Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: a role in cell survival and neurite outgrowth

NGF (nerve growth factor) and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. The role of TG2 in NGF-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-L-valinyl-L-prolinyl-L-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF

Evidence of oxidative stress and mitochondrial dysfunction in spinocerebellar ataxia type 2 (SCA2) patient fibroblasts: Effect of coenzyme Q10 supplementation on these parameters

Spinocerebellar ataxia type 2 (SCA2) is a rare neurodegenerative disorder caused by a CAG repeat expansion in the ataxin-2 gene. We show increased oxidative stress, abnormalities in the antioxidant system, changes in complexes involved in oxidative phosphorylation and changes in mitochondrial morphology in SCA2 patient fibroblasts compared to controls, and we show that treatment with CoQ10 can partially reverse these changes. Together, our results suggest that oxidative stress and mitochondrial dysfunction may be contributory factors to the pathophysiology of SCA2 and that therapeutic strategies involving manipulation of the antioxidant system could prove to be of clinical benefit

Time-saving opportunities in knee osteoarthritis: T2 mapping and structural imaging of the knee using a single 5-min MRI scan.

OBJECTIVES: To assess the discriminative power of a 5-min quantitative double-echo steady-state (qDESS) sequence for simultaneous T2 measurements of cartilage and meniscus, and structural knee osteoarthritis (OA) assessment, in a clinical OA population, using radiographic knee OA as reference standard. METHODS: Fifty-three subjects were included and divided over three groups based on radiographic and clinical knee OA: 20 subjects with no OA (Kellgren-Lawrence grade (KLG) 0), 18 with mild OA (KLG2), and 15 with moderate OA (KLG3). All patients underwent a 5-min qDESS scan. We measured T2 relaxation times in four cartilage and four meniscus regions of interest (ROIs) and performed structural OA evaluation with the MRI Osteoarthritis Knee Score (MOAKS) using qDESS with multiplanar reformatting. Between-group differences in T2 values and MOAKS were calculated using ANOVA. Correlations of the reference standard (i.e., radiographic knee OA) with T2 and MOAKS were assessed with correlation analyses for ordinal variables. RESULTS: In cartilage, mean T2 values were 36.1 ± SD 4.3, 40.6 ± 5.9, and 47.1 ± 4.3 ms for no, mild, and moderate OA, respectively (p < 0.001). In menisci, mean T2 values were 15 ± 3.6, 17.5 ± 3.8, and 20.6 ± 4.7 ms for no, mild, and moderate OA, respectively (p < 0.001). Statistically significant correlations were found between radiographic OA and T2 and between radiographic OA and MOAKS in all ROIs (p < 0.05). CONCLUSION: Quantitative T2 and structural assessment of cartilage and meniscus, using a single 5-min qDESS scan, can distinguish between different grades of radiographic OA, demonstrating the potential of qDESS as an efficient tool for OA imaging. KEY POINTS: • Quantitative T 2values of cartilage and meniscus as well as structural assessment of the knee with a single 5-min quantitative double-echo steady-state (qDESS) scan can distinguish between different grades of knee osteoarthritis (OA). • Quantitative and structural qDESS-based measurements correlate significantly with the reference standard, radiographic degree of OA, for all cartilage and meniscus regions. • By providing quantitative measurements and diagnostic image quality in one rapid MRI scan, qDESS has great potential for application in large-scale clinical trials in knee OA

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1–10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs

Smart homes and their users:a systematic analysis and key challenges

Published research on smart homes and their users is growing exponentially, yet a clear understanding of who these users are and how they might use smart home technologies is missing from a field being overwhelmingly pushed by technology developers. Through a systematic analysis of peer-reviewed literature on smart homes and their users, this paper takes stock of the dominant research themes and the linkages and disconnects between them. Key findings within each of nine themes are analysed, grouped into three: (1) views of the smart home-functional, instrumental, socio-technical; (2) users and the use of the smart home-prospective users, interactions and decisions, using technologies in the home; and (3) challenges for realising the smart home-hardware and software, design, domestication. These themes are integrated into an organising framework for future research that identifies the presence or absence of cross-cutting relationships between different understandings of smart homes and their users. The usefulness of the organising framework is illustrated in relation to two major concerns-privacy and control-that have been narrowly interpreted to date, precluding deeper insights and potential solutions. Future research on smart homes and their users can benefit by exploring and developing cross-cutting relationships between the research themes identified

Co-designing a theory-informed, multicomponent intervention to increase vaccine uptake with Congolese migrants: A qualitative, community-based participatory research study (LISOLO MALAMU).

INTRODUCTION: Disparities in the uptake of routine and COVID-19 vaccinations have been observed in migrant populations, and attributed to issues of mistrust, access and low vaccine confidence. Participatory research approaches and behaviour change theory hold the potential for developing tailored vaccination interventions that address these complex barriers in partnership with communities and should be explored further. METHODS: This study used a theory-informed, community-based participatory research approach to co-design a culturally tailored behaviour change intervention aimed at increasing COVID-19 vaccine uptake among Congolese migrants in London, United Kingdom (2021-2022). It was designed and led by a community-academic partnership in response to unmet needs in the Congolese community as the COVID-19 pandemic started. Barriers and facilitators to COVID-19 vaccination, information and communication preferences, and intervention suggestions were explored through qualitative in-depth interviews with Congolese migrants, thematically analysed, and mapped to the theoretical domains framework (TDF) and the capability, opportunity, motivation, behaviour model to identify target behaviours and strategies to include in interventions. Interventions were co-designed and tailored in workshops involving Congolese migrants. RESULTS: Thirty-two Congolese adult migrants (24 (75%) women, mean 14.3 (SD: 7.5) years in the United Kingdom, mean age 52.6 (SD: 11.0) years) took part in in-depth interviews and 16 (same sample) took part in co-design workshops. Fourteen barriers and 10 facilitators to COVID-19 vaccination were identified; most barrier data related to four TDF domains (beliefs about consequences; emotion; social influences and environmental context and resources), and the behavioural diagnosis concluded interventions should target improving psychological capability, reflective and automatic motivations and social opportunities. Strategies included culturally tailored behaviour change techniques based on education, persuasion, modelling, enablement and environmental restructuring, which resulted in a co-designed intervention comprising community-led workshops, plays and posters. Findings and interventions were disseminated through a community celebration event. CONCLUSIONS: Our study demonstrates how behavioural theory can be applied to co-designing tailored interventions with underserved migrant communities through a participatory research paradigm to address a range of health issues and inequalities. Future research should build on this empowering approach, with the goal of developing more sensitive vaccination services and interventions which respond to migrant communities' unique cultural needs and realities. PATIENT OR PUBLIC CONTRIBUTION: Patient and public involvement (PPI) were embedded in the participatory study design and approach, with community members co-producing all stages of the study and co-authoring this paper. An independent PPI board (St George's Migrant Health Research Group Patient and Public Involvement Advisory Board) comprising five adult migrants with lived experience of accessing healthcare in the United Kingdom were also consulted at significant points over the course of the study

Preparation of Large Monodisperse Vesicles

Preparation of monodisperse vesicles is important both for research purposes and for practical applications. While the extrusion of vesicles through small pores (∼100 nm in diameter) results in relatively uniform populations of vesicles, extrusion to larger sizes results in very heterogeneous populations of vesicles. Here we report a simple method for preparing large monodisperse multilamellar vesicles through a combination of extrusion and large-pore dialysis. For example, extrusion of polydisperse vesicles through 5-µm-diameter pores eliminates vesicles larger than 5 µm in diameter. Dialysis of extruded vesicles against 3-µm-pore-size polycarbonate membranes eliminates vesicles smaller than 3 µm in diameter, leaving behind a population of monodisperse vesicles with a mean diameter of ∼4 µm. The simplicity of this method makes it an effective tool for laboratory vesicle preparation with potential applications in preparing large monodisperse liposomes for drug delivery