification Associated with Neurological and Developmental Dysfunctions in Caenorhabditis elegans Elongator Mutants

Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5′methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development

Revisiting the Myths of Protein Interior: Studying Proteins with Mass-Fractal Hydrophobicity-Fractal and Polarizability-Fractal Dimensions

A robust marker to describe mass, hydrophobicity and polarizability distribution holds the key to deciphering structural and folding constraints within proteins. Since each of these distributions is inhomogeneous in nature, the construct should be sensitive in describing the patterns therein. We show, for the first time, that the hydrophobicity and polarizability distributions in protein interior follow fractal scaling. It is found that (barring ‘all-α’) all the major structural classes of proteins have an amount of unused hydrophobicity left in them. This amount of untapped hydrophobicity is observed to be greater in thermophilic proteins, than that in their (structurally aligned) mesophilic counterparts. ‘All-β’(thermophilic, mesophilic alike) proteins are found to have maximum amount of unused hydrophobicity, while ‘all-α’ proteins have been found to have minimum polarizability. A non-trivial dependency is observed between dielectric constant and hydrophobicity distributions within (α+β) and ‘all-α’ proteins, whereas absolutely no dependency is found between them in the ‘all-β’ class. This study proves that proteins are not as optimally packed as they are supposed to be. It is also proved that origin of α-helices are possibly not hydrophobic but electrostatic; whereas β-sheets are predominantly hydrophobic in nature. Significance of this study lies in protein engineering studies; because it quantifies the extent of packing that ensures protein functionality. It shows that myths regarding protein interior organization might obfuscate our knowledge of actual reality. However, if the later is studied with a robust marker of strong mathematical basis, unknown correlations can still be unearthed; which help us to understand the nature of hydrophobicity, causality behind protein folding, and the importance of anisotropic electrostatics in stabilizing a highly complex structure named ‘proteins’

Complete Genome Sequence of Crohn's Disease-Associated Adherent-Invasive E. coli Strain LF82

International audienceBACKGROUND: Ileal lesions of Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages. PRINCIPAL FINDINGS: We report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohn's disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome. CONCLUSION: LF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82