ATP Reception and Chemosensory Adaptation in \u3c/i\u3eTetrahymena thermophila\u3c/i\u3e

Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog β- γ -methylene ATP are both effective depolarizing chemorepellents in Tetrahymena thermophila. Chemorepellent behavior consists of repeated bouts of backward swimming (avoidance reactions) that can easily be quantified to provide a convenient bioassay for purinergic reception studies. Chemosensory adaptation occurs following prolonged exposure (10 min) to the repellents, and cells regain normal swimming behavior. Adaptation is specific since cells that are behaviorally adapted to either ATP or β- γ -methylene ATP still retain full responsiveness to the chemorepellents GTP and lysozyme. However, cross adaptation occurs between ATP and β- γ -methylene ATP, suggesting that they involve the same receptor. Behavioral sensitivity to both ATP and β- γ -methylene ATP is increased by the addition of Na+, but addition of either Ca2+ or Mg2+ dramatically decreases the response to ATP. These ionic effects are correlated with in vivo ATP hydrolysis, suggesting that divalent ions decrease purinergic sensitivity by activating a Ca2+- or Mg2+-dependent ecto-ATPase to hydrolyze the ATP signal. In vivo [32P]ATP binding studies and Scatchard analysis suggest that the behavioral adaptation is due to a decrease in the number of surface binding sites, as represented by decreased Bmax values. All these changes are reversible (de-adaptation) after 12 min in a repellent-free buffer. Electrophysiological analysis showed that both β- γ -methylene ATP (10 micromol l-1) and ATP (500 micromol l-1) elicited sustained, reversible depolarizations while GTP (10 micromol l-1) produced a transient depolarization, suggesting that the chemosensory response pathways for ATP and GTP reception may differ. There may be separate ATP and GTP receptors since ATP and GTP responses do not cross-adapt and ‘cold’ (unlabeled) GTP is not a good inhibitor of [32P]ATP binding. These results suggests that T. thermophila possess high-affinity surface receptors for ATP that are down-regulated during chemosensory adaptation. These ATP receptors may act as chemorepellent receptors to enable T. thermophila to recognize recently lysed cells and avoid a possibly deleterious situation. This is the simplest eukaryotic organism to show an electrophysiological response to external ATP

ACUTE AND SUB CHRONIC TOXICITY STUDIES OF PURIFIED WITHANIA SOMNIFERA EXTRACT IN RATS

Objective: The objective of the present study was to evaluate the acute and sub-chronic (90 d; repeated dose) toxicity of Withania somnifera (ashwagandha) extract in rats.Methods: The acute toxicity was evaluated as per OECD (Organisation for Economic Co-operation and Development) guidelines 423. Purified ashwagandha extract (PAE) was fed at 2000 mg/kg body weight (bw) to overnight fasted female rats. The animals were observed daily for clinical signs of abnormality/mortality. After 14 d, animals were sacrificed and gross pathological changes were recorded. Sub-chronic toxicity of PAE was studied by feeding the extract at 100, 500 and 1000 mg/kg bw daily to rats as per OECD guidelines 408. After 90 d feeding, heamatological and biochemical parameters of treated rats were compared with control animals. Histopathology of all the major organs was also studied.Results: In the acute toxicity study, no mortality or clinical signs of toxicity were observed in any of the animals at maximum recommended dose level of 2000 mg/kg bw; therefore the LD50 is>2000 mg/kg bw in rats. The repeated administration of PAE for 90 d in rats at the maximum dose level of 1000 mg/kg bw did not induce any observable toxic effects, when compared to its corresponding control animals. The hematology and biochemistry profile of treated rats was similar to control animals and difference was non-significant (p>0.05). The histopathology of major organs of all the control and treated animals was normal. In this study the NOAEL (No Observed Adverse Effect Level) was calculated as 1000 mg/kg bw daily for rats.Conclusion: The present study clearly indicates that PAE does not have any toxic effects in animals at the dose evaluated as evidenced by acute and sub chronic toxicity studies in rats

Netrin-3 and Netrin-4-Like Proteins are Secreted from \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are signaling proteins, acting as chemorepellants or chemoattractants, and their role is especially important in early growth in organisms. In studies involving Tetrahymena thermophila, netrin proteins often act as chemorepellants, so research centered around verifying if this was also true for Netrin-4 protein. Since Netrin-1 and Netrin-3 have been shown to influence neurological and developmental growth in organisms, the implications for discovering the cellular effects of Netrin-4 are significant for human health and research. Through behavioral assays, we were able to confirm that Netrin4 does act as a chemorepellant. In addition, our ELISA and Western blots also helped substantiate the idea that Tetrahymena produce Netrin-4 for physiological functions, as they possess receptors for these proteins. The exact purposes of Netrin-4 for this organism is unknown up to this point, so further testing is needed to determine the cellular mechanisms with which Netrin-4 is involved

Mapping Netrin Signaling in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrin family of proteins, found throughout the animal kingdom, are well known for their roles in developmental signaling. Netrin-1, the best-studied member of this family, signals through four receptor types in vertebrates: the UNC-5 family, DCC, neogenin, and DSCAM. We have previously characterized a netrin-1-like protein in the ciliated protozoan, Tetrahymena thermophila. This protein is secreted from Tetrahymena, and functions as a chemorepellent. Since a netrin-like protein is produced by this organism, we hypothesized that some components of the vertebrate netrin signaling pathway might also be present in Tetrahymena. Through immunolocalization on the plasma membrane of the cell, we have found that Tetrahymena appear to have a UNC-5 like protein, as well as proteins that are immunologically similar to neogenin. A homolog of src-1, a tyrosine kinase involved in vertebrate netrin-1, is also present in Tetrahymena. Future experiments will allow us to make more comparisons between netrin signaling in Tetrahymena with netrin signaling in the animal kingdom, and will allow us to determine the suitability of Tetrahymena as a model system for this particular pathway

How the Western Was Won: Evidence for Netrin Signaling Machinery in Tetrahymena thermophila

Netrins are pleiotropic signaling molecules with diverse roles in animal development. Netrin signals through a number of receptors in animals, including the UNC-5 family, neogenin, DSCAM, and DCC. Previous studies have shown that netrin-1-peptide, netrin-3-peptide, and recombinant netrin-4 all act as chemorepellents in Tetrahymena (Kuruvilla et al., 2016, Khol et al., 2018; Bradley and Kuruvilla, 2020). In addition, netrin-1 peptide appears to signal through a tyrosine kinase in this organism (Kuruvilla et al., 2016), similar to vertebrate signaling through UNC-5, which uses the tyrosine kinase, src. In light of these data, we hypothesized that Tetrahymena thermophila possess netrin signaling machinery, including a tyrosine kinase. In order to investigate this hypothesis, we searched for various netrin receptors, as well as a src homologue, in Tetrahymena using immunofluorescence (Khol et al., 2018). We found that anti-UNC-5 and anti-neogenin antibodies showed fluorescence, while anti-DCC and anti-DSCAM antibodies did not. In addition, an anti-src antibody showed significant fluorescence in Tetrahymena (Khol et al., 2018. In our current study, we searched the Tetrahymena Genome Database for homologs of UNC-5, neogenin, and src. We also used Western blotting to screen for potential homologues of these proteins. At the present time, there are several proteins of interest which we would like to study further

Biochemical Evidence for Netrin-Signaling Homologues in Tetrahymena thermophila

Netrins are pleiotropic guidance proteins that are involved in developmental signaling of branched structures within vertebrates. However, like many developmental pathways, dysregulation of the netrin pathway has been implicated in cancer progression and metastasis. Since Tetrahymena respond to guidance proteins, showing chemoattractant and chemorepellent behavior, we hypothesized that we could use these organisms as a model system for cancer signaling. We have previously found that netrin-1-peptided, netrin-3-peptide, and recombinant netrin-4 are all chemorepellents in this organism. Since netrin-1-peptide signals through a tyrosine kinase in Tetrahymena, we hypothesized that Tetrahymena might possess tyrosine kinases as well as a receptor homologous to UNC-5, a netrin receptor which relays signals via tyrosine kinases in vertebrates. Using immunoprecipitation with a polyclonal anti-UNC-5-B antibody, we purified a 250 kD protein from Tetrahymena whole cell extract. Similarly, we immunoprecipitated several proteins, including a 60 kD protein and a 75 kD protein using a polyclonal anti-src-antibody. Our purified samples were sent out for identification by mass spectroscopy. Mass spectroscopy indicated that we have purified a number of novel peptides not currently found in the Tetrahymena Genome Database. Our data indicate that the proteome database in this organism is incomplete, and that there are additional proteins waiting to be discovered in this organism

The feasibility of determining the impact of primary health care research projects using the Payback Framework

<p>Abstract</p> <p>Background</p> <p>Primary health care research is under pressure to be accountable to funders in terms of benefits for practice and policy. However, methods to assess the impact of primary health care research must be appropriate to use with the diverse topics, settings and approaches of this sector. This project explored the feasibility of using the Buxton and Hanney Payback Framework to determine the impact of a stratified random sample (n = 4) of competitively funded, primary health care research projects.</p> <p>Methods</p> <p>The project conducted telephone interviews based on the Payback Framework with leaders of the research teams and nominated users of their research, used bibliometric methods for assessing impact through publication outputs and obtained documentary evidence of impact where possible. The purpose was to determine the effectiveness of the data collection methods and the applicability of the Payback Framework, and any other issues which arose around the assessment of impact of primary health care research.</p> <p>Results and discussion</p> <p>The thirteen interviews were resource intensive to organise conduct and analyse but provided better information about impact than bibliometric analysis or documentary analysis. Bibliometric analysis of the papers published from the four projects was hampered by the inclusion of only one of the journals in major citation indexes. Document analysis provided more evidence of dissemination than of impact.</p> <p>The payback framework and logic model were a sound basis for assessing impact. Chief investigators and nominated users of research provided substantial information relevant to the impact categories closest to their spheres of influence and awareness, but less about the impact their research had on the wider health sector, population health or economic benefits. An additional category of impact emerged from the interviews, that of strengthening research networks which could enhance the impact of later work. The framework provided rich information about the pathways to impact, better understanding of which may enhance impact.</p> <p>Conclusion</p> <p>It is feasible to use the Buxton and Hanney Payback framework and logic model to determine the proximal impacts of primary health care research. Though resource intensive, telephone interviews of chief investigators and nominated users provided rich information.</p

Microglial activation correlates <em>in vivo</em> with both tau and amyloid in Alzheimer’s disease

Alzheimer’s disease is characterised by the histopathological presence of β-amyloid plaques and tau containing neurofibrillary tangles. Microglial activation is also a recognised pathological component. The relationship between microglial activation and protein aggregation is still debated. We investigated the relationship between amyloid plaques, tau tangles and activated microglia using PET imaging. Fifty-one subjects (nineteen healthy controls, sixteen mild cognitive impairment (MCI) and sixteen Alzheimer’s disease subjects) participated in the study. All subjects had neuropsychometric testing, magnetic resonance imaging (MRI), amyloid (18F-flutemetamol), and microglial (11C-PBR28) PET. All MCI and Alzheimer’s disease (AD) subjects and eight of the controls had tau (18F-AV1451) PET. 11CPBR28 PET was analysed using Logan graphical analysis with an arterial plasma input function, while 18F-flutemetamol and 18F-AV1451 PET were analysed as target: cerebellar ratios to create parametric Standardised Uptake Value Ratio (SUVR) maps. Biological parametric mapping (BPM) in the Statistical Parametric Mapping platform was used to examine correlations between uptake of tracers at a voxel-level. There were significant widespread clusters of positive correlation between levels of microglial activation and tau aggregation in both the MCI (amyloid positive and amyloid negative) and AD subjects. The correlations were stronger in AD than in MCI, suggesting that these pathologies increase together as disease progresses. Levels of microglial activation and amyloid deposition were also correlated, although in a different spatial distribution; correlations were stronger in MCI than Alzheimer’s subjects, in line with a plateauing of amyloid load with disease progression. Clusters of positive correlations between microglial activation and protein aggregation often targeted similar areas of association cortex, indicating that all three processes are present in specific vulnerable brain areas. For the first time using PET imaging, we show that microglial activation can correlate with both tau aggregation and amyloid deposition. This confirms the complex relationship between these processes. These results suggest that preventative treatment for Alzheimer’s disease should target all three processes

Who needs what from a national health research system: Lessons from reforms to the English Department of Health's R&D system

This article has been made available through the Brunel Open Access Publishing Fund.Health research systems consist of diverse groups who have some role in health research, but the boundaries around such a system are not clear-cut. To explore what various stakeholders need we reviewed the literature including that on the history of English health R&D reforms, and we also applied some relevant conceptual frameworks. We first describe the needs and capabilities of the main groups of stakeholders in health research systems, and explain key features of policymaking systems within which these stakeholders operate in the UK. The five groups are policymakers (and health care managers), health professionals, patients and the general public, industry, and researchers. As individuals and as organisations they have a range of needs from the health research system, but should also develop specific capabilities in order to contribute effectively to the system and benefit from it. Second, we discuss key phases of reform in the development of the English health research system over four decades - especially that of the English Department of Health's R&D system - and identify how far legitimate demands of key stakeholder interests were addressed. Third, in drawing lessons we highlight points emerging from contemporary reports, but also attempt to identify issues through application of relevant conceptual frameworks. The main lessons are: the importance of comprehensively addressing the diverse needs of various interacting institutions and stakeholders; the desirability of developing facilitating mechanisms at interfaces between the health research system and its various stakeholders; and the importance of additional money in being able to expand the scope of the health research system whilst maintaining support for basic science. We conclude that the latest health R&D strategy in England builds on recent progress and tackles acknowledged weaknesses. The strategy goes a considerable way to identifying and more effectively meeting the needs of key groups such as medical academics, patients and industry, and has been remarkably successful in increasing the funding for health research. There are still areas that might benefit from further recognition and resourcing, but the lessons identified, and progress made by the reforms are relevant for the design and coordination of national health research systems beyond England.This article is available through the Brunel Open Access Publishing Fund