Direct visualization of newly synthesized target proteins in situ

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ

Application of a developed tool to visualize newly synthesized AMPA receptor components in situ

The information flow between neurons happens at contact points, the synapses. One underlying mechanism of learning and memory is the change in the strength of information flow in selected synapses. In order to match the huge demand in membranes and proteins to build and maintain the neurites' complex architecture, neurons use decentralized protein synthesis. Many candidate proteins for local synthesis are known, and the need of de novo synthesis for memory formation is well established. The underlying mechanisms of how somatic versus dendritic synthesis is regulated are yet to be elucidated. Which proteins are newly synthesized in order to allow learning? In this thesis protein synthesis is studied in hippocampal neurons. The fractional distribution of somatic and dendritic synthesis for candidate proteins and their subsequent transport to their destination are investigated using a newly developed technique. In the first part of this study we describe the development of this technique and use it in the second part to answer biological questions. We focus here on AMPA receptor subunits, the key players in fast excitatory transmission. AMPA receptors contain multiple subunits with diverse functions. It remains to be understood, when and where in a neuron these subunits come together to form a protein complex and how the choice of subunits is regulated. The investigation of the subunits' site of synthesis and redistribution kinetics in this study will help us to understand how neurons are able to change their synaptic strength in an input specific manner which eventually allows learning and memory. Key questions which are addressed in this study: How can specific newly synthesized endogenous proteins be visualized in situ? What are the neuron's abilities to locally synthesize and fully assemble AMPA receptor complexes? How fast do different AMPA receptor subunits redistribute within neurons after synthesis

The supportive care needs of carers of people affected by cancer stationed in British Forces Germany

Carers are central to the effective support of people affected by cancer. Little is known however about the specific support needs of carers of military personnel. This study aimed to determine the supportive care needs of unpaid carers within British Forces (including military/civilian personnel, their families and dependent adults) currently stationed in Germany. Qualitative semi-structured interviews were conducted with seven carers (six men and one woman) in three British army garrisons across Germany, to identify core aspects of the experience of caregiving in this environment. Analysis of interviews with carers was based on qualitative thematic and content analysis. Three key themes were identified: (1) impact on carers of their partners being diagnosed with cancer; (2) provision of support; and (3) challenges to accessing and obtaining support. Central issues pivot around the context of working and living in the armed forces and a lack of supportive care infrastructure. While drawing on a small sample size, this study nonetheless identifies that carers of people with cancer within the armed forces, who are stationed in Germany require a specialised and contextually specific set of services to address their supportive care needs

Local and global influences on protein turnover in neurons and glia

Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from 20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover

Synaptic Control of Secretory Trafficking in Dendrites

SummaryLocalized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER) from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. Here, we monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor KIF17 by Ca2+/calmodulin-dependent protein kinases (CaMK). Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport

Cross-national comparison of prescribing patterns in Australian and Dutch nursing homes

Background: Prescribing quality is a major issue in nursing home patients. Few cross-national comparisons of prescribing patterns have been carried out in this population. Objectives: To compare prevalence of medication use in nursing home patients between Australia and The Netherlands. Methods: An analysis of medication use based on pharmacy dispensing data was undertaken for residents in nursing homes in Australia (AU) and the Netherlands (NL). The data included residents >65 years old who remained in a high care nursing home in 2009 in AU (n = 1,560) or NL (n = 2,037). Annual prevalence was defined as the dispensing of 1 or more prescriptions for a drug during the study year expressed as percentage of all residents. Multiple logistic regression was used to calculate the prevalence odds ratios (OR) and associated 95% confidence intervals (CI), adjusted for gender and age. Results: The mean age of residents was 85.8 (SD 7.5) (AU) and 82.8 (SD 7.5) (NL), the majority were female (AU: 70.3%, NL: 68.2%). Residents used a mean of 11.4 (SD 5.3) (AU) and 10.8 (SD 7.0) (NL) drugs. The prevalence of medication use was similar in the two countries for most ATC groups. Major differences were observed in the use of benzodiazepines (anxiolytics: AU: 14.1%, NL: 27.8%, OR 0.41 (0.37-0.53)), osteoporosis medication (AU: 51.2%, NL: 28.9; OR 2.56 (2.22-2.96)). Overall use of antipsychotics (AU: 37.7%, NL: 40.3%; OR 0.91 (0.79-1.04)) was similar, but choice of individual drugs differed, e.g. haloperidol (AU: 8.2%, NL: 19.7%; OR 0.34 (0.27-0.42)) and risperidone (AU: 17.4%, NL: 7.3%; OR 2.86 (2.30-2.57)). Systemic antibacterials (AU: 66.8%, NL 62.4%; OR 1.08 (0.93-1.24)) and cardiovascular system drugs (AU: 73.8%, NL: 72.9%; OR 1.05 (0.90-1.23)) overall were similar but major differences were also found in the choice of agents. Conclusions: There are many similarities, but also striking differences in prescribing patterns for nursing home patients between Australia and the Netherlands. Differences in policies, guidelines, education/training and cultural beliefs are possible explanations. Investigating this further should improve our understanding of the various influences on prescribing in nursing homes.1 page(s