Measuring Endocytosis During Proliferative Cell Quiescence

Quiescence (also called "G0") is the state in which cells have exited the cell cycle but are capable to reenter as required. Though poorly understood, it represents one of the most prevalent cell states across all life. Many biologically important cell types reside in quiescence including mature hepatocytes, endothelial cells, and dormant adult stem cells. Furthermore, the quiescence program occurs in both short- and long-term varieties, depending on the physiological environments. A barrier slowing our understanding of quiescence has been a scarcity of available in vitro model systems to allow for the exploration of key regulatory pathways, such as endocytosis. Endocytosis, the internalization of extracellular material into the cell, is a fundamental and highly regulated process that impacts many cell biological functions. Accordingly, we have developed an in vitro model of deep quiescence in hTERT-immortalized RPE1 cells, combining both long-term contact inhibition and mitogen removal, to measure endocytosis. In addition, we present an analytical approach employing automated high-throughput microscopy and image analysis that yields high-content data allowing for meaningful and statistically robust interpretation. Importantly, the methods presented herein provide a suitable platform that can be easily adapted to investigate other regulatory processes across the cell cycle

Effects of SlowMo, a Blended Digital Therapy Targeting Reasoning, on Paranoia Among People With Psychosis: A Randomized Clinical Trial.

IMPORTANCE: Persistent paranoia is common among patients with psychosis. Cognitive-behavioral therapy for psychosis can be effective. However, challenges in engagement and effectiveness remain. OBJECTIVE: To investigate the effects on paranoia and mechanisms of action of SlowMo, a digitally supported reasoning intervention, plus usual care compared with usual care only. DESIGN, SETTING, AND PARTICIPANTS: This parallel-arm, assessor-blinded, randomized clinical trial recruited participants at UK community health services from May 1, 2017, to May 14, 2019. Eligible participants consisted of a referral sample with schizophrenia-spectrum psychosis and distressing, persistent (≥3 months) paranoia. INTERVENTIONS: Individuals were randomized 1:1 to SlowMo, consisting of 8 digitally supported face-to-face sessions and a mobile app, plus usual care (n = 181) and usual care only (n = 181). MAIN OUTCOMES AND MEASURES: The primary outcome was paranoia, measured by the Green et al Paranoid Thoughts Scale (GPTS) total score at 24 weeks. Secondary outcomes included GPTS total score at 12 weeks and GPTS Part A (reference) and Part B (persecutory) scores, the Psychotic Symptom Rating Scales (PSYRATS Delusion subscale), reasoning (belief flexibility, possibility of being mistaken [Maudsley Assessment of Delusions, rated 0%-100%]), and jumping to conclusions (Beads Task). RESULTS: A total of 361 participants were included in intention-to-treat analysis, of whom 252 (69.8%) were male and 249 (69.0%) were White; the mean (SD) age was 42.6 (11.6) years. At 24 weeks, 332 participants (92.0%) provided primary outcome data. Of 181 participants in the SlowMo group, 145 (80.1%) completed therapy. SlowMo plus usual care was not associated with greater reductions than usual care in GPTS total score at 24 weeks (Cohen d, 0.20; 95% CI, −0.02 to 0.40; P = .06). There were significant effects on secondary paranoia outcomes at 12 weeks, including GPTS total score (Cohen d, 0.30; 95% CI, 0.09-0.51; P = .005), Part A score (Cohen d, 0.22; 95% CI, 0.06-0.39; P = .009), and Part B score (Cohen d, 0.32; 95% CI, 0.08-0.56; P = .009), and at 24 weeks, including Part B score (Cohen d, 0.25; 95% CI, 0.01-0.49; P = .04) but not Part A score (Cohen d, 0.12; 95% CI, −0.05 to 0.28; P = .18). Improvements were observed in an observer-rated measure of persecutory delusions (PSYRATS delusion) at 12 weeks (Cohen d, 0.47; 95% CI, 0.17-0.78; P = .002) and 24 weeks (Cohen d, 0.50; 95% CI, 0.20-0.80; P = .001) and belief flexibility at 12 weeks (Cohen d, 0.29; 95% CI, 0.09-0.49; P = .004) and 24 weeks (Cohen d, 0.28; 95% CI, 0.08-0.49; P = .005). There were no significant effects on jumping to conclusions. Improved belief flexibility and worry mediated paranoia change (range mediated, 36%-56%). CONCLUSIONS AND RELEVANCE: SlowMo did not demonstrate significant improvements in the primary measure of paranoia at 24 weeks; however, a beneficial effect of SlowMo on paranoia was indicated by the results on the primary measure at an earlier point and on observer-rated paranoia and self-reported persecution at 12 and 24 weeks. Further work to optimize SlowMo’s effects is warranted. TRIAL REGISTRATION: isrctn.org Identifier: ISRCTN 3244867

Impact of insulin signaling and proteasomal activity on physiological 2 output of a neuronal circuit in aging D. melanogaster

The insulin family of growth factors plays an important role in development and function of the nervous system. Reduced insulin and insulin-growth-factor signaling (IIS), however, can improve symptoms of neurodegenerative diseases in laboratory model organisms and protect against age-associated decline in neuronal function. Recently, we showed that chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber escape response circuit. Here, we expand our initial findings by demonstrating that reduced functional output in the giant fiber system of aging flies can be prevented by increasing proteasomal activity within the circuit. Manipulations of IIS in neurons can also affect longevity, underscoring the relevance of the nervous system for aging

The impact of Patient and Public Involvement in the SlowMo study: Reflections on peer innovation

BACKGROUND: The SlowMo study demonstrated the effects of SlowMo, an eight-session digitally supported reasoning intervention, on paranoia in a large-scale randomized-controlled trial with 362 participants with schizophrenia-spectrum psychosis. AIM: The current evaluation aimed to investigate the impact of Patient and Public Involvement (PPI) in the SlowMo study. METHOD: PPI members were six women and three men from Sussex, Oxford and London with experience of using mental health services for psychosis. They received training and met at least 3-monthly throughout the project. The impact of PPI was captured quantitatively and qualitatively through (i) a PPI log of recommendations and implementation; (ii) written subjective experiences of PPI members; (iii) meeting minutes; and (iv) outputs produced. RESULTS: The PPI log revealed 107 recommendations arising from PPI meetings, of which 87 (81%) were implemented. Implementation was greater for recruitment-, data collection- and organization-related actions than for dissemination and emergent innovations. Qualitative feedback revealed impacts on study recruitment, data collection, PPI participants' confidence, knowledge, career aspirations and society more widely. Outputs produced included a film about psychosis that aired on BBC primetime television, novel webpages and journal articles. Barriers to PPI impact included geography, travel, funding, co-ordination and well-being. DISCUSSION: A future challenge for PPI impact will be the extent to which peer innovation (innovative PPI-led ideas) can be supported within research study delivery. PATIENT AND PUBLIC CONTRIBUTION: Planned Patient and Public Contribution in SlowMo comprised consultation and collaboration in (i) design, (ii) recruitment, (iii) qualitative interviews and analysis of service users' experiences of SlowMo therapy and (iv) dissemination

The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in Normal Rat Kidney cells

Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family

SlowMo, a digital therapy targeting reasoning in paranoia, versus treatment as usual in the treatment of people who fear harm from others: study protocol for a randomised controlled trial

Background: Paranoia is one of the most common symptoms of schizophrenia-spectrum disorders, and is associated with significant distress and disruption to the person’s life. Developing more effective and accessible psychological interventions for paranoia is a clinical priority. Our research team has approached this challenge in two main ways: firstly, by adopting an interventionist causal approach to increase effectiveness and secondly, by incorporating user-centred inclusive design methods to enhance accessibility and usability. Our resultant new digital intervention, SlowMo, intensively targets a reasoning style associated with paranoia, fast thinking, characterised by jumping to conclusions and belief inflexibility. It consists of an easy-to-use, enjoyable and memorable digital interface. An interactive web-based app facilitates delivery of face-to-face meetings which is then synchronised with an innovative mobile app for use in daily life. Methods/Design: We aim to test the clinical efficacy of SlowMo over 24 weeks to determine the mechanisms through which it reduces paranoia, and to identify participant characteristics that moderate its effectiveness. In a parallel-group randomised controlled trial, with 1:1 allocation, 360 participants with distressing persecutory beliefs will be independently randomised to receive either the SlowMo intervention added to treatment as usual (TAU) or TAU, using randomly varying permuted blocks, stratified by paranoia severity and site. Research workers will be blind to therapy allocation. The primary outcome is paranoia severity over 24 weeks; our hypothesised mechanism of change is reasoning; moderators include negative symptoms and working memory; and secondary outcomes include wellbeing, quality of life, and service use. The accessibility, usability and acceptability of the digital platform will be assessed. Discussion: SlowMo has been developed as the first blended digital therapy to target fears of harm from others through an inclusive design approach. In addition to testing its efficacy, this trial will add to our understanding of psychological mechanisms in paranoia. The study will examine the usability and adherence of a novel digital therapy, including an app for self-management, in a large sample of people affected by severe mental health difficulties

Assessment of carbon in woody plants and soil across a vineyard-woodland landscape

<p>Abstract</p> <p>Background</p> <p>Quantification of ecosystem services, such as carbon (C) storage, can demonstrate the benefits of managing for both production and habitat conservation in agricultural landscapes. In this study, we evaluated C stocks and woody plant diversity across vineyard blocks and adjoining woodland ecosystems (wildlands) for an organic vineyard in northern California. Carbon was measured in soil from 44 one m deep pits, and in aboveground woody biomass from 93 vegetation plots. These data were combined with physical landscape variables to model C stocks using a geographic information system and multivariate linear regression.</p> <p>Results</p> <p>Field data showed wildlands to be heterogeneous in both C stocks and woody tree diversity, reflecting the mosaic of several different vegetation types, and storing on average 36.8 Mg C/ha in aboveground woody biomass and 89.3 Mg C/ha in soil. Not surprisingly, vineyard blocks showed less variation in above- and belowground C, with an average of 3.0 and 84.1 Mg C/ha, respectively.</p> <p>Conclusions</p> <p>This research demonstrates that vineyards managed with practices that conserve some fraction of adjoining wildlands yield benefits for increasing overall C stocks and species and habitat diversity in integrated agricultural landscapes. For such complex landscapes, high resolution spatial modeling is challenging and requires accurate characterization of the landscape by vegetation type, physical structure, sufficient sampling, and allometric equations that relate tree species to each landscape. Geographic information systems and remote sensing techniques are useful for integrating the above variables into an analysis platform to estimate C stocks in these working landscapes, thereby helping land managers qualify for greenhouse gas mitigation credits. Carbon policy in California, however, shows a lack of focus on C stocks compared to emissions, and on agriculture compared to other sectors. Correcting these policy shortcomings could create incentives for ecosystem service provision, including C storage, as well as encourage better farm stewardship and habitat conservation.</p

Interactive effects of pesticide exposure and habitat structure on behavior and predation of a marine larval fish

Coastal development has generated multiple stressors in marine and estuarine ecosystems, including habitat degradation and pollutant exposure, but the effects of these stressors on the ecology of fishes remain poorly understood. We studied the separate and combined effects of an acute 4 h sublethal exposure of the pyrethroid pesticide esfenvalerate and structural habitat complexity on behavior and predation risk of larval topsmelt (Atherinops affinis). Larvae were exposed to four nominal esfenvalerate concentrations (control, 0.12, 0.59, 1.18 μg/L), before placement into 12 L mesocosms with a three-spine stickleback (Gasterosteus aculeatus) predator. Five treatments of artificial eelgrass included a (1) uniform and (2) patchy distribution of eelgrass at a low density (500 shoots per m(2)), a (3) uniform and (4) patchy distribution of eelgrass at a high density (1,000 shoots per m(2)), and (5) the absence of eelgrass. The capture success of predators and aggregative behavior of prey were observed in each mesocosm for 10 min of each trial, and mortality of prey was recorded after 60 min. Exposure to esfenvalerate increased the proportion of larvae with swimming abnormalities. Surprisingly, prey mortality did not increase linearly with pesticide exposure but increased with habitat structure (density of eelgrass), which may have been a consequence of compensating predator behavior. The degree of prey aggregation decreased with both habitat structure and pesticide exposure, suggesting that anti-predator behaviors by prey may have been hampered by the interactive effects of both of these factors