Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.     

Oral microcirculation in post-menopause: a possible correlation with periodontitis

doi: 10.1111/j.1741‐2358.2011.00608.x Oral microcirculation in post‐menopause: a possible correlation with periodontitis Objectives: The reduction in the level of oestrogen, typical in menopause, has some effect on the health of the oral cavity. In fact, post‐menopausal women present more severe periodontal disease than pre‐menopausal women. Numerous factors can be held to be responsible for this increase, among which are the effects of oestrogens on the oral epithelium, on the salivary glands, on bone tissue and on the endothelium. Our double blind study aims to evaluate the possible variations in oral microcirculation in post‐menopausal women. Methods: Twenty‐seven women in post‐menopause (age: Mean ± SD: 57.3 ± 8.73) and 27 women in pre‐menopause (age: Mean ± SD: 27.77 ± 3.56) were examined. Oral microcirculation was investigated using oral videocapillaroscopy. Results: The study showed significant differences between cases and controls for the following parameters: decrease in diameter of loops (mean ± SD: 0.038 ± 0.008; 0.045 ± 0.005), increase in tortuosity (mean ± SD: 3.83 ± 1.13; 1.83 ± 1.06) in labial mucosa and decrease in density in periodontal mucosa (Mean ± SD: 28.86 ± 10.92; 89.62 ± 17.83). Conclusion: The decrease in periodontal density may compromise the epithelium tropism, making it prone to inflammation. The tortuosity may indicate a greater permanence of inflammatory factors, increased in post‐menopausal women.     

The recovery of polyphenols from olive mill waste using two adsorbing vegetable matrices

Olive mill wastewater (OMW) is considered one of the most pollutive waste materials in the Mediterranean basin. However, its phenolic fraction should be recovered, since it has been shown to have incredible benefits for health. In the present study, the adsorbent and desorbent capacities of Azolla and granular activated carbon (GAC) were investigated. The GAC was found to be more efficient than Azolla in both the adsorption and the desorption of phenols. The total characterization of two powder products obtained from Azolla and GAC desorption is reported, together with their antioxidant and antiradical activities. In the Azolla powder product, total polyphenols were more than twice as numerous as those found in the GAC powder product. The GAC powder contained hydroxytyrosol in concentrations that were 3.5 times higher than those of Azolla. On the other hand, both powder products showed great antiradical activities: the IC?? was found to be 102 mg ml?1 for the Azolla and 199 mg ml?1 for the GAC powders respectively. The oxygen radical absorbance capacity was very high: 4097 μmol TE g?1 Azolla powder product and 1277 μmol TE g?1 of GAC powder products.     

LC-MS based global metabolite profiling of grapes: solvent extraction protocol optimisation

Optimal solvent conditions for grape sample preparation were investigated for the purpose of metabolite profiling studies, with the aim of obtaining as many features as possible with the best analytical repeatability. Mixtures of water, methanol and chloroform in different combinations were studied as solvents for the extraction of ground grapes. The experimental design used a two stage study to find the optimum extraction medium. The extracts obtained were further purified using solid phase extraction and analysed using a UPLC full scan TOF MS with both reversed phase and hydrophilic interaction chromatography. The data obtained were processed using data extraction algorithms and advanced statistical software for data mining. The results obtained indicated that a fairly broad optimal area for solvent composition could be identified, containing approximately equal amounts of methanol and chloroform and up to 20% water. Since the water content of the samples was variable, the robustness of the optimal conditions suggests these are appropriate for large scale profiling studies for characterisation of the grape metabolome.     

Rupture of osteocyte processes across microcracks: the effect of crack length and stress

Bone cells are connected to one another in a network, via their dendritic cellular processes. Previously, we hypothesized that these processes could be ruptured by microcracks. We proposed this as a mechanism by which osteoctyes could detect the presence of microcracks. In order for this mechanism to be effective, the number of ruptured processes would have to increase with microcrack length and also with the applied cyclic stress. This paper presents for the first time experimental data, which shows that this is indeed the case. We examined samples of bovine, ovine and murine bone ex vivo and observed processes passing across crack faces: some were still intact whilst others had ruptured. The number of intact processes per unit crack length decreased significantly with increasing crack length and also decreased in samples, which had been tested in vitro at higher stress levels. A theoretical model that we had developed previously was able to predict the overall magnitude and general trends in the experimental data. This work has provided further support for our "scissors" model, which proposes that microcracks can be detected because they disturb the osteocyte network, specifically by rupturing cellular processes where they pass across the crack faces.     

Identification of New Autoantigens by Protein Array Indicates a Role for IL4 Neutralization in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients'' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.Autoantibodies specific for proteins or nonprotein antigens (dsDNA, snRNP, carbohydrates) are often the serological hallmark of autoimmune diseases. Autoantibodies can be simply an epiphenomenon secondary to a chronic inflammatory milieu (1), but they can also play a direct pathogenetic role, as antithyroglobulin antibodies do in Hashimoto''s thyroiditis (2).Autoimmune hepatitis (AIH)¹ is a chronic necro-inflammatory disease of unknown etiology that affects predominantly women with an incidence of 1 to 2 per 100,000 per year and a prevalence of 10 to 20 out of 100,000 (3, 4). AIH is subdivided into two major types on the basis of autoantibody reactivity (5). Antibodies to nuclei and/or to smooth muscle characterize type 1 AIH, whereas antibodies to a liver-kidney microsomal constituent define patients with type 2 AIH. Because the detection of these autoantibodies is done by means of immunofluorescence on rodent multi-organ sections (liver, kidney, stomach), there are problems with the standardization and interpretation of the immunostaining patterns (6). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an international committee to define guidelines and develop procedures and reference standards for more reliable testing (7, 8). Although ELISA and bead assays with purified or recombinant autoantigens are under development (9), they actually represent a complementary, rather than alternative, approach to traditional immunofluorescence. Moreover, serological overlap is frequently observed between AIH and other non-autoimmune liver diseases such as chronic viral hepatitis (10). Therefore, new, highly specific markers represent an unmet medical need for the more accurate diagnosis and classification of AIH.Besides the potential diagnostic application, the discovery of novel AIH autoantigens could provide insights into the disease pathogenicity mechanism. Although some AIH target-autoantigens have been identified and characterized, little is known about their pathogenetic role, and other autoantigens are probably still unknown. Autoantibodies, to be considered pathogenetic, must have at least two features: (i) the target-autoantigen should be either expressed on the plasma membrane of target cells or secreted by cells (i.e. should be exposed to autoantibodies), and (ii) binding of the autoantibodies to the target antigen should disturb a cellular function directly or indirectly. A possible pathogenetic role in AIH has been put forward for autoantibodies specific for cytochrome P450 2D6 (CYP2D6) or Asialoglycoprotein receptor 1 (AGPR-1), which are both present on the hepatocyte cell membrane (10).Protein microarrays are a powerful technology, as they allow the simultaneous screening of thousands of analytes (11). In the present study, to identify new autoantigens with potential diagnostic and/or pathogenetic roles in AIH, we printed a microarray with 1626 human proteins whose main feature was being either secreted or membrane associated (i.e. potentially exposed to autoantibody recognition). We used this microarray to screen panels of sera from patients with AIH and identified six new protein antigens that are recognized with high sensitivity and specificity. One of these six autoantigens is the interleukin-4 (IL4) receptor fibronectin type III (FNIII) domain of the IL4 receptor (CD124), and, interestingly, patients'' autoantibodies specific for CD124 neutralize IL4 signaling, suggesting a possible pathogenetic role for IL4 neutralization in AIH.