Intracellular localization and interaction of mRNA binding proteins as detected by FRET

<p>Abstract</p> <p>Background</p> <p>A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the <it>in vivo </it>composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.</p> <p>Results</p> <p>All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, <it>in vitro </it>transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1.</p> <p>Conclusions</p> <p>Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.</p

Adjustment to short-term imprisonment under low prison staffing

Aims and method To understand experience of early imprisonment in one prison under low staffing levels. A researcher, independent of the prison, interviewed each prisoner soon after reception and 3–4 weeks later. The first question of the second interview was: ‘I’d like to start by asking you about your experience of the last 3–4 weeks in prison'. Data are verbatim answers to this. Narratives were brief, so responses from all 130 participants were analysed, using grounded theory methods. Results The core experience was of ‘routine’ – characterised by repetitive acts of daily living and basic work, and little reference to life outside prison – generally resolved passively, towards boredom and ‘entrapment’. Clinical implications This ‘routine’ seems akin to the ‘institutionalism’ described in the end days of the 1960s’ mental hospitals. In an earlier study of similar men at a similar stage of imprisonment, under higher staff:prisoner ratios, experience was initially more distressing, but resolved actively and positively, suggesting that staff loss may have affected rehabilitative climate

Remorse in psychotic violent offenders: an overvalued idea?

Expressing remorse – or not – appears to influence criminal justice outcomes, but preliminary exploration of both judicial and psychological concepts suggests they lack clarity. We asked the following questions: does psychosis impair capacity for, or expression of, remorse for a homicide or other serious harm to others? Is failure to express remorse for an offence associated with recidivism? We conducted systematic reviews of empirical literature on remorse for serious violence while psychotic, and on relationships between remorse and reoffending regardless of mental state. No articles on remorse for homicide or other serious violence while psychotic were identified. There is weak evidence that lack of remorse is associated with reoffending generally, but nothing specific to psychosis. The literature is strong enough to support a case for research into valid measurement of remorse for offending, associations of such measures with recidivism, and whether a change in remorse can be effected – or matters. It is not strong enough to support reliance on perceptions of the presence or absence of remorse as a basis for judicial decisions

How stigma impacts on people with psychosis: The mediating effect of self-esteem and hopelessness on subjective recovery and psychotic experiences

This study aimed to examine how stigma impacts on symptomatic and subjective recovery from psychosis, both concurrently and longitudinally. We also aimed to investigate whether self-esteem and hopelessness mediated the observed associations between stigma and outcomes. 80 service-users with psychosis completed symptom (Positive and Negative Syndrome Scale) and subjective recovery measures (Process of Recovery Questionnaire) at baseline and 6-months later, and also completed the King Stigma Scale, the Self-Esteem Rating Scale and the Beck Hopelessness Scale at baseline. In cross sectional regression and multiple mediation analyses of the baseline data, we found that stigma predicted both symptomatic and subjective recovery, and the effects of stigma on these outcomes were mediated by hopelessness and self-esteem. When the follow-up data were examined, stigma at baseline continued to predict recovery judgements and symptoms. However, self-esteem only mediated the effect of stigma on PANSS passive social withdrawal. Self-esteem and hopelessness should be considered in interventions to reduce the effects of stigma. Interventions that address the current and long-term effects of stigma may positively affect outcome for people being treated for psychosis

Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration

BACKGROUND: Progressive neuron loss in the frontal and temporal lobes of the cerebral cortex typifies frontotemporal lobar degeneration (FTLD). FTLD sub types are classified on the basis of neuronal aggregated protein deposits, typically containing either aberrantly phosphorylated TDP-43 or tau. Our recent work demonstrated that tau tubulin kinases 1 and 2 (TTBK1/2) robustly phosphorylate TDP-43 and co-localize with phosphorylated TDP-43 in human postmortem neurons from FTLD patients. Both TTBK1 and TTBK2 were initially identified as tau kinases and TTBK1 has been shown to phosphorylate tau epitopes commonly observed in Alzheimer's disease and other tauopathies. METHODS: To further elucidate how TTBK1/2 activity contributes to both TDP-43 and tau phosphorylation in the context of the neurodegeneration seen in FTLD, we examined the consequences of elevated human TTBK1/2 kinase expression in transgenic animal models of disease. RESULTS: We show that C. elegans co-expressing tau/TTBK1 tau/TTBK2, or TDP-43/TTBK1 transgenes in combination exhibit synergistic exacerbation of behavioral abnormalities and increased pathological protein phosphorylation. We also show that C. elegans co-expressing tau/TTBK1 or tau/TTBK2 transgenes in combination exhibit aberrant neuronal architecture and neuron loss. Surprisingly, the TTBK2/TDP-43 transgenic combination showed no exacerbation of TDP-43 proteinopathy related phenotypes. Additionally, we observed elevated TTBK1/2 protein expression in cortical and hippocampal neurons of FTLD-tau and FTLD-TDP cases relative to normal controls. CONCLUSIONS: Our findings suggest a possible etiology for the two most common FTLD subtypes through a kinase activation driven mechanism of neurodegeneration

Benefits and risks of conjugal visits in prison: A systematic literature review

Background Imprisonment impacts on lives beyond the prisoner's. In particular, family and intimate relationships are affected. Only some countries permit private conjugal visits in prison between a prisoner and community living partner. Aims Our aim was to find evidence from published international literature on the safety, benefits or harms of such visits. Methods A systematic literature review was conducted using broad search terms, including words like ‘private’ and ‘family’, to maximise search sensitivity but strict criteria for inclusion – of visits unobserved by prison staff and away from other prisoners. All included papers were quality assessed. Two of us independently extracted data from included papers, according to a prepared checklist. Meta-analysis was considered. Results Seventeen papers were identified from 12 independent studies, all but three of them from North America. The only study of health benefits found a positive association with maintaining sexual relationships. The three before-and-after study of partnership qualities suggested benefit, but conjugal visiting was within a wider family-support programme. Studies with in-prison behaviour as a possible outcome suggest small, if any, association, although one US-wide study found significantly fewer in-prison sexual assaults in states allowing conjugal visiting than those not. Other studies were of prisoner, staff or partner attitudes. There is little evidence of adverse effects, although two qualitative studies raise concerns about the visiting partner's sense of institutionalisation or coercion. Conclusions The balance of evidence about conjugal visiting is positive, but there is little of it. As stable family relationships have, elsewhere, been associated with desistance from crime, the contribution of conjugal visiting to these should be better researched

Induced ferroelectric phases in SrTiO3 by a nanocomposite approach

Inducing new phases in thick films via vertical lattice strain is one of the critical advantages of vertically aligned nanocomposites (VANs). In SrTiO3 (STO), the ground state is ferroelastic, and the ferroelectricity in STO is suppressed by the orthorhombic transition. Here, we explore whether vertical lattice strain in three-dimensional VANs can be used to induce new ferroelectric phases in SrTiO3:MgO (STO:MgO) VAN thin films. The STO:MgO system incorporates ordered, vertically aligned MgO nanopillars into a STO film matrix. Strong lattice coupling between STO and MgO imposes a large lattice strain in the STO film. We have investigated ferroelectricity in the STO phase, existing up to room temperature, using piezoresponse force microscopy, phase field simulation and second harmonic generation. We also serendipitously discovered the formation of metastable TiO nanocores in MgO nanopillars embedded in the STO film matrix. Our results emphasize the design of new phases via vertical epitaxial strain in VAN thin films