Modeling RNA tertiary structure motifs by graph-grammars

A new approach, graph-grammars, to encode RNA tertiary structure patterns is introduced and exemplified with the classical sarcin–ricin motif. The sarcin–ricin motif is found in the stem of the crucial ribosomal loop E (also referred to as the sarcin–ricin loop), which is sensitive to the α-sarcin and ricin toxins. Here, we generate a graph-grammar for the sarcin-ricin motif and apply it to derive putative sequences that would fold in this motif. The biological relevance of the derived sequences is confirmed by a comparison with those found in known sarcin–ricin sites in an alignment of over 800 bacterial 23S ribosomal RNAs. The comparison raised alternative alignments in few sarcin–ricin sites, which were assessed using tertiary structure predictions and 3D modeling. The sarcin–ricin motif graph-grammar was built with indivisible nucleotide interaction cycles that were recently observed in structured RNAs. A comparison of the sequences and 3D structures of each cycle that constitute the sarcin–ricin motif gave us additional insights about RNA sequence–structure relationships. In particular, this analysis revealed the sequence space of an RNA motif depends on a structural context that goes beyond the single base pairing and base-stacking interactions

Vers une gestion intégrée de l'eau: portrait et diagnostic du bassin versant de la rivière Gatineau

Le présent document comporte la première partie du futur Plan directeur de l’eau du bassin versant de la rivière Gatineau, soit l’analyse du bassin versant. Cette tâche a été abordée sous deux volets : la description des différentes composantes des milieux naturels et humain et l’établissement d’un diagnostic caractérisant les sources de pollution et dressant un bilan quantitatif et qualitatif des ressources en eau du bassin versant. Globalement, le vaste territoire du bassin versant de la rivière Gatineau présente une nature riche et variée. La population qui y habite est concentrée dans le sud du bassin versant. Les activités économiques s’y déroulant sont principalement le secteur des services, la foresterie et le récréotourisme, un peu d’agriculture et de production hydroélectrique. Les principales pressions sur le milieu aquatique proviennent d’abord de la forte villégiature, ensuite, des activités domestiques et de gestion municipale, et enfin des activités forestières et de l’agriculture. Les principales répercussions observées sont l’enrichissement et l’ensablement des plans d’eau, des contaminations microbiennes et toxiques locales des eaux de surface et des eaux souterraines

Osteopontin: an early innate immune marker of Escherichia coli mastitis harbors genetic polymorphisms with possible links with resistance to mastitis

<p>Abstract</p> <p>Background</p> <p>Mastitis is the most important disease in dairy cows and it causes significant lost of profit to producers. Identification of the genes, and their variants, involved in innate immune responses is essential for the understanding of this inflammatory disease and to identify potential genetic markers for resistance to mastitis. The progeny of dairy cows would benefit from receiving favourable alleles that support greater resistance to infection, thus reducing antibiotic use. This study aims to identify a key gene in the innate immune response to mastitis, led us to evaluate its genetic association with somatic cell score (SCS), which is an indicator of clinical mastitis, and to evaluate its impact on other traits related to milk production.</p> <p>Results</p> <p>The osteopontin transcript (<it>SPP1</it>) was identified in the somatic cells from cows experimentally infected with <it>Escherichia coli</it>. By selecting bulls with extreme estimated breeding values (EBVs) for SCS, which is an indicator of mammary gland health, four DNA polymorphisms in the <it>SPP1 </it>genomic sequence were found. Statistical analysis revealed that the SNP <it>SPP1c.-1301G>A </it>has an impact on EBV for SCS (<it>P </it>< 0.001) Using an allele substitution model, <it>SPP1c.-1251C>T</it>, <it>SPP1c.-430G>A</it>, and <it>SPP1c.*40A>C </it>have an impact on SCS whereas <it>SPP1c.-1301G>A </it>has an effect on the EBVs for milk yield (second and third lactations), fat and protein percentages (all three lactations). Analysis revealed statistically significant differences between haplotype groups at a comparison-wise level with sire EBVS for SCS for the first (<it>P </it>= 0.012), second (<it>P </it>< 0.001), and third (<it>P </it>< 0.001) lactations.</p> <p>Conclusion</p> <p>This study reports the link between DNA polymorphisms of <it>SPP1</it>, the number of milk immune cells and, potentially, the susceptibility to mastitis. These SNPs were identified by <it>in silico </it>search to be located in transcription factor recognition sites which factors are presumably involved in the Th1 immune response and in the Th2 regulation pathway. Indeed, one SNP abolished the SP1 recognition site, whereas another SNP affected the transcription binding factor IKAROS. All together, these findings support the genetic potential of these variants in terms of selection for the improvement of mastitis resistance in dairy cows.</p

Head-to-tail cyclization of side chain-protected linear peptides to recapitulate genetically-encoded cyclized peptides

Genetically‐encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution‐phase macrocyclization of side chain‐protected linear peptides obtained from standard solid‐phase peptide synthesis. Cyclic peptide targets, including cyclo‐[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically‐encoded counterparts. Synthetic products were characterized by tandem high‐resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation‐induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost‐effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells

Inhibition of the Integrin/FAK Signaling Axis and c-Myc Synergistically Disrupts Ovarian Cancer Malignancy

Integrins, a family of heterodimeric receptors for extracellular matrix, are promising therapeutic targets for ovarian cancer, particularly high-grade serous-type (HGSOC), as they drive tumor cell attachment, migration, proliferation and survival by activating focal adhesion kinase (FAK)-dependent signaling. Owing to the potential off-target effects of FAK inhibitors, disruption of the integrin signaling axis remains to be a challenge. Here, we tackled this barrier by screening for inhibitors being functionally cooperative with small-molecule VS-6063, a phase II FAK inhibitor. From this screening, JQ1, a potent inhibitor of Myc oncogenic network, emerged as the most robust collaborator. Treatment with a combination of VS-6063 and JQ1 synergistically caused an arrest of tumor cells at the G2/M phase and a decrease in the XIAP-linked cell survival. Our subsequent mechanistic analyses indicate that this functional cooperation was strongly associated with the concomitant disruption of activation or expression of FAK and c-Myc as well as their downstream signaling through the PI3K/Akt pathway. In line with these observations, we detected a strong co-amplification or upregulation at genomic or protein level for FAK and c-Myc in a large portion of primary tumors in the TCGA or a local HGSOC patient cohort. Taken together, our results suggest that the integrin–FAK signaling axis and c-Myc synergistically drive cell proliferation, survival and oncogenic potential in HGSOC. As such, our study provides key genetic, functional and signaling bases for the small-molecule-based co-targeting of these two distinct oncogenic drivers as a new line of targeted therapy against human ovarian cancer

CF2 Represses Actin 88F Gene Expression and Maintains Filament Balance during Indirect Flight Muscle Development in Drosophila

The zinc finger protein CF2 is a characterized activator of muscle structural genes in the body wall muscles of the Drosophila larva. To investigate the function of CF2 in the indirect flight muscle (IFM), we examined the phenotypes of flies bearing five homozygous viable mutations. The gross structure of the IFM was not affected, but the stronger hypomorphic alleles caused an increase of up to 1.5X in the diameter of the myofibrils. This size increase did not cause any disruption of the hexameric arrangement of thick and thin filaments. RT-PCR analysis revealed an increase in the transcription of several structural genes. Ectopic overexpression of CF2 in the developing IFM disrupts muscle formation. While our results indicate a role for CF2 as a direct negative regulator of the thin filament protein gene Actin 88F (Act88F), effects on levels of transcripts of myosin heavy chain (mhc) appear to be indirect. This role is in direct contrast to that described in the larval muscles, where CF2 activates structural gene expression. The variation in myofibril phenotypes of CF2 mutants suggest the CF2 may have separate functions in fine-tuning expression of structural genes to insure proper filament stoichiometry, and monitoring and/or controlling the final myofibril size

Modulation of Drosophila Retinal Epithelial Integrity by the Adhesion Proteins Capricious and Tartan

Background The development of the Drosophila eye imaginal disc requires complex epithelial rearrangements. Cells of the morphogenetic furrow are apically constricted and this leads to a physical indentation in the epithelium. Posterior to the furrow, cells start to rearrange into distinct clusters and eventually form a precisely patterned array of ommatidia. These morphogenetic processes include regulated changes of adhesion between cells. Methodology/Principal Findings Here, we show that two transmembrane adhesion proteins, Capricious and Tartan, have dynamic and complementary expression patterns in the eye imaginal disc. We also describe novel null mutations in capricious and double null mutations in capricious and tartan. We report that they have redundant functions in regulating the architecture of the morphogenetic furrow and ommatidial spacing. Conclusions/Significance We conclude that Capricious and Tartan contribute to the adhesive properties of the cells in the morphogenetic furrow and that this regulated adhesion participates in the control of spacing ommatidial clusters

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4