Formulation and characterization of a 0.1% rapamycin cream for the treatment of Tuberous Sclerosis Complex-related angiofibromas

International audience

Stability of fruit bases and chocolate fillings

Syrups with high sugar content and dehydrated fruits in its composition can be added to chocolate fillings to reduce the need of artificial flavor and dyes attributing a natural appeal to the product. Fruit bases were produced with lyophilized strawberry, passion fruit, and sliced orange peel. Rheological dynamic oscillatory tests were applied to determine the products stability and tendency of shelf life. Values of G´< G´´ were observed for strawberry and passion fruit flavor, whereas values of G´ > G´´ were found for orange flavor during the 90 days of storage. It was observed that shear stress values did not vary significantly suggesting product stability during the studied period. For all fillings, it was found a behavior similar to the fruit base indicating that it has great influence on the filling behavior and its stability. The use of a sugar matrix in fillings provided good shelf life for the fruit base, which could be kept under room temperature conditions for a period as long as one year. The good stability and storage conditions allow the use of fruit base for handmade products as well as for industrialized products.

Multidimensional cluster stability analysis from a Brazilian Bradyrhizobium sp RFLP/PCR data set

The taxonomy of the N(2)-fixing bacteria belonging to the genus Bradyrhizobium is still poorly refined, mainly due to conflicting results obtained by the analysis of the phenotypic and genotypic properties. This paper presents an application of a method aiming at the identification of possible new clusters within a Brazilian collection of 119 Bradryrhizobium strains showing phenotypic characteristics of B. japonicum and B. elkanii. The stability was studied as a function of the number of restriction enzymes used in the RFLP-PCR analysis of three ribosomal regions with three restriction enzymes per region. The method proposed here uses Clustering algorithms with distances calculated by average-linkage clustering. Introducing perturbations using sub-sampling techniques makes the stability analysis. The method showed efficacy in the grouping of the species B. japonicum and B. elkanii. Furthermore, two new clusters were clearly defined, indicating possible new species, and sub-clusters within each detected cluster. (C) 2008 Elsevier B.V. All rights reserved.

Citrate and Phosphate Influence on Green Fluorescent Protein Thermal Stability

Protein structure and function can be regulated by no specific interactions, such as ionic interactions in the presence of salts. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. The aim of this study was to evaluate the thermal stability of GFP in the presence of different salts at several concentrations and exposed to constant temperatures, in a range of 70-95 degrees C. Thermal stability was expressed in decimal reduction time. It was observed that the D-values obtained were higher in the presence of citrate and phosphate, when compared with that obtained in their absence, indicating that these salts stabilized the protein against thermal denaturation. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 269-272, 2011

Investigation of charged polymer influence on green fluorescent protein thermal stability

Methods of stabilization and formulation of proteins are important in both biopharmaceutical and biocatalysis industries. Polymers are often used as modifiers of characteristics of biological macromolecules to improve the biochemical activity and stability of proteins or drug bioavailability. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Relative thermal stability was undertaken by incubation of GFP at varying temperatures and GFP fluorescence was used as a reporter for unfolding. At 80 degrees C, DEAE-dextran did not have any effect on GFP fluorescence, indicating that it does not confer stability.

Thermal Cycling Stability of Silica Membranes for Gas Separation

Hydrogen is being seen as an alternative energy carrier to conventional hydrocarbons to reduce greenhouse gas emissions. High efficiency separation technologies to remove hydrogen from the greenhouse gas, carbon dioxide, are therefore in growing demand. Traditional thermodynamic separation systems utilise distillation, absorption and adsorption, but are limited in efficiency at compact scales. Molecular sieve silica (MSS) membranes can perform this separation as they have high permselectivity of hydrogen to carbon dioxide, but their stability under thermal cycling is not well reported. In this work we exposed a standard MSS membrane and a carbonised template MSS (CTMSS) membrane to thermal cycling from 100 to 450°C. The standard MSS and carbonised template CTMSS membranes both showed permselectivity of helium to nitrogen dropping from around 10 to 6 in the first set of cycles, remaining stable until the last test. The permselectivity drop was due to small micropore collapse, which occurred via structure movement during cycling. Simulating single stage membrane separation with a 50:50 molar feed of H2:CO2, H2 exiting the permeate stream would start at 79% and stabilise at 67%. Higher selectivity membranes showed less of a purity drop, indicating the margin at which to design a stable membrane separation unit for CO2 capture.

Stability of the Transthyretin Molecule as a Key Factor in

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/ABeta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4- (3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Phenotypic stability and genetic gains in six-year girth growth of Hevea clones

Rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell. Arg.] budgrafts of seven clones were evaluated on five contrasting sites in the plateau region of the São Paulo State, Brazil. The objective of this work was to study the phenotypic stability for girth growth. The experimental design was a randomized block design with three replications and seven treatments. Analysis of variance of girth at six-year plant growth indicated a highly significant clone x site interaction. Only linear sites and clone x site components of clone x year interaction were significant, indicating that the performance of clones over sites for this trait could be predicted. The clones GT 1 and PB 235 showed the greatest stability in relation to girth growth, with foreseen responses to change, introduced in the sites. The clones PB 235 and IAN 873 showed significative difference in relation to regression coefficient, representing clones with specific adaptability on favorable and unfavorable sites respectively. The clone GT 1 became the most promissory one in the study of stability and adaptability even showing low girth growth. Expected genetic gains from planting sites, along with estimates of clonal variance and repeatability of clonal means are generally greatest or close to the greatest when selection is done at the same site.

Different methods to assess yield temporal stability in rubber

The objective of this work was to assess, during six years, the temporal stability of natural rubber yield of 25 superior Hevea brasiliensis genotypes, using the Wricke, Eberhart & Russell, Lin & Binns, additive main effect and multiplicative interaction (AMMI) analysis, and harmonic mean of the relative performance of the genetic values (HMRPGV) methods. The IAC 40 and IAC 300 genotypes were identified as stable and high yielding by the Eberhart & Russell, Lin & Binns, HMRPGV, and AMMI Biplot methods. The ranking of the other more stable genotypes identified by these analyses was altered. The observed results in the AMMI Biplot agreed with those observed in the Wricke method for identifying stable, but lower yielding genotypes. The simultaneous use of different methods allows a more accurate indication of stable genotypes. Stability analyses based on different principles show agreement in indicating stable genotypes.

Stability and adaptability of runner peanut genotypes based on nonlinear regression and AMMI analysis

The objective of this work was to estimate the stability and adaptability of pod and seed yield in runner peanut genotypes based on the nonlinear regression and AMMI analysis. Yield data from 11 trials, distributed in six environments and three harvests, carried out in the Northeast region of Brazil during the rainy season were used. Significant effects of genotypes (G), environments (E), and GE interactions were detected in the analysis, indicating different behaviors among genotypes in favorable and unfavorable environmental conditions. The genotypes BRS Pérola Branca and LViPE‑06 are more stable and adapted to the semiarid environment, whereas LGoPE‑06 is a promising material for pod production, despite being highly dependent on favorable environments.

Stability of cognitive performance in children with mild intellectual disability.

AIM: Longitudinal studies that have examined cognitive performance in children with intellectual disability more than twice over the course of their development are scarce. We assessed population and individual stability of cognitive performance in a clinical sample of children with borderline to mild non-syndromic intellectual disability. METHOD: Thirty-six children (28 males, eight females; age range 3-19y) with borderline to mild intellectual disability (Full-scale IQ [FSIQ] 50-85) of unknown origin were examined in a retrospective clinical case series using linear mixed models including at least three assessments with standardized intelligence tests. RESULTS: Average cognitive performance remained remarkably stable over time (high population stability, drop of only 0.38 IQ points per year, standard error=0.39, p=0.325) whereas individual stability was at best moderate (intraclass correlation of 0.58), indicating that about 60% of the residual variation in FSIQ scores can be attributed to between-child variability. Neither sex nor socio-economic status had a statistically significant impact on FSIQ. INTERPRETATION: Although intellectual disability during childhood is a relatively stable phenomenon, individual stability of IQ is only moderate, likely to be caused by test-to-test reliability (e.g. level of child's cooperation, motivation, and attention). Therefore, clinical decisions and predictions should not rely on single IQ assessments, but should also consider adaptive functioning and previous developmental history.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting thatXIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance ofXIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

Stability of fruit bases and chocolate fillings

Syrups with high sugar content and dehydrated fruits in its composition can be added to chocolate fillings to reduce the need of artificial flavor and dyes attributing a natural appeal to the product. Fruit bases were produced with lyophilized strawberry, passion fruit, and sliced orange peel. Rheological dynamic oscillatory tests were applied to determine the products stability and tendency of shelf life. Values of G´< G´´ were observed for strawberry and passion fruit flavor, whereas values of G´ > G´´ were found for orange flavor during the 90 days of storage. It was observed that shear stress values did not vary significantly suggesting product stability during the studied period. For all fillings, it was found a behavior similar to the fruit base indicating that it has great influence on the filling behavior and its stability. The use of a sugar matrix in fillings provided good shelf life for the fruit base, which could be kept under room temperature conditions for a period as long as one year. The good stability and storage conditions allow the use of fruit base for handmade products as well as for industrialized products.

Sensory acceptability and physical stability evaluation of a prebiotic soy-based dessert developed with passion fruit juice

The aim of this study was to use a factorial design approach for developing a palatable and stable soy-based dessert with the addition of Soy Protein (SP), oligofructose, and Passion Fruit Juice (PFJ). Panelists (n = 50) used a seven-point hedonic scale to assess the overall liking, degree of liking of creaminess, taste, and color of the desserts. In addition, the samples were submitted to a preference ranking test in order to evaluate the products' preference. Water Holding Capacity (WHC) and backscattering (BS) measures were also determined to assess the physical stability of the trials. Sample F3 (35% PFJ and 2% SP) was the only one that presented a WHC index of 94.8%; moreover, none of the developed samples had synerisis after 72 hours of storage indicating adequate physical stability of the emulsion process. Samples F2 (25% PFJ, and 3.0% SP), F4 (35% PFJ, and 3.0% SP), and F5 (30% PFJ, and 2.5% SP) presented mean hedonic scores above 'slightly liked' for all sensory attributes. The acceptance index of samples varied from 62.50 to 88% showing the great sensory potential of such products.