Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.     

Synthesis and in vitro anti Mycobacterium tuberculosis activity of a series of phthalimide derivatives

New phthalimide derivatives were easily prepared through condensation of phthalic anhydride and selected amines with variable yields (70–90%). All compounds (3a–l) were evaluated against Mycobacterium tuberculosis H₃₇Rv using Alamar Blue susceptibility. The compounds 3c, 3i, and 3l have the minimum inhibitory concentrations (MICs) of 3.9, 7.8, and 5.0 μg/mL, respectively, and could be considered new lead compounds in the treatment of tuberculosis and multi-drug resistant tuberculosis.     

Changes in chromosomes and in development of cells of Sciara ocellaris induced by microsporidian infections

Two species of microsporidia distinguished by the shape of their spores were found to infect cells of various tissues of Sciara ocellaris. Although the infection affects profoundly the development of the infected cells, the interaction between the infective agents and the host cells are often well-balanced and the infected cells may survive longer than the uninfected of the same tissue. The infected cells, including their nuclei and chromosomes, increase greatly. The general reaction of the chromosomes of cells of S. ocellaris infected by microsporidia is the increase of their volume by an increase in the polyteny and the accumulation of chromosomal products between their chromonemata. The cells of the fat bodies, on the other hand, have peculiar types of reactions. Some show an increase in polyteny, frequently showing asynapsis of the entire or parts of the chromosome. Other cells show increased chromosome polyteny associated with different degrees of polyploidy. Still other infected cells develop a new type of chromosome morphology called brachy-polytene which may or may not be associated with polyploidy. Special emphasis must be given to the fact that in Diptera, which have polytene chromosomes, the relationships of the infection and the host cell may in many cases be studied thoroughly, starting with the reaction of the genes to the infective agent. It was shown that in the infected cells of the salivary gland of S. ocellaris the pattern of puffs is greatly changed; hence, this change may be the probable cause of their change in development. Infected cells of the salivary gland with enlarged and active polytene chromosomes were found in adult flies, a situation which never occurs in non-infected animals. Since the microsporidia when entering the cells of S. ocellaris do not cause degeneration of the infected cells but determine a new pattern of development, their association with host cells offers great possibilities in the study of basic problems in cell biology, mainly related to chromosomal morphology, physiology and differentiation.This paper is dedicated to our dear friend Professor Sally Hughes-Schrader for her outstanding contribution to Biology.Work supported by Grants of the Public Health Service (GM 15769), Oonselho Nacional de Pesquisas and Pundação de Amparo á Pesquisa do Estado de São Paulo Brazil.     

Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.     

Effect of time delay in specific growth rate on the periodic operation of bioreactors with input multiplicities

The effect of time delay in specific growth rate () on the periodic operation of bioreactors with input multiplicities is theoretically analyzed for productivity improvement. A periodic rectangular pulse is applied either in feed substrate concentration (S_f) or in dilution rate (D). Periodic operation under feed substrate concentration cycling gives improvement in productivity at lower value of ¯S_f of the two steady-state multiplicities of S_f only when the time delay in is larger. Whereas the larger value of ¯S_f gives improvement in average productivity for all values of time delay. Dilution rate (D) cycling gives an improvement in average productivity particularly for larger time delay in . This improvement in average productivity is obtained only at smaller value of dilution rate out of the two steady-state input multiplicities of D.List of Symbols D 1/h dilution rate - F memory function - g dummy variable - K_i g/l substrate inhibition constant - K_m g/l substrate saturation constant - P g/l product concentration - P_m g/l product saturation constant - Q g/(hl) product cell produced per unit time - S g/l substrate concentration - S_f g/l feed substrate concentration - S_f,p g/l feed substrate concentration during fraction of a period - X g/l biomass concentration - Y_X/S g/g cell mass yield - w variable either S or Z - Z g/l weighted average of substrate concentration Greek Letters 1/h time delay parameter - ₁ , ₂ product yield parameters, g/g and 1/h - pulse width expressed as a fraction of a period - 1/h specific growth rate - _m 1/h maximum specific growth rate - h period of oscillation - – average value     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

Computer simulation of antibody binding specificity

A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives⁸) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form. © 1993 Wiley-Liss, Inc.