Comparison of Immune Responses to Different Versions of VLP Associated Stabilized RSV Pre-Fusion F Protein

Efforts to develop a vaccine for respiratory syncytial virus (RSV) have primarily focused on the RSV fusion protein. The pre-fusion conformation of this protein induces the most potent neutralizing antibodies and is the focus of recent efforts in vaccine development. Following the first identification of mutations in the RSV F protein (DS-Cav1 mutant protein) that stabilized the pre-fusion conformation, other mutant stabilized pre-fusion F proteins have been described. To determine if there are differences in alternate versions of stabilized pre-fusion F proteins, we explored the use, as vaccine candidates, of virus-like particles (VLPs) containing five different pre-fusion F proteins, including the DS-Cav1 protein. The expression of these five pre-F proteins, their assembly into VLPs, their pre-fusion conformation stability in VLPs, their reactivity with anti-F monoclonal antibodies, and their induction of immune responses after the immunization of mice, were characterized, comparing VLPs containing the DS-Cav1 pre-F protein with VLPs containing four alternative pre-fusion F proteins. The concentrations of anti-F IgG induced by each VLP that blocked the binding of prototype monoclonal antibodies using two different soluble pre-fusion F proteins as targets were measured. Our results indicate that both the conformation and immunogenicity of alternative VLP associated stabilized pre-fusion RSV F proteins are different from those of DS-Cav1 VLPs

TA treatment of depression : a hermeneutic single-case efficacy design study - ‘Deborah’

This study is the third of a series of seven, and belongs to the second Italian systematic replication of findings from two previous series (Widdowson 2012a, 2012b, 2012c, 2013; Benelli, 2016a, 2016b, 2016c) that investigated the effectiveness of a manualised transactional analysis treatment for depression through Hermeneutic Single-Case Efficacy Design (HSCED). Major Depression and Subthreshold Depression are often in comorbidity with Anxiety disorders in childhood and adolescence and represent a risk factor for ongoing mental health problems in adulthood. The therapist was a white Italian woman with 15 years of clinical experience and the client, Deborah, was a 15-year old white Italian female adoloscent who attended sixteen sessions of transactional analysis psychotherapy. The conclusion of the judges was that this was a good-outcome case: the depressive and anxious symptomatology clinically and reliably improved over the course of the therapy and these improvements were maintained throughout the duration of the follow-up intervals. Furthermore, the client reported significant change in her post-treatment interview and these changes were directly attributed to the therapy. In this case study, the transactional analysis manualised treatment for depression in adulthood has demonstrated its effectiveness also in treating depressive and anxiety symptoms in adolescence

TA treatment of depression : a hermeneutic single-case efficacy design study - ‘Deborah’

This study is the third of a series of seven, and belongs to the second Italian systematic replication of findings from two previous series (Widdowson 2012a, 2012b, 2012c, 2013; Benelli, 2016a, 2016b, 2016c) that investigated the effectiveness of a manualised transactional analysis treatment for depression through Hermeneutic Single-Case Efficacy Design (HSCED). Major Depression and Subthreshold Depression are often in comorbidity with Anxiety disorders in childhood and adolescence and represent a risk factor for ongoing mental health problems in adulthood. The therapist was a white Italian woman with 15 years of clinical experience and the client, Deborah, was a 15-year old white Italian female adoloscent who attended sixteen sessions of transactional analysis psychotherapy. The conclusion of the judges was that this was a good-outcome case: the depressive and anxious symptomatology clinically and reliably improved over the course of the therapy and these improvements were maintained throughout the duration of the follow-up intervals. Furthermore, the client reported significant change in her post-treatment interview and these changes were directly attributed to the therapy. In this case study, the transactional analysis manualised treatment for depression in adulthood has demonstrated its effectiveness also in treating depressive and anxiety symptoms in adolescence

eTOX ALLIES:an automated pipeLine for linear interaction energy-based simulations

Abstract Background Computational methods to predict binding affinities of small ligands toward relevant biological (off-)targets are helpful in prioritizing the screening and synthesis of new drug candidates, thereby speeding up the drug discovery process. However, use of ligand-based approaches can lead to erroneous predictions when structural and dynamic features of the target substantially affect ligand binding. Free energy methods for affinity computation can include steric and electrostatic protein–ligand interactions, solvent effects, and thermal fluctuations, but often they are computationally demanding and require a high level of supervision. As a result their application is typically limited to the screening of small sets of compounds by experts in molecular modeling. Results We have developed eTOX ALLIES, an open source framework that allows the automated prediction of ligand-binding free energies requiring the ligand structure as only input. eTOX ALLIES is based on the linear interaction energy approach, an efficient end-point free energy method derived from Free Energy Perturbation theory. Upon submission of a ligand or dataset of compounds, the tool performs the multiple steps required for binding free-energy prediction (docking, ligand topology creation, molecular dynamics simulations, data analysis), making use of external open source software where necessary. Moreover, functionalities are also available to enable and assist the creation and calibration of new models. In addition, a web graphical user interface has been developed to allow use of free-energy based models to users that are not an expert in molecular modeling. Conclusions Because of the user-friendliness, efficiency and free-software licensing, eTOX ALLIES represents a novel extension of the toolbox for computational chemists, pharmaceutical scientists and toxicologists, who are interested in fast affinity predictions of small molecules toward biological (off-)targets for which protein flexibility, solvent and binding site interactions directly affect the strength of ligand-protein binding

The D647N mutation of FGFR1 induces ligand-independent receptor activation

The activation loop (A-loop) of kinases, a key regulatory region, is recurrently mutated in several kinase proteins in cancer resulting in dysregulated kinase activity and response to kinase inhibitors. FGFR1 receptor tyrosine kinase represents an important oncogene and therapeutic target for solid and hematological tumors. Here we investigate the biochemical and molecular effects of D647N mutation lying in the A-loop of FGFR1.When expressed in normal and tumoral in vitro cell models, FGFR1D647N is phosphorylated also in the absence of ligands, and this is accompanied by the activation of intracellular signaling. The expression of FGFR1D647N significantly increases single and collective migration of cancer cells in vitro and in vivo, when compared to FGFR1WT. FGFR1D647N expression exacerbates the aggressiveness of cancer cells, increasing their invasiveness in vitro and augmenting their pro-angiogenic capacity in vivo.Remarkably, the D647N mutation significantly increases the sensitivity of FGFR1 to the ATP-competitive inhibitor Erdafitinib suggesting the possibility that this mutation could become a specific target for the development of new inhibitors. Although further efforts are warranted for an exhaustive description of the activation mechanisms, for the identification of more specific inhibitors and for confirming the clinical significance of mutated FGFR1D647N, overall our data demonstrate that the D647N substitution of FGFR1 is a novel pro-oncogenic activating mutation of the receptor that, when found in cancer patients, may anticipate good response to erdafitinib treatment

A patient-centered multidisciplinary cardiac rehabilitation program improves glycemic control and functional outcome in coronary artery disease after percutaneous and surgical revascularization

Background: Cardiac rehabilitation (CR) is strongly associated with all-cause mortality reduction in patients with coronary artery disease (CAD). The impact of CR on pathological risk factors, such as impaired glucose tolerance (IGT), and functional recovery remains under debate. The aim of the present study is to determine whether CR has a positive effect on physical exercise improvement and on pathological risk factors in IGT and diabetic patients with CAD. Methods: One hundred and seventy-one consecutive patients participating in a 3-month CR from January 2014 to June 2015 were enrolled. The primary endpoint was defined as an improvement of peak workload and VO2-peak; glycated hemoglobin (HbA1c) reduction was considered as a secondary endpoint. Results: Euglycemic patients presented a significant improvement in peak workload compared to diabetic patients (from 5.75 ± 1.45 to 6.65 ± 1.84 METs, p = 0.018 vs. 4.8 ± 0.8 to 4.9 ± 1.4 METs). VO2-peak improved in euglycemic patients (VO2-peak from 19.3 ± 5.3 mL/min/kg to 22.5 ± 5.9, p = 0.003), while diabetic patients did not present  a  statistically significant trend (VO2-peak from 16.9 ± 4.4 mL/min/kg to 18.0 ± 3.8, p < 0.056). Diabetic patients have benefited more in terms of blood glucose control compared to IGT patients (HbA1c from 7.7 ± 1.0 to 7.4 ± 1.1 compared to 5.6 ± 0.4 to 5.9 ± 0.5, p = 0.02, respectively). Conclusions: A multidisciplinary CR program improves physical functional capacity in CAD setting, particularly in euglycemic patients. IGT patients as well as diabetic patients may benefit from a CR program, but long-term outcome needs to be clarified in larger studies

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

Expression of genes involved in the T cell signalling pathway in circulating immune cells of cattle 24 months following oral challenge with Bovine Amyloidotic Spongiform Encephalopathy (BASE)

BACKGROUND: Bovine Amyloidotic Spongiform Encephalopathy (BASE) is a variant of classical BSE that affects cows and can be transmitted to primates and mice. BASE is biochemically different from BSE and shares some molecular and histo-pathological features with the MV2 sub-type of human sporadic Creutzfeld Jakob Disease (sCJD). RESULTS: The present work examined the effects of BASE on gene expression in circulating immune cells. Ontology analysis of genes differentially expressed between cattle orally challenged with brain homogenate from cattle following intracranial inoculation with BASE and control cattle identified three main pathways which were affected. Within the immune function pathway, the most affected genes were related to the T cell receptor-mediated T cell activation pathways. The differential expression of these genes in BASE challenged animals at 10,12 and 24 months following challenge, vs unchallenged controls, was investigated by real time PCR. CONCLUSIONS: The results of this study show that the effects of prion diseases are not limited to the CNS, but involve the immune system and particularly T cell signalling during the early stage following challenge, before the appearance of clinical signs.Andrea Trovato, Simona Panelli, Francesco Strozzi, Caterina Cambulli, Ilaria Barbieri, Nicola Martinelli, Guerino Lombardi, Rossana Capoferri and John L William

A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors

Reaction profiling of a set of acrylamide-based human tissue transglutaminase inhibitors

The major function of the enzyme human tissue transglutaminase (TG2) is the crosslinking of proteins via a transamidation between the γ-carboxamide of a glutamine and the ε-amino group of a lysine. Overexpression of TG2 can lead to undesirable outcomes and has been linked to conditions such as fibrosis, celiac disease and neurodegenerative diseases. Accordingly, TG2 is a tempting drug target. The most effective TG2 inhibitors to date are small-molecule peptidomimetics featuring electrophilic warheads that irreversibly modify the active site catalytic cysteine (CYS277). In an effort to facilitate the design of such TG2 inhibitors, we undertook a quantum mechanical reaction profiling of the Michael reaction between a set of six acrylamide-based known TG2 inhibitors and the TG2 CYS277. The inhibitors were docked into the active site and the coordinates were refined by MD simulations prior to modelling the covalent modification of the CYS277 thiolate. The results of QM/MM MD umbrella sampling applied to reaction coordinates driving the Michael reaction are presented for two approximations of the Michael reaction: a concerted reaction (simultaneous thiolate attack onto the acrylamide warhead and pronation from the adjacent HIS335) and a two-stage reaction (consecutive thiolate attack and protonation). The two-stage approximation of the Michael reaction gave the better results for the evaluation of acrylamide-based potential TG2 inhibitors in silico. Good correlations were observed between the experimental TG2 IC50 data and the calculated activation energies over the range 0.0061 – 6.3 µM (three orders of magnitude) and we propose that this approach may be used to evaluate acrylamide-based potential TG2 inhibitors