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.     

Study of Siphonaptera in Somalia

In the period 1982-1984 samples of fleas were collected from wild animals of the Middle Scebeli, Low Scebeli and Bay Regions of Somalia. In total 1,335 specimens (486 males and 849 females) were obtained from 17 species of mammalian hosts out of the 19 examined. The following species of fleas were identified: Echidnophaga gallinacea, E. larina, E. murina, Ctenocephalides felis strongylus, Synosternus burtoni, S. somalicus, S. burtoni, S. somalicus, C. felis strongylus and E. larina are known to be widespread in Somalia; on the contrary, the presence of E. gallinacea in this country has not been reported in the literature, though the flea collection of the Institute of Parasitology of the University of Rome owns five females of this species which were collected by Zavattari in South Somalia during the year 1933. Moreover, as far as it is known, E. murina has not been reported in Somalia until now. The spermatheca of the females identified as S. burtoni is described in detail as it shows characteristics which have not been apparently reported before.     

HIV viral sequences in seronegative people at risk detected by in situ hybridisation and polymerase chain reaction.

A study was conducted to assess the occurrence of latent infection with the human immunodeficiency virus (HIV) among seronegative people at high risk of infection. The presence of HIV genomes was analysed by molecular techniques in two seronegative children born to mothers infected with HIV and in three regular sexual partners of seropositive drug addicts. The adults were selected from a seronegative cohort at high risk of infection because of their sexual contacts and the children selected because of impaired growth. HIV retroviral sequences were detected in four of the five subjects directly at the cellular level by in situ hybridisation in peripheral blood mononuclear cells. HIV genomic sequences were confirmed by in vitro amplification of viral DNA with the polymerase chain reaction technique. The existence of a latent viral infection state in these seronegative subjects indicates the unreliability of standard serological analysis in people who have been in regular contact with infected patients.     

Effects of a stable prostacyclin analogue on platelet activity and on host immunocompetence in mice

The stable prostacyclin (PGI₂) analogue, iloprost, is a potent inhibitor of both tumor cell-induced platelet aggregation and of experimental metastasis in mice. To explore possible mechanisms of antimetastatic effect of iloprost, we measured the effect of this drug on both platelet aggregation and immunocompetence in the mouse. Iloprost (4×10⁻⁸M) inhibited platelet aggregation as induced by a mixture of collagen and epinephrine for at least 180 minutes of incubation, and completely reversed platelet aggregation when added during the second wave of aggregation. In addition, aggregation of platelets obtained from iloprost-treated mice (0.2 mg/kg) was completely inhibited for at least 90 minutes of incubation. Moreover, iloprost pretreatment counteracted tumor cell-induced thrombocytopenia. Thus, mouse platelets were equally sensitive to the inhibitory effect of iloprost on aggregation as platelets of other species including humans. Effects of iloprost on parameters of host immunocompetence that may influence tumor growth and metastasis formation were also evaluated. Iloprost treatment increased significantly macrophage cytostasis to tumor cells, natural killer (NK) lytic activity of spleen cells and T-cell mediated cytotoxicity . These results suggested that the antimetastatic effect of iloprost in the mouse may be attributable to multiple mechanisms including inhibition of platelet aggregation and stimulation of certain host immune functions.     

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.