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 .     

Kinetics of cooperative ligand–lattice binding: Fast Monte Carlo integration

A fast Monte Carlo integration algorithm with varying time step is described for cooperative binding of ligands of arbitrary length to a one-dimensional lattice. This algorithm is particularly suitable for strongly cooperative or anticooperative systems, i.e., when the time scales for different kinetic events are very different. As an application, the kinetics of a bimodal two-ligand system are briefly discussed.     

Osmotic water permeability of cell membranes from Mesembryanthemum crystallinum leaves: effects of age and salinity

The osmotic water permeability ( P _os) of cell membranes isolated from leaves of 40-, 50- and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P _os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P _os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 m M NaCl caused a decline in the P _os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P _os of the leaf-cell membranes. These variations in the P _os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.     

Oligosyndactyly: A lethal mutation in the mouse that results in mitotic arrest very early in development

The mutation, oligosyndactyly, results in syndactyly, muscle anomalies, and diabetes insipidus in heterozygous mice. When homozygous, the mutation is lethal early in development. Although homozygous embryos are able to form blastocyst outgrowths (the in vitro equivalent to implantation), cells begin to accumulate in mitosis as early as the blastocyst stage. Even though the cytologic appearance is that of mitotic cells treated with a microtubule inhibitor such as colcemid, the homozygous embryos do, in fact, have normal appearing mitotic spindles. These results define the Os mutation as one which, in the homozygous state, prevents the movement of chromosomes from the metaphase plate. It is the first mammalian developmental mutation to be so defined and is unique among all mitotic arrest mutations thus far described in higher eucaryotes.     

Nd-YAG Laser in Lung Cancer

We administered 45 Nd-YAG laser treatments in 29 patients (18 men) aged 39 to 82 years who had lung malignancy; 26 patients had primary non-oat cell lung cancer and three had metastatic airway malignancy. In all, 25 of the patients had been previously treated with combination(s) of surgical procedure, radiation therapy and chemotherapy. Indications for laser treatment included endobronchial airway obstruction with uncontrolled cough, hemoptysis, dyspnea or unresolved atelectasis-pneumonia. Of 15 patients with partially occluded tracheobronchial airway tumors, immediate palliative relief was achieved in 13 patients and lasted one to six months after a single treatment. In this group there was one postoperative death related to respiratory failure and two patients subsequently died of massive pulmonary hemorrhage. However, of 14 patients with totally obstructed airways, immediate palliative relief was achieved in only five patients and this lasted three weeks to three months after a single treatment. In this group there were two postoperative deaths related to progressive respiratory failure; in one case it was associated with endobronchial combustion of the fiberoptic bronchoscope. All three patients in both groups who died of respiratory failure were in acute respiratory distress and terminally ill before the procedure. These findings suggest that Nd-YAG laser therapy may be most beneficial in patients with partially rather than totally occluded airways due to lung malignancy.     

Mutant HLA-A2 antigens as restricting elements for virus-specific cytotoxic T cells

Mutants of the EB virus-transformed cell line T5-1 (HLA-Al, 2; B8, 27), bearing well-characterized alterations in HLA-A2 antigen expression and unable to bind the HLA-A2-specific monoclonal antibody 13137.2, have been tested for their susceptibility to EB virus-specific cytolysis using effector T-cell preparations functionally restricted through relevant HLA antigens. Initial experiments first confirmed that the parent line T5-1 was susceptible to cytolysis by both common A2-restricted and 1327-restricted effector cells. While those T5-1 mutants with little or no surface A2 expression were not lysed by A2-restricted effectors, those targets with quantitatively normal expression of mutant A2 molecules were as susceptible to A2-restricted lysis as the parent line itself. In contrast, all the T5-1 mutant lines were susceptible to B27-restricted cytolysis. The results demonstrate that experimentally induced mutations of HLA-A2 antigen structure, affecting a serologically defined site on the molecule, can occur without altering that same molecule's expression of the T cell-restricting determinant(s). Such experimentally induced mutations are quite different from the naturally occurring variant A2 antigens which are present within the serologically defined A2 antigen group and which show changes at the T cell-restricting site.     

Hypoxanthine transport in normal and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient diploid human lymphoblasts

We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of K_m. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process.     

A study of communication specificity between cells in culture

We have examined the specificity of communication between cells in culture by co-culturing cells derived from mammalian, avian, and arthropod organisms. Both mammalian and avian culture cells have similar gap junctional phenotypes, while the insect (arthropod) cell lines have a significantly different gap junctional structure. Electrophysiological and ultrastructural methods were used to examine ionic coupling and junctional interactions between homologous and heterologous cell types. In homologous cell systems, gap junctions and ionic coupling are present at a high incidence. Also, heterologous vertebrate cells in co-culture can communicate readily. By contrast, practically no coupling (0-8%) is detectable between heterologous insect cell lines (Homopteran or Lepidopteran) and vertebrate cells (mammalian myocardial or 3T3 cells). No gap junctions have been observed between arthropod and vertebrate cell types, even though the heterologous cells may be separated by less than 10 nm. In additional studies, a low incidence of coupling was found between heterologous insect cell lines derived from different arthropod orders. However, extensive coupling was detected between insect cell lines that are derived from the same order (Homoptera). These observations suggest that there is little or no apparent specificity for communication between vertebrate cells in culture that express the same gap junctional phenotype, while there is a definite communication specificity that exists between arthropod cells in culture.     

The Hunter syndrome in females: is there an autosomal recessive form of iduronate sulfatase deficiency?

Profound iduronate sulfatase deficiency, characteristic of the Hunter syndrome, has been found in cultured fibroblasts, serum, lymphocytes, and tissues of two clinically affected girls. The patients are karyotypically normal and have normal fathers; cloning of the mothers' fibroblasts did not reveal the mosaicism expected of carriers of an X-linked disease. Homozygosity for a previously unsuspected autosomal recessive gene for iduronate sulfatase is considered the most likely explanation, although heterozygosity for the X-linked gene and subsequent selection cannot be completely excluded.