The cyclotides and related macrocyclic peptides as scaffolds in drug design

The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Cyclotides are macrocyclic cystine-knotted mini-proteins that have a broad range of bioactivities and are exceptionally stable, being resistant to chemical, thermal and enzymatic degradation. The general limitations of peptides as drugs can potentially be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides and related peptide scaffolds for drug design is evaluated herein, with reference to increasing knowledge of the structures and sequence diversity of natural cyclotides and the emergence of new approaches in protein engineering.

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin, hnRNP A2 bound A2RE in the latter site with a K-d near 50 nM, whereas the K-d for hnRNP A1 was above 10 muM. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 muM for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.

Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2 alpha may signal skeletal muscle atrophy in weight-losing cancer patients

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the alpha-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2alpha have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2alpha were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2alpha (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2alpha. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2alpha (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients.

Attenuation of skeletal muscle atrophy in cancer cachexia by d-myo-inositol 1,2,6-triphosphate

PURPOSE: To determine the effectiveness of the polyanionic, metal binding agent D-myo-inositol-1,2,6-triphosphate (alpha trinositol, AT), and its hexanoyl ester (HAT), in tissue wasting in cancer cachexia. METHODS: The anti-cachexic effect was evaluated in the MAC16 tumour model. RESULTS: Both AT and HAT attenuated the loss of body weight through an increase in the nonfat carcass mass due to an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. The decrease in protein degradation was associated with a decrease in activity of the ubiquitin-proteasome proteolytic pathway and caspase-3 and -8. Protein synthesis was increased due to attenuation of the elevated autophosphorylation of double-stranded RNA-dependent protein kinase, and of eukaryotic initiation factor 2alpha together with hyperphosphorylation of eIF4E-binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2. In vitro, AT completely attenuated the protein degradation in murine myotubes induced by both proteolysis-inducing factor and angiotensin II. CONCLUSION: These results show that AT is a novel therapeutic agent with the potential to alleviate muscle wasting in cancer patients.

Characterizing the hierarchical structures of bioactive sol-gel silicate glass and hybrid scaffolds for bone regeneration

Bone is the second most widely transplanted tissue after blood. Synthetic alternatives are needed that can reduce the need for transplants and regenerate bone by acting as active temporary templates for bone growth. Bioactive glasses are one of the most promising bone replacement/regeneration materials because they bond to existing bone, are degradable and stimulate new bone growth by the action of their dissolution products on cells. Sol-gel-derived bioactive glasses can be foamed to produce interconnected macropores suitable for tissue ingrowth, particularly cell migration and vascularization and cell penetration. The scaffolds fulfil many of the criteria of an ideal synthetic bone graft, but are not suitable for all bone defect sites because they are brittle. One strategy for improving toughness of the scaffolds without losing their other beneficial properties is to synthesize inorganic/organic hybrids. These hybrids have polymers introduced into the sol-gel process so that the organic and inorganic components interact at the molecular level, providing control over mechanical properties and degradation rates. However, a full understanding of how each feature or property of the glass and hybrid scaffolds affects cellular response is needed to optimize the materials and ensure long-term success and clinical products. This review focuses on the techniques that have been developed for characterizing the hierarchical structures of sol-gel glasses and hybrids, from atomicscale amorphous networks, through the covalent bonding between components in hybrids and nanoporosity, to quantifying open macroporous networks of the scaffolds. Methods for non-destructive in situ monitoring of degradation and bioactivity mechanisms of the materials are also included. © 2012 The Royal Society.

Copper(II) complexes with unsymmetrical pentadentate ed3a-type diamino-tricarboxylate ligands. Crystal structures, configurational analysis and DFT study of complexes

The O–O–N–N–O-type pentadentate ligands H3ed3a, H3pd3a and H3pd3p (H3ed3a stands ethylenediamine-N,N,N′-triacetic acid; H3pd3a stands 1,3-propanediamine-N,N,N′-triacetic acid and H3pd3p stands 1,3-propanediamine-N,N,N′-tri-3-propionic acid) and the corresponding novel octahedral or square-planar/trigonal-bipyramidal copper(II) complexes have been prepared and characterized. H3ed3a, H3pd3a and H3pd3p ligands coordinate to copper(II) ion via five donor atoms (three deprotonated carboxylate atoms and two amine nitrogens) affording octahedral in case of ed3a3− and intermediate square-pyramidal/trigonal-bipyramidal structure in case of pd3a3− and pd3p3−. A six coordinate, octahedral geometry has been established crystallographically for the [Mg(H2O)6][Cu(ed3a)(H2O)]2 · 2H2O complex and five coordinate square-pyramidal for the [Mg(H2O)5Cu(pd3a)][Cu(pd3a)] · 2H2O. Structural data correlating similar chelate Cu(II) complexes have been used for the better understanding the pathway: octahedral → square-pyramidal ↔ trigonal- bipyramid geometry. An extensive configuration analysis is discussed in relation to information obtained for similar complexes. The infra-red and electronic absorption spectra of the complexes are discussed in comparison with related complexes of known geometries. Molecular mechanics and density functional theory (DFT) programs have been used to model the most stable geometric isomer yielding, at the same time, significant structural data. The results from density functional studies have been compared with X-ray data.

Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies

This work was supported by a Knowledge Transfer Network BBSRC Industrial Case (#414 BB/L502467/1) studentship in association Zoetis Inc.

Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control

<p>Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. </p>

samExploreR: exploring reproducibility and robustness of RNA-seq results based on SAM files

<p>MOTIVATION: Data from RNA-seq experiments provide us with many new possibilities to gain insights into biological and disease mechanisms of cellular functioning. However, the reproducibility and robustness of RNA-seq data analysis results is often unclear. This is in part attributed to the two counter acting goals of (a) a cost efficient and (b) an optimal experimental design leading to a compromise, e.g., in the sequencing depth of experiments.</p><p>RESULTS: We introduce an R package called samExploreR that allows the subsampling (m out of n bootstraping) of short-reads based on SAM files facilitating the investigation of sequencing depth related questions for the experimental design. Overall, this provides a systematic way for exploring the reproducibility and robustness of general RNA-seq studies. We exemplify the usage of samExploreR by studying the influence of the sequencing depth and the annotation on the identification of differentially expressed genes.</p><p>AVAILABILITY: Availability: samExploreR is available as an R package from Bioconductor (after acceptance of the paper, download link: http://www.bio-complexity.com/samExploreR_1.0.0.tar.gz).</p><p><br/></p>

The identification of a novel role for BRCA1 in regulating RNA Polymerase I transcription

The unrestrained proliferation of cancer cells requires a high level of ribosome biogenesis. The first stage of ribosome biogenesis is the transcription of the large ribosomal RNAs (rRNAs); the structural and functional components of the ribosome. Transcription of rRNA is carried out by RNA Polymerase I (Pol-I) and its associated holoenzyme complex. Here we report that BRCA1, a nuclear phosphoprotein, and a known tumour suppressor involved in variety of cellular processes such as DNA damage response, transcriptional regulation, cell cycle control and ubiquitylation, is associated with rDNA repeats, in particular with the regulatory regions of the rRNA gene. We demonstrate that BRCA1 interacts directly with the basal Pol-I transcription factors; upstream binding factor (UBF), selectivity factor-1 (SL1) as well as interacting with RNA Pol-I itself. We show that in response to DNA damage, BRCA1 occupancy at the rDNA repeat is decreased and the observed BRCA1 interactions with the Pol-I transcription machinery are weakened. We propose, therefore, that there is a rDNA associated fraction of BRCA1 involved in DNA damage dependent regulation of Pol-I transcription, regulating the stability and formation of the Pol-I holoenzyme during initiation and/or elongation in response to DNA damage.

Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.

Études d'ingénierie du ribozyme VS de Neurospora

Les ribozymes sont des ARN catalytiques fréquemment exploités pour le développement d’outils biochimiques et d’agents thérapeutiques. Ils sont particulièrement intéressants pour effectuer l’inactivation de gènes, en permettant la dégradation d’ARNm ou d’ARN viraux associés à des maladies. Les ribozymes les plus utilisés en ce moment pour le développement d’agents thérapeutiques sont les ribozymes hammerhead et hairpin, qui permettent la reconnaissance spécifique d’ARN simple brin par la formation de structures secondaires stables. In vivo, la majorité des ARN adoptent des structures secondaires et tertiaires complexes et les régions simples brins sont parfois difficiles d’accès. Il serait intéressant de pouvoir cibler des ARN repliés et un motif d’ARN intéressant à cibler est la tige-boucle d’ARN qui peut être importante dans le repliement global des ARN et pour accomplir des fonctions biologiques. Le ribozyme VS de Neurospora fait la reconnaissance de son substrat replié en tigeboucle de façon spécifique par une interaction kissing-loop, mais il n’a jamais été exploité pour faire la reconnaissance d’un ARN cible très différent de son substrat naturel. Le but des travaux présentés dans cette thèse est de déterminer si le ribozyme VS possède l’adaptabilité nécessaire pour l’ingénierie de ribozymes qui clivent des ARN cibles différents du substrat naturel. Dans le cadre de cette thèse, le ribozyme VS a été modifié pour l’adapter à différents substrats et des études de cinétiques ont été réalisées pour évaluer l’impact de ces modifications sur l’activité de clivage du ribozyme. Dans un premier temps, le ribozyme a été modifié pour faire la reconnaissance et le clivage de substrats possédant différentes longueurs de tiges Ib. Le ribozyme a été adapté avec succès à ces substrats de différentes longueurs de tige Ib, avec une activité qui est similaire à celle du ribozyme avec un substrat sans modification. Dans un deuxième temps, c’est l’interaction kissing-loop I/V du ribozyme qui a été substituée de façon rationnelle, dans le but de savoir si un ribozyme VS mutant peut reconnaitre et cliver un substrat ayant une boucle différente de celle de son substrat naturel. L’interaction kissing-loop I/V a été substituée pour les interactions kissing-loop TAR/TAR* de l’ARN du VIH-1 et L22/L88 de l’ARN 23S de Deinococcus radiodurans. La réaction de iii clivage des ribozymes comportant ces nouvelles interactions kissing-loop est toujours observée, mais avec une activité diminuée. Finalement, la sélection in vitro (SELEX) de ribozymes a été effectuée pour permettre un clivage plus efficace d’un substrat mutant avec une nouvelle boucle. Le SELEX a permis la sélection d’un ribozyme qui clive un substrat avec une boucle terminale mutée pour celle de l’ARN TAR du VIH-1 et cela avec une activité de clivage très efficace. L’ensemble de ces études démontre que le ribozyme VS peut être modifié de diverses façons pour la reconnaissance spécifique de différents substrats, tout en conservant une bonne activité de clivage. Ces résultats montrent le grand potentiel d’ingénierie du ribozyme VS et sont prometteurs pour la poursuite d’études d’ingénierie du ribozyme VS, en vue du clivage d’ARN cibles repliés en tige-boucle complètement différents du substrat naturel du ribozyme VS.

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal+) or galactose-non-metabolising (Gal-) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). Sc. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal+ or Gal-). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that Sc. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.

Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.