Use of GPI-anchored proteins to study biomolecular interactions by surface plasmon resonance

AbstractSurface plasmon resonance is a powerful tool to examine the kinetics of cell surface receptor-ligand interactions and requires only small amounts of protein. For these studies, one component is required in highly purified form to be coupled to the biosensor surface. The second component does not need to be purified. The human high affinity receptor for immunoglobulin G, FcγRI, presents a problem as the receptor itself cannot readily be produced in large amounts for purification and, as there are eight potential ligands for the receptor (human IgG1–4 and mouse IgG1, 2a, 2b and 3), it is difficult to immobilise the ligand. Using a previously established method for generating GPI-anchored proteins, we have produced and captured a soluble version of FcγRI and shown that it retains its affinity for human IgG1 and specificity for the different IgG subclasses. In addition, we also produced and captured a GPI-anchored version of the cell adhesion molecule CD2. This system circumvents the need for extensive receptor purification and is very rapid as solubilised receptors can be transferred from the cell surface to the sensor chip in 2 h. This system may be generally applicable for biosensor studies to other type I membrane proteins, and/or naturally occurring GPI-anchored proteins, especially where the interaction between a ligand and a panel of variant receptors is to be studied

A beginner's guide to gene editing

Genome editing enables precise changes to be made in the genome of living cells. The technique was originally developed in the 1980′s but largely limited to use in mice. The discovery that a targeted double stranded break (DSB) at a unique site in the genome, close to the site to be changed, could substantially increase the efficiency of editing raised the possibility of using the technique in a broader range of animal models and potentially human cells. But the challenge was to identify reagents that could create targeted breaks at a unique genomic location with minimal off-target effects. In 2005, the demonstration that programmable zinc finger nucleases (ZFNs) could perform this task, led to a number of proof-of-concept studies, but a limitation was the ease with which effective ZFNs could be produced. In 2009, the development of TAL-effector nucleases (TALENs) increased the specificity of gene editing and the ease of design and production. However, it wasn't until 2013 and the development of the CRISPR Cas9/guideRNA that gene editing became a research tool that any lab could use

Metaplastic and Energy-Efficient Biocompatible Graphene Artificial Synaptic Transistors for Enhanced Accuracy Neuromorphic Computing

CMOS-based computing systems that employ the von Neumann architecture are relatively limited when it comes to parallel data storage and processing. In contrast, the human brain is a living computational signal processing unit that operates with extreme parallelism and energy efficiency. Although numerous neuromorphic electronic devices have emerged in the last decade, most of them are rigid or contain materials that are toxic to biological systems. In this work, we report on biocompatible bilayer graphene-based artificial synaptic transistors (BLAST) capable of mimicking synaptic behavior. The BLAST devices leverage a dry ion-selective membrane, enabling long-term potentiation, with ~50 aJ/m^2 switching energy efficiency, at least an order of magnitude lower than previous reports on two-dimensional material-based artificial synapses. The devices show unique metaplasticity, a useful feature for generalizable deep neural networks, and we demonstrate that metaplastic BLASTs outperform ideal linear synapses in classic image classification tasks. With switching energy well below the 1 fJ energy estimated per biological synapse, the proposed devices are powerful candidates for bio-interfaced online learning, bridging the gap between artificial and biological neural networks

AAV2-mediated in vivo immune gene therapy of solid tumours

Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour vasculature and immune cell recruitment

Optimal outcomes from cardiac rehabilitation are associated with longer-term follow-up and risk factor status at 12 months : An observational registry-based study

AIM: The purpose of Cardiac Rehabilitation (CR) is to promote and reduce risk factors in the short and long term, however, the latter has, to date, been poorly evaluated. We explored characteristics associated with provision and outcomes of a long-term assessment in CR. METHOD: Data from the UK National Audit of CR between April 2015 and March 2020 was used. Programmes were selected if they had an established mechanism and routine methodology to collect the 12-month assessments. Risk factors pre and post phase II CR and at the 12-month assessment were explored; BMI ≤30, ≥150 min of physical activity per week, hospital anxiety and depression scale (HADS) scores <8. The data came from 32 programmes, 24,644 patients with coronary heart disease. Patients being in at least one optimal risk factor stage throughout phase II CR (OR = 1.43 95% CI 1.28 to 1.59) or successfully reaching an optimal stage during phase II CR (OR = 1.61 95% CI 1.44 to 1.80) had an increased likelihood of being assessed at 12 months compared to those who did not. Patients being in the optimal stage upon completion of phase II CR had an increased likelihood of still being in the optimal stage at 12 months. Most prominent was BMI; (OR = 14.6 (95% CI 11.1 to 19.2) for patients reaching an optimal stage throughout phase II CR. CONCLUSION: Being in an optimal stage upon routine CR completion could be an overlooked predictor in the provision of a long-term CR service and prediction of longer-term risk factor status

Imaging of X-Ray-Excited Emissions from Quantum Dots and Biological Tissue in Whole Mouse

Optical imaging in clinical and preclinical settings can provide a wealth of biological information, particularly when coupled with targetted nanoparticles, but optical scattering and absorption limit the depth and resolution in both animal and human subjects. Two new hybrid approaches are presented, using the penetrating power of X-rays to increase the depth of optical imaging. Foremost, we demonstrate the excitation by X-rays of quantum-dots (QD) emitting in the near-infrared (NIR), using a clinical X-ray system to map the distribution of QDs at depth in whole mouse. We elicit a clear, spatially-resolved NIR signal from deep organs (brain, liver and kidney) with short (1 second) exposures and tolerable radiation doses that will permit future in vivo applications. Furthermore, X-ray-excited endogenous emission is also detected from whole mouse. The use of keV X-rays to excite emission from QDs and tissue represent novel biomedical imaging technologies, and exploit emerging QDs as optical probes for spatial-temporal molecular imaging at greater depth than previously possible

Cerium dioxide, a Jekyll and Hyde nanomaterial, can increase basal and decrease elevated inflammation and oxidative stress

It was hypothesized that the catalyst nanoceria can increase oxidative stress/inflammation from the basal state and reduce it from the elevated state . Nanoceria are cleared by macrophages. To test the hypothesis, M0 (non-polarized), M1- (classically activated, pro-inflammatory), and M2-like (alternatively activated, regulatory phenotype) RAW 264.7 macrophages were nanoceria exposed. Responses were quantified by arginase activity, IL-1ß level, cell oxygen consumption rate (OCR), the glycolysis stress test (GST), morphology determined by light microscopy, macrophage phenotype marker expression and morphology using a novel three dimensional immunohistochemical method, and RT-qPCR. Nanoceria blocked arginase and IL-1ß effects, increased M0 cell OCR and GST toward the M2 phenotype and altered multiple M1- and M2-like cell endpoints toward the M0 level. M1-like cells had greater volume and less circularity/roundness, and the M2-like cells had greater volume than M0 macrophages. Nanoceria converted M1- and M2-like cells toward M0 morphology. The results are overall consistent with the hypothesis

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction

Objective: The three main types of killer cells – CD8+ T cells, NK cells and NKT cells – have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. Methods: Peripheral CD8+ T cells (CD8+CD3+CD56−), NK cells (CD56+CD3−) and NKT-like cells (CD56+CD3+) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥0.7), and 9 healthy individuals were studied. Results: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. Conclusions: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction

Comparative hazards of chrysotile asbestos and its substitutes: A European perspective.

Although the use of amphibole asbestos (crocidolite and amosite) has been banned in most European countries because of its known effects on the lung and pleura, chrysotile asbestos remains in use in a number of widely used products, notably asbestos cement and friction linings in vehicle brakes and clutches. A ban on chrysotile throughout the European Union for these remaining applications is currently under consideration, but this requires confidence in the safety of substitute materials. The main substitutes for the residual uses of chrysotile are p-aramid, polyvinyl alcohol (PVA), and cellulose fibers, and it is these materials that are evaluated here. Because it critically affects both exposure concentrations and deposition in the lung, diameter is a key determinant of the intrinsic hazard of a fiber; the propensity of a material to release fibers into the air is also important. It is generally accepted that to be pathogenic to the lung or pleura, fibers must be long, thin, and durable; fiber chemistry may also be significant. These basic principles are used in a pragmatic way to form a judgement on the relative safety of the substitute materials, taking into account what is known about their hazardous properties and also the potential for uncontrolled exposures during a lifetime of use (including disposal). We conclude that chrysotile asbestos is intrinsically more hazardous than p-aramid, PVA, or cellulose fibers and that its continued use in asbestos-cement products and friction materials is not justifiable in the face of available technically adequate substitutes