Probing the interaction of Hepatitis C virus glycoproteins with putative receptors and neutralising antibodies

Hepatitis C virus (HCV) is a hepatotropic blood-borne virus which causes chronic hepatitis in the majority of cases and represents a global health burden. In order for HCV to enter cells, proteins on the surface of the virus must interact and bind to receptors on target cells. HCV surface molecules involved with receptor binding, and cellular entry, as well as immune escape, are the glycoproteins E1 and E2. The cellular receptors SRBI, CD81, CLDNs and most recently occludin have been shown to facilitate HCV entry into hepatocytes. Several conservative regions on E1E2 have, through substitution mutagenesis, proven to be important for receptor binding and antibody neutralisation. We aimed to characterise one discontinuous region, amino acid residues 611, 613-619 and 621, and its role in the interaction with CD81 by single alanine substitution mutagenesis. Mutant plasmids were transfected into HEK 293FT cells and assessed for protein expression and binding by conformation-sensitive, CD81-inhibiting antibodies. Also, to investigate whether a conformational change of the E1E2 occurs upon SRB1 binding, rendering the CD81 binding domains accessible, two assays have been compared. A plate based experiment, exclusive of SRBI and a cell based assay, including SRBI was designed to examine the antigenic exposure of the CD81 binding regions to targeting monoclonal antibodies. Additionally, Huh-7 cells expressing different levels of SRBI were used to investigate whether some HCVpp isolates rely on high SRB1 levels for infectivity and sensitivity to neutralising antibodies. These studies were performed to help elucidate the regions and residues important in HCV E1E2: receptor interaction and their interplay with each other and with neutralising antibodies

Probing the interaction of Hepatitis C virus glycoproteins with putative receptors and neutralising antibodies

Hepatitis C virus (HCV) is a hepatotropic blood-borne virus which causes chronic hepatitis in the majority of cases and represents a global health burden. In order for HCV to enter cells, proteins on the surface of the virus must interact and bind to receptors on target cells. HCV surface molecules involved with receptor binding, and cellular entry, as well as immune escape, are the glycoproteins E1 and E2. The cellular receptors SRBI, CD81, CLDNs and most recently occludin have been shown to facilitate HCV entry into hepatocytes. Several conservative regions on E1E2 have, through substitution mutagenesis, proven to be important for receptor binding and antibody neutralisation. We aimed to characterise one discontinuous region, amino acid residues 611, 613-619 and 621, and its role in the interaction with CD81 by single alanine substitution mutagenesis. Mutant plasmids were transfected into HEK 293FT cells and assessed for protein expression and binding by conformation-sensitive, CD81-inhibiting antibodies. Also, to investigate whether a conformational change of the E1E2 occurs upon SRB1 binding, rendering the CD81 binding domains accessible, two assays have been compared. A plate based experiment, exclusive of SRBI and a cell based assay, including SRBI was designed to examine the antigenic exposure of the CD81 binding regions to targeting monoclonal antibodies. Additionally, Huh-7 cells expressing different levels of SRBI were used to investigate whether some HCVpp isolates rely on high SRB1 levels for infectivity and sensitivity to neutralising antibodies. These studies were performed to help elucidate the regions and residues important in HCV E1E2: receptor interaction and their interplay with each other and with neutralising antibodies

Mining method for cancer and pre-cancer detection caused by mutant codon 248 in TP53

Process of prediction has a substantial function in detecting and efficient protection of cancer. The tumor suppressor P53 is approximately near 50% of all human beings tumors due to the mutations which is appear in the TP53 gene to the cells within updated UMD TP53 Mutation Database Oct. 2017 [1], it is so difficult working with prime data (in excel) to predict and diagnosis cancers. In this research a functional model of mining approach and Artificial Neural Network which is proposed to predict cancer and pre-cancer caused by specific codon mutation (each codon has hundreds mutations cause cancers) of tumor protein P53, and applied this approach on mutability of hotspot codon 248 (exon 7), CGG. The Quick Propagation mechanism has been used for training and testing the Neural Network structure to determine the accuracy of the proposed architecture. This research procedure demonstrates that Neural Network based prediction of Cancer and Premalignant Disease (pre-cancer) of mutated codon 248 and manifests perfect performance in the prognosis of the mutation situation to pre-cancer or cancer in general. Using of data mining preprocessing steps and pattern extraction to construct the prediction model by selecting (8) out of (132) new TP53 gene database fields in order to classify the cases to the target class pathology (Cancer, Pre-cancer) using these fields. A high professional Neural Network software simulation (Alyuda NeuroIntellegence) is used to build the classifier and Neural Network, the testing and experimental results from the proposed architecture shows that using Quick Propagation algorithm is very accurate in term of accuracy and minimum error rates showing the results of accuracy (99.97%, 100%, 99.85%) for (Train, Validation and Test) phases respectively with error rate of (0.0003, 0, 0.0015) for (Train, Validation and Test) phases respectively

The CoroPrevention-SDM Approach : A Technology-supported Shared Decision Making Approach for a Comprehensive Secondary Prevention Program for Cardiac Patients

After a cardiac event, secondary prevention is recommended to foster recovery and reduce the risk of recurrent events. European guidelines and EAPC position statements on prevention of cardiovascular diseases recommend a holistic approach that actively engages patients by using shared decision making (SDM). It has been demonstrated that telerehabilitation can be a feasible and effective add-on or alternative compared to conventional in-hospital secondary prevention. However, till date, there is no eHealth solution that offers a holistic approach for secondary prevention that includes SDM. In this paper, we present the CoroPrevention-SDM approach, a technology-supported shared decision making approach for a comprehensive secondary prevention program for cardiac patients. The CoroPrevention Tool Suite consists of three applications that support patients and caregivers in following this approach: 1) a caregiver dashboard that includes decision support systems and supports SDM, 2) a patient mobile application that supports patients in making behaviour changes in their daily life, and 3) an extended ePRO application that collects patient reported outcomes and patient preferences. In a formative usability study, we assessed patients’ and caregivers’ opinion about our approach. The study indicated that both are willing to use our proposed approach to collaboratively set behavioural goals during SDM encounters.publishedVersionPeer reviewe

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codonswitching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes

A mobile application to perform the Six-Minute Walk Test (6MWT) at home : a random walk in the park is as accurate as a standardized 6MWT

The six-minute walk test (6MWT) provides an objective measurement of a person’s functional exercise capacity. In this study, we developed a smartphone application that allows cardiac patients to do a self-administered 6MWT at home on a random trajectory. In a prospective study with 102 cardiovascular disease patients, we aimed to identify the optimal circumstances to perform a smartphone-measured 6MWT, i.e., the best algorithm and the best position to wear the smartphone during the test. Furthermore, we investigated if a random walk is as accurate as a standardized 6MWT. When considering both the reliability and accuracy of the distance walked, the best circumstances to perform a standardized smartphone-measured 6MWT are wearing the smartphone in a strap around the patient’s arm and using an algorithm that relies on the processed step count data acquired from Google Fit. Furthermore, we demonstrated that a smartphone-measured walk along a random trajectory is as accurate to determine a cardiac patient’s functional exercise capacity as a standardized (smartphone-measured) 6MWT. We conclude this paper by presenting how our 6MWT application can be used in a home setting to remotely follow up on cardiac patients’ functional exercise capacity