Functional analysis of a human A1 adenosine receptor/green fluorescent protein/Gi1α fusion protein following stable expression in CHO cells

AbstractFusion proteins between the human A1 adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein α subunit Gi1α (A1/Gi), and between the human A1 adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly Gi1α (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [35S]GTPγS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [35S]GTPγS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein α subunit

Process development for improved Car-t production utilizing an automated single use perfusion stirred-tank bioreactor

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T-cells (CAR-T), have generated significant clinical and commercial outcomes due to their unparalleled response rates against refractory/relapsed blood cancers. However, the development and manufacture of these advanced therapies face a number of translational bottlenecks that must be addressed to ensure long-term commercial viability. The cost and variability associated with these personalized advanced therapies presents a critical manufacturing and translational challenge. Due to the nature of cell therapies, single-use technologies are typically used for their production. This work demonstrates the importance of determining critical manufacturing process parameters using single-use technologies and incorporating automation into the process. The work builds off of previous proof of concept work completed in the group for the production of CAR-T cells in automated, stirred tank, single-use ambr250® bioreactors. These follow-on experiments utilize concepts of quality by design and design of experiments (DoE) to systematically determine the impact of process parameters on CAR-T production. Please click Additional Files below to see the full abstract

Process development for improved Car-t production utilizing an automated perfusion stirred-tank bioreactor

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T-cells (CAR-T), have generated significant clinical and commercial outcomes due to their unparalleled response rates against refractory/relapsed blood cancers. However, the development and manufacture of these advanced therapies face a number of translational bottlenecks that must be addressed to ensure long-term commercial viability. Please click Additional Files below to see the full abstract

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours

Purpose: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials. Experimental design: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules. Results: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma. Conclusions: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly

One naive T cell, multiple fates in CD8+ T cell differentiation

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR–pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets

Tomato: a crop species amenable to improvement by cellular and molecular methods

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

Synthetic Social Support: Theorizing Lay Health Worker Interventions

Levels of social support are strongly associated with health outcomes and inequalities. The use of lay health workers (LHWs) has been suggested by policy makers across the world as an intervention to identify risks to health and to promote health, particularly in disadvantaged communities. However, there have been few attempts to theorize the work undertaken by LHWs to understand how interventions work. In this article, the authors present the concept of 'synthetic socialsupport' and distinguish it from the work of health professionals or the spontaneous social support received from friends and family. The authors provide new empirical data to illustrate the concept based on qualitative, observational research, using a novel shadowing method involving clinical and non-clinical researchers, on the everyday work of 'pregnancy outreach workers' (POWs) in Birmingham, UK. The service was being evaluated as part of a randomized controlled trial. These LHWs provided instrumental, informational, emotional and appraisal support to the women they worked with, which are all key components of social support. The social support was 'synthetic' because it was distinct from the support embedded in spontaneous social networks: it was non-reciprocal; it was offered on a strictly time-limited basis; the LHWs were accountable for the relationship, and the social networks produced were targeted rather than spontaneous. The latter two qualities of this synthetic form of social support may have benefits over spontaneous networks by improving the opportunities for the cultivation of new relationships (both strong and weak ties) outside the women's existing spontaneous networks that can have a positive impact on them and by offering a reliable source of health information and support in a chaotic environment. The concept of SSS can help inform policy makers about how deploying lay workers may enable them to achieve desired outcomes, specify their programme theories and evaluate accordingly. [Abstract copyright: Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Shared genetic contribution to ischemic stroke and Alzheimer's disease

Objective Increasing evidence suggests epidemiological and pathological links between Alzheimer's disease (AD) and ischemic stroke (IS). We investigated the evidence that shared genetic factors underpin the two diseases. Methods Using genome-wide association study (GWAS) data from METASTROKE + (15,916 IS cases and 68,826 controls) and the International Genomics of Alzheimer's Project (IGAP; 17,008 AD cases and 37,154 controls), we evaluated known associations with AD and IS. On the subset of data for which we could obtain compatible genotype-level data (4,610 IS cases, 1,281 AD cases, and 14,320 controls), we estimated the genome-wide genetic correlation (rG) between AD and IS, and the three subtypes (cardioembolic, small vessel, and large vessel), using genome-wide single-nucleotide polymorphism (SNP) data. We then performed a meta-analysis and pathway analysis in the combined AD and small vessel stroke data sets to identify the SNPs and molecular pathways through which disease risk may be conferred. Results We found evidence of a shared genetic contribution between AD and small vessel stroke (rG [standard error] = 0.37 [0.17]; p = 0.011). Conversely, there was no evidence to support shared genetic factors in AD and IS overall or with the other stroke subtypes. Of the known GWAS associations with IS or AD, none reached significance for association with the other trait (or stroke subtypes). A meta-analysis of AD IGAP and METASTROKE + small vessel stroke GWAS data highlighted a region (ATP5H/KCTD2/ICT1) associated with both diseases (p = 1.8 × 10-8). A pathway analysis identified four associated pathways involving cholesterol transport and immune response. Interpretation Our findings indicate shared genetic susceptibility to AD and small vessel stroke and highlight potential causal pathways and loci. Ann Neurol 2016;79:739-74

The contribution of musculoskeletal disorders in multimorbidity: Implications for practice and policy

People frequently live for many years with multiple chronic conditions (multimorbidity) that impair health outcomes and are expensive to manage. Multimorbidity has been shown to reduce quality of life and increase mortality. People with multimorbidity also rely more heavily on health and care services and have poorer work outcomes. Musculoskeletal disorders (MSDs) are ubiquitous in multimorbidity because of their high prevalence, shared risk factors, and shared pathogenic processes amongst other long-term conditions. Additionally, these conditions significantly contribute to the total impact of multimorbidity, having been shown to reduce quality of life, increase work disability, and increase treatment burden and healthcare costs. For people living with multimorbidity, MSDs could impair the ability to cope and maintain health and independence, leading to precipitous physical and social decline. Recognition, by health professionals, policymakers, non-profit organisations, and research funders, of the impact of musculoskeletal health in multimorbidity is essential when planning support for people living with multimorbidity