Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus.

We have previously shown that gp65 (E3) is a virion structural protein which varies widely in quantity among different strains of mouse hepatitis virus (MHV). In this study, the biosynthetic pathway and possible biological activities of this protein were examined. The glycosylation of gp65 in virus-infected cells was inhibited by tunicamycin but not by monensin, suggesting that it contains an N-glycosidic linkage. Glycosylation is cotranslational and appears to be complete before the glycoprotein reaches the Golgi complex. Pulse-chase experiments showed that this protein decreased in size after 30 min of chase, suggesting that the carbohydrate chains of gp65 undergo trimming during its transport across the Golgi. This interpretation is supported by the endoglycosidase treatment of gp65, which showed that the peptide backbone of gp65 did not decrease in size after pulse-chase periods. This maturation pathway is distinct from that of the E1 or E2 glycoproteins. Partial endoglycosidase treatment indicated that gp65 contains 9 to 10 carbohydrate side chains; thus, almost all of the potential glycosylation sites of gp65 were glycosylated. In vitro translation studies coupled with protease digestion suggest that gp65 is an integral membrane protein. The presence of gp65 in the virion is correlated with the presence of an acetylesterase activity. No hemagglutinin activity was detected

Competing interactions in two dimensional Coulomb systems: Surface charge heterogeneities in co-assembled cationic-anionic incompatible mixtures

A binary mixture of oppositely charged components confined to a plane such as cationic and anionic lipid bilayers may exhibit local segregation. The relative strength of the net short range interactions, which favors macroscopic segregation, and the long range electrostatic interactions, which favors mixing, determines the length scale of the finite size or microphase segregation. The free energy of the system can be examined analytically in two separate regimes, when considering small density fluctuations at high temperatures, and when considering the periodic ordering of the system at low temperatures (F. J. Solis and M. Olvera de la Cruz, J. Chem. Phys. 122, 054905 (2000)). A simple Molecular Dynamics simulation of oppositely charged monomers, interacting with a short range Lennard Jones potential and confined to a two dimensional plane, is examined at different strengths of short and long range interactions. The system exhibits well-defined domains that can be characterized by their periodic length-scale as well as the orientational ordering of their interfaces. By adding salt, the ordering of the domains disappears and the mixture macroscopically phase segregates in agreement with analytical predictions.Comment: 8 pages, 5 figures, accepted for publication in J. Chem. Phys, Figure 1 include

Ion condensation on charged patterned surfaces

We study ion condensation onto a patterned surface of alternating charges. The competition between self-energy and ion-surface interactions leads to the formation of ionic crystalline structures at low temperatures. We consider different arrangements of underlying ionic crystals, including single ion adsorption, as well as the formation of dipoles at the interface between charged domains. Molecular dynamic simulation illustrates existence of single and mixed phases. Our results contribute to understanding pattern recognition, and molecular separation and synthesis near patterned surfaces.Comment: 3 figure

Multiple effects of CDK4/6 inhibition in cancer: From cell cycle arrest to immunomodulation

Dysregulation of the cell cycle is a hallmark of cancer that leads to aberrant cellular proliferation. CDK4/6 are cyclin-dependent kinases activated in response to proliferative signaling, which induce RB hyper-phosphorylation and hence activation of E2F transcription factors, thus promoting cell cycle progression through the S phase. Pharmacologic inhibition of CDK4/6 by palbociclib, ribociclib, or abemaciclib has been showing promising activity in multiple cancers with the best results achieved in combination with other agents. Indeed, CDK4/6 inhibitors are currently approved in combination with endocrine therapy for the treatment of estrogen receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer. Moreover, a number of clinical trials are currently underway to test the efficacy of combining CDK4/6 inhibitors with different drugs not only in breast but also in other types of cancer. Beyond the inhibition of cell proliferation, CDK4/6 inhibitors have recently revealed new effects on cancer cells and on tumor microenvironment. In particular, it has been reported that these agents induce a senescent-like phenotype, impact on cell metabolism and exert both immunomodulatory and immunogenic effects. Here we describe recent data on the anti-tumor effects of CDK4/6 inhibitors as single agents or in combined therapies, focusing in particular on their metabolic and immunomodulatory activities

Complexity of qualitative timeline-based planning

The timeline-based approach to automated planning was originally developed in the context of space missions. In this approach, problem domains are expressed as systems consisting of independent but interacting components whose behaviors over time, the timelines, are governed by a set of temporal constraints, called synchronization rules. Although timeline-based system descriptions have been successfully used in practice for decades, the research on the theoretical aspects only started recently. In the last few years, some interesting results have been shown concerning both its expressive power and the computational complexity of the related planning problem. In particular, the general problem has been proved to be EXPSPACE-complete. Given the applicability of the approach in many practical scenarios, it is thus natural to ask whether computationally simpler but still expressive fragments can be identified. In this paper, we study the timeline-based planning problem with the restriction that only qualitative synchronization rules, i.e., rules without explicit time bounds in the constraints, are allowed. We show that the problem becomes PSPACE-complete

Thesaurus: un database per il patrimonio culturale sommerso

Thesaurus Project aims at promoting the knowledge of the underwater cultural heritage, ancient and modern, through the application of several typologies of tools: underwater autonomous vehicles, which will be able to explore the sea bottom in teams communicating with each other; a database, which will be useful to store and manage all the information referring to archaeological or historical objects, shipwrecks and sites. This paper aims to explain the logic structure of the database indicating the particular needs of the research, the different typologies of items which have to be managed (archaeological and historical objects; ancient, medieval or modern shipwrecks; underwater sites; written or figurative sources, etc.), the relation with other similar databases and projects. The main task of this part of Thesaurus is to plan and organize an IT system, which will allow archaeologists to describe information in detail, in order to make an efficient managing and retrieving data system available

Antiproliferative Activity Predictor: A New Reliable In Silico Tool for Drug Response Prediction against NCI60 Panel

In vitro antiproliferative assays still represent one of the most important tools in the anticancer drug discovery field, especially to gain insights into the mechanisms of action of anticancer small molecules. The NCI-DTP (National Cancer Institute Developmental Therapeutics Program) undoubtedly represents the most famous project aimed at rapidly testing thousands of compounds against multiple tumor cell lines (NCI60). The large amount of biological data stored in the National Cancer Institute (NCI) database and many other databases has led researchers in the fields of computational biology and medicinal chemistry to develop tools to predict the anticancer properties of new agents in advance. In this work, based on the available antiproliferative data collected by the NCI and the manipulation of molecular descriptors, we propose the new in silico Antiproliferative Activity Predictor (AAP) tool to calculate the GI50 values of input structures against the NCI60 panel. This ligand-based protocol, validated by both internal and external sets of structures, has proven to be highly reliable and robust. The obtained GI50 values of a test set of 99 structures present an error of less than ±1 unit. The AAP is more powerful for GI50 calculation in the range of 4–6, showing that the results strictly correlate with the experimental data. The encouraging results were further supported by the examination of an in-house database of curcumin analogues that have already been studied as antiproliferative agents. The AAP tool identified several potentially active compounds, and a subsequent evaluation of a set of molecules selected by the NCI for the one-dose/five-dose antiproliferative assays confirmed the great potential of our protocol for the development of new anticancer small molecules. The integration of the AAP tool in the free web service DRUDIT provides an interesting device for the discovery and/or optimization of anticancer drugs to the medicinal chemistry community. The training set will be updated with new NCI-tested compounds to cover more chemical spaces, activities, and cell lines. Currently, the same protocol is being developed for predicting the TGI (total growth inhibition) and LC50 (median lethal concentration) parameters to estimate toxicity profiles of small molecules

Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector

Adenovirus (Ad) and adeno-associated virus(AAV) have attractive and complementary properties that canbe exploited for gene transfer purposes. Ad vectors are probablythe most efficient vehicles to deliver foreign genes both invitro and in vivo. AAV exhibits the unique ability to establishlatency by efficiently integrating at a specific locus of humanchromosome 19 (AAVS1). Two viral elements are necessaryfor the integration at AAVS1: Rep68y78 and the invertedterminal repeats (AAV-ITRs). In this study, we report thedevelopment of two helper-dependent adenoviral (HD) vectors,one carrying the Rep78 gene, the other an AAV-ITRflankedtransgene. Although Rep proteins have been demonstratedto interfere with Ad replication, HD Rep78 vector wassuccessfully amplified on serial passages in 293CRE4 cellswith a yield of 50–100 transducing units per cell. DNAintegration at the AAVS1 site also was demonstrated inhepatoma cells coinfected with the HD-expressing Rep78 andwith the second HD vector carrying a transgene flanked byAAV-ITRs. The high transduction efficiency, large cloningcapacity, and high titer of the HD, combined with the sitespecificintegration machinery provided by AAV-derived components,make the AdyAAV hybrid viruses a promising vehiclefor gene therap

Dual inhibition of CDK4/6 and PI3K/AKT/mTOR signaling impairs energy metabolism in MPM cancer cells

Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated to asbestos exposure. One of the most frequent genetic alteration in MPM patients is CDKN2A/ARF loss, leading to aberrant activation of the Rb pathway. In MPM cells, we previously demonstrated the therapeutic efficacy of targeting this signaling with the CDK4/6 inhibitor palbociclib in combination with PI3K/mTOR inhibitors. Here, we investigated whether such combination may have an impact on cell energy metabolism. Methods: The study was performed in MPM cells of different histotypes; metabolic analyses were conducted by measuring GLUT-1 expression and glucose uptake/consumption, and by SeaHorse technologies. Results: MPM cell models differed for their ability to adapt to metabolic stress conditions, such as glucose starvation and hypoxia. Independently of these differences, combined treatments with palbociclib and PI3K/mTOR inhibitors inhibited cell proliferation more efficaciously than single agents. The drugs alone reduced glucose uptake/consumption as well as glycolysis, and their combination further enhanced these effects under both normoxic and hypoxic conditions. Moreover, the drug combinations significantly impaired mitochondrial respiration as compared with individual treatments. These metabolic effects were mediated by the concomitant inhibition of Rb/E2F/c-myc and PI3K/AKT/mTOR signaling. Conclusions: Dual blockade of glycolysis and respiration contributes to the anti-tumor efficacy of palbociclib-PI3K/mTOR inhibitors combination