Membrane domain localization of HIV-1 subtype C gp41 and receptor proteins in cultured HIV-1 target cell lines

MSc (Med) Molecular Medicine and Haematology, Faculty of Health Sciences, University of the WitwatersrandIn recent years there has been much progress in understanding and defining the key protein structure-function relationships that mediate Human Immunodeficiency Virus (HIV-1) entry into host CD4+ cells. This process involves fusion of the virus and host cell membranes, following engagement of corresponding viral (gp120) and target (CD4) receptor proteins. Binding of gp120 to CD4 triggers extensive conformational changes in gp120, exposing binding sites for the co-receptor proteins (CCR5 or CXCR4), and facilitating insertion of gp41, the viral fusion protein, into the target cell membrane. Following insertion of gp41, oligomerisation of fusogenic domains on gp41 is thought to drive the juxtaposition of the virus and host cell and fusion of their membranes. Recent reports suggest that detergent-resistant membrane domains, known as lipid rafts, play a crucial role in orientating the receptor molecules during this step of HIV-1 infection. Lipid rafts are typically rich in cholesterol, sphingolipids and GPI-anchored proteins, and are biophysically distinct from the glycerophosolipid bilayer, which constitutes the bulk of mammalian cell membranes. The role of lipid rafts in virus entry, however, is still controversial, and further experimentation is needed to define their importance in this regard. To provide insight into the role of lipid rafts during HIV-1 entry, we evaluated the natural distribution of the host receptor proteins in HIV-1 target cells (U87.CD4.CCR5/CXCR4). CD4 was detected in membrane samples fractionated by sucrose density gradient centrifugation using immunoblotting techniques, while CCR5 and CXCR4 were detected on whole cells by fluorescence microscopy. We then used a primary CCR5-utilising subtype C HIV-1 isolate (FV5) to characterise dynamic changes in the distribution of these receptors and gp41 during viral entry in real-time. Viral fusion assays were set up by inoculating v target cells with FV5 at 23 ºC, a temperature that allows HIV-1 attachment, but is nonpermissive for advancement of the fusion reaction. This prefusogenic form of the virus-host receptor complex is defined as the temperature-arrested state (TAS). We found that, under normal, uninfected conditions, CD4, CCR5 and CXCR4 are distributed throughout both raft and non-raft microdomains on the U87 cell surface, and there is little evidence for any significant redistribution of these receptors into lipid rafts during the HIV-mediated fusion reaction. Interestingly, we observed a change in the structure of CD4 during the fusion process, which could describe a functionally important event in HIV-1 entry, or be related to compromises in the integrity of the virally-infected membranes. Moreover, we discovered that gp41 is capable of membrane insertion and oligomerisation at TAS, in contrast to previous reports that suggest the fusion peptide is not capable of breaching the membrane at this temperature. Our results provide valuable novel insights into the HIV-1 subtype C entry process, and the involvement of lipid rafts in this stage of the viral lifecycle, that may be relevant to novel therapy and immunogen design

Theory and the breadth-and-depth method of analysing large amounts of qualitative data:A research note

This research note builds on a previously published discussion of the ‘breadth-and-depth’ method for working with extensive amounts of secondary qualitative data, to consider the way that theory can be used and developed as part of this method. We illustrate potential deductive, inductive, and abductive logics of the relationship between theory and data that can be pursued using the method, but note that in reality research analysis rarely proceeds along such clear categorical lines. Rather, qualitative researchers are more likely to pursue an approach akin to a retroductive logic and analytic practice, which the breadth-and-depth method also can accommodate

Twenty + Futures

In a period of heightened awareness of global threats to orderly and predictable futures for people and planet – recession, climate change, peak oil, loss of biodiversity, terrorism – does this uncertainty impact on how young adults in their twenties think about their futures, particularly partnering and parenting? Exploratory interviews with childless young men and women in their twenties sought to investigate

Analysing large volumes of complex qualitative data:Reflections from a group of international experts

This working paper brings together the reflections of a wide range of international researchers to explore, showcase and reflect critically on the potentials and challenges of analysing large volumes of complex qualitative, and qualitative longitudinal (QLR) data, including archived material. Big Qual analysis is a new area for qualitative work and there is little guidance on how best to work with masses of qualitative material. The working paper comprises a set of blogs housed in the ‘Big Qual Analysis Resource Hub’ (http://bigqlr.ncrm.ac.uk/). We created this website to map the progress of our ESRC National Centre for Research Methods research project ‘Working across qualitative longitudinal studies: a feasibility study looking at care and intimacy’ (2015-2019). As part of the project we developed procedures for working with multiple sets of in-depth temporal qualitative data (see Davidson et al. 2019; Edwards et al. 2019 for discussion of our methodological findings). We have gathered together and made available the 27 blog post reflections from 32 authors in this working paper form because accounts of data management and analysis in qualitative research are often sanitised by the time they reach academic journals. Here, our contributors document and share publicly the trials and tribulations, intellectual commitments, contingencies and decision-making processes underlying such analysis, contributing to debates around good practice. We hope that this collection of reflections will promote further conversations about analysis/secondary analysis across large scale and/or multiple qualitative data sets. With guest posts from international scholars, from early career through to established researchers, on topics as varied as the ethics of using Big Qual data, using secondary qualitative material and computer-assisted qualitative data analysis software, this collection of reflections profiles the diversity of work taking place internationally

Identification of a novel class of autotaxin inhibitors through cross-screening

Three novel series were generated in order to mimic the pharmacophoric features displayed by lead compound AM095, a Lysophosphatidic acid (LPA1) receptor antagonist. Biological evaluation of this array of putative LPA1 antagonists led us to the discovery of three novel series of inhibitors of the ecto-enzyme Autotaxin (ATX), responsible for LPA production in blood, with potencies in the range 1 – 4 μM accompanied with good (> 100 μg/mL) solubility

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer‐related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1‐Cre KrasG12D/+ Trp53R172H/+ (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor‐free survival in KPC mice with early‐stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease

Vascularized tissue‐engineered chambers promote survival and function of transplanted islets and improve glycemic control

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154402/1/fsb2fj054879fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154402/2/fsb2fj054879fje-sup-0001.pd

Least restrictive practice: its role in patient independence and recovery

One of thefive overarching principles of the Mental Health Act: Code of Practice is to provide patients with care and treatment which is least restrictive whilst encouraging recovery and promoting independence. However, there is limited research which explores the application of these principles within a medium secure unit. The aims of the research were to explore what are patient’sexperi- ences of least restrictive practices and to what extent do they perceive that least restrictive practices maximise their independence and recovery. Semi-structured interviews were carried out with 12 male inpatients within a medium secure unit. Five themes were evident: Positive Changes, Perceived Lack of Transparency, Social Isolation, Institutionalisation and Normality. It was found that patient’s perceived that there was lack of shared understanding between staffand patients of what is considered least restrictive. Patient recovery was promoted through positive risk-taking, the reduction in the use of seclusion and through the promo- tion of meaningful activities that resembled life in the community. Nevertheless, patients perceived that there was a lack of opportunities to socialise with patients from other wards. Due to the security level of the hospital patients perceived that independence was not achievable