Replacement of dichloromethane within chromatographic purification : a guide to alternative solvents

Replacement of dichloromethane as the bulk medium within chromatographic purification has been evaluated with a broad range of molecules containing functionality common within Medicinal Chemistry programmes. Analysis of the data set has generated a set of general guidelines to assist in the selection of alternative solvents for CH2Cl2 as the bulk media in these ubiquitously employed processes

Rotenone-insensitive NAD(P)H dehydrogenases in plants: Immunodetection and distribution of native proteins in mitochondria.

Antisera produced against peptides deduced from potato nda1 and ndb1, homologues of yeast genes for mitochondrial rotenone-insensitive NADH dehydrogenases, recognise respective proteins upon expression in Escherichiacoli. In western blots of potato (Solanum tuberosum L.) mitochondrial proteins, the NDB and NDA antibodies specifically detect polypeptides of 61 and 48 kDa, respectively. The proteins are found in mitochondria of flowers, leaves and tubers. Different signal intensities are seen relative to other respiratory chain components when organs are compared, indicating variations in relative abundance of dehydrogenases within the plant. The antibodies detect single polypeptides, of similar size as in potato, in mitochondria from several plant species. No specific cross-reaction was found in chloroplasts, but a weak NDA signal of 50 kDa was found in microsomes, possibly associated with peroxisomes. Two- dimensional native/SDS-PAGE analyses indicate that both NDA and NDB proteins reside as higher molecular mass forms, possibly oligomeric. The NDB immunoreactive protein is released by sonication of mitochondria, but resistant to extraction by digitonin and partially to Triton X-100. In comparison, the NDA protein remains bound to the inner membrane at sonication or digitonin treatment, but can be solubilised with Triton. Investigation of a beetroot (Beta vulgaris L.) induction system for external NADH dehydrogenase indicates that the NDB antibody does not recognise the induced external NADH dehydrogenase in this species, but possibly an external NADPH dehydrogenase

A mixed-methods process evaluation of Early signs Monitoring to Prevent relapse in psychosis and prOmote Well-being, Engagement, and Recovery (EMPOWER) – a blended digital intervention for relapse prevention in schizophrenia tested with a feasibility cluster randomised controlled trial in Scotland and Australia

Introduction: schizophrenia is described as a severe mental illness affecting individuals, their families, and society. Many people with schizophrenia across the world do not have access to evidence-based care. User-led digital interventions that deliver treatment to patients in the community may help upscale provision. However, this will only happen if the interventions are implemented, and digital interventions frequently face implementation barriers. Standard clinical trials do not always generate evidence which can help answer questions related to implementation which means additional studies are required. EMPOWER was a user-led relapse prevention mobile app trialled in a feasibility cluster randomised control trial both the UK and Australia. This provided an opportunity to conduct implementation research using process evaluations. Researchers conducting process evaluations are encouraged to engage in cumulative science and build upon previous work conducted in interventions underpinned by similar intervention theory. Methods: Seven studies were conducted. Following two introductory chapters, a systematic review (Chapter 3) summarised what user-led interventions exist and what intervention theory underpinned them. None were like EMPOWER which justified developing a novel process evaluation framework (Chapter 6) underpinned by the qualitative work from Chapters 4 and 5. Chapter 6 revealed key uncertainties such as the recruitment process which resulted in the ethnography conducted for Chapter 7, understanding end user experiences which resulted in the qualitative interviews in Chapter 8, and a need to more fully understand the underlying intervention theory using temporal methods which resulted in the multilevel vector autoregression of ecological momentary assessment data in Chapter 9. Results: Chapter 3 suggested user-led interventions target a variety of problems faced by people with schizophrenia, but the field is young and there is high risk of bias. Additionally, there is low adherence and high dropout suggesting a key need to understand implementation. Chapters 4 and 5 analysed data from focus groups to understand how early warning signs of relapse are managed in clinical care and implementation expectations and the data were used to develop the novel process evaluation framework in Chapter 6. The findings from Chapter 7 suggest trial recruitment process was complex and the patient participants who took part are likely to be a highly selective sample. Two overarching themes were constructed in Chapter 8 that were relevant for understanding end-user experiences within the EMPOWER trial: Affordances and Change Processes. Affordances described the processes underpinning how and why participants interacted with or avoided the various components of the intervention. Affordances spanned all EMPOWER components, including self-monitoring, peer support workers, clinical triaging, self-management messages and diary function. The affordances were Access to Social Connection, Access to Digital, Access to Mental Health Support, the Ability to Gauge Mental Health and Access to Mental Health Information. The affordances framework helped explain the multitude of engagement trajectories observed within the main EMPOWER trial. Chapter 9 found that experiencing fear of relapse predicted next day experiences of depression and anxiety which supports the assumptions of the underlying intervention theory. However, observed effect sizes were very small. Discussion: The findings present a holistic process evaluation of EMPOWER. Across all studies, implementation issues were found to be complex. Conducting qualitative research was essential for developing theory to explain and understand the implementation processes within the EMPOWER trial and highlights the value of conducting implementation research in parallel with RCTs. A revised logic model was created and is presented in Chapter 10 which can be used to evaluate a future full-scale trial

NAD(P) turnover in plant mitochondria

An analytical procedure based on alkaline extraction and HPLC analysis was adapted for quantification of pyridine nucleotides in plant mitochondria. The amounts of NAD and NADP extracted from seven different species varied from 1.0 to 3.7 and 0 to 0.5 nmol (mg protein) –1 , respectively. Although NADP was found in four species, its reduced form was in all cases below the detection limit of 0.1 nmol (mg protein) –1 . The NAD pool was mainly oxidized in the absence of substrates. However, oxidation of substrates followed by anaerobiosis caused 50–92% NAD pool reduction, indicating that the majority of the NAD+ was metabolically active. The NAD reduction level in potato tuber mitochondria oxidizing malate varied with assay conditions. The highest level of reduction (>80%) was reached at anaerobiosis, at pH 6.5 and 7.2, conditions favouring malic enzyme (ME), whereas the lowest reduction level (0%) was observed at pH 7.5, conditions favouring malate dehydrogenase (MDH). Mitochondria incubated at 0°C without respiratory substrate showed a loss of endogenous NAD + which correlated with a decline in the rate of oxidation of NAD+ -linked substrates. The lost NAD+ was mainly recovered as breakdown products in both the surrounding medium and the mitochondria. When submitochondrial fractions were incubated with NAD + or NADP + , the highest rate of NAD(P)+metabolism was detected in the outer membrane fraction. The metabolites detected, adenosine monophosphate (AMP), nicotinamide mononucleotide (NMN) and adenosine, imply that several enzymes involved in pyridine nucleotide degradation, including an NAD pyrophosphatase, are localized to the outer membrane

Global landscape of mouse and human cytokine transcriptional regulation

Cytokines are cell-to-cell signaling proteins that play a central role in immune development, pathogen responses, and diseases. Cytokines are highly regulated at the transcriptional level by combinations of transcription factors (TFs) that recruit cofactors and the transcriptional machinery. Here, we mined through three decades of studies to generate a comprehensive database, CytReg, reporting 843 and 647 interactions between TFs and cytokine genes, in human and mouse respectively. By integrating CytReg with other functional datasets, we determined general principles governing the transcriptional regulation of cytokine genes. In particular, we show a correlation between TF connectivity and immune phenotype and disease, we discuss the balance between tissue-specific and pathogen-activated TFs regulating each cytokine gene, and cooperativity and plasticity in cytokine regulation. We also illustrate the use of our database as a blueprint to predict TF–disease associations and identify potential TF–cytokine regulatory axes in autoimmune diseases. Finally, we discuss research biases in cytokine regulation studies, and use CytReg to predict novel interactions based on co-expression and motif analyses which we further validated experimentally. Overall, this resource provides a framework for the rational design of future cytokine gene regulation studies.National Institutes of Health (NIH) [R00 GM114296 and R35 GM128625 to J.I.F.B., 5T32HL007501-34 to J.A.S.]; National Science Foundation [NSF-REU BIO-1659605 to M.M.]. Funding for open access charge: NIH [R35 GM128625]. (R00 GM114296 - National Institutes of Health (NIH); R35 GM128625 - National Institutes of Health (NIH); 5T32HL007501-34 - National Institutes of Health (NIH); NSF-REU BIO-1659605 - National Science Foundation; R35 GM128625 - NIH)Published versio

Comparative Metabarcoding and Metatranscriptomic Analysis of Microeukaryotes Within Coastal Surface Waters of West Greenland and Northwest Iceland

Climate change alters environmental conditions that are expected to have a profound effect on the biodiversity, community composition, and metabolic processes of microeukaryotic plankton in Arctic and Subarctic coastal waters. The molecular biodiversity [large subunit (LSU) rRNA gene] of three plankton size-fractions (micro-, nano-, and picoplankton) from coastal waters of ice-influenced west Greenland was compared with fractions from ice-free northwest Iceland within their summer environmental context. Putative metabolic functions were determined by differentially expressed mRNA (metatranscriptomics) of the microplankton. Temperature and salinity variations were more closely correlated than inorganic macronutrients with metabolic functions and community composition. Temperature explained much of the community variance, approximately 20% among micro- and nanoplankton, whereas other environmental factors accounted for rather low fractional variance (<7%). Species of smaller cell-size were more evenly distributed (Pielou’s evenness index J) across regions, with a higher diversity and total abundance, and thereby indicating high plasticity. The metatranscriptomic profiles in these respective microeukaryotic communities revealed that diatoms were more plastic in their gene expression than dinoflagellates, but dinoflagellates had a more diverse, albeit homogeneously expressed, gene pool. This could be interpreted as expression of alternative lifestyle strategies, whereby the functionally more conservative diatoms fill their niches primarily through variable resource use, whereas dinoflagellates apparently differentiate their niches through more diverse lifestyles. Patterns of microeukaryotic diversity are thus primarily associated with differences in metabolic function and activity of diatom- versus dinoflagellate-dominated communities in Arctic and Subarctic waters during summer

Effects of Montmorency tart cherry and blueberry juice on cardiometabolic outcomes in healthy individuals: protocol for a 3-arm placebo randomized controlled trial

© This is an open access article distributed under the Creative Commons Attribution License. https://creativecommons.org/licenses/by/4.0/Cardiometabolic disease is recognized as the predominant cause of global mortality and healthcare expenditure. Whilst pharmaceutical interventions are effective in the short term, their long-term efficacy remain equivocal and their associated side-effects are concerning. Owing to their high levels of anthocyanins, Montmorency tart cherries and blueberries have been cited as potentially important natural treatment/preventative modalities for cardiometabolic disease. This study proposed a randomized controlled trial, aims to test the effects of consumption of Montmorency tart cherry and blueberry juice on cardiometabolic outcomes compared to placebo. This 20-day, parallel, single-blind, randomized, placebo-controlled trial will recruit 45 individuals, who will be assigned to receive 60 mL per day of either Montmorency tart cherry juice, blueberry juice or a cherry/blueberry flavoured placebo. The primary study outcome is the between-group difference in systolic blood pressure from baseline to post-intervention. Secondary outcome measures will be between-group differences in anthropometric, energy expenditure and substrate oxidation (during rest and physical activity), haematological, blood pressure/resting heart rate, psychological wellbeing and sleep efficacy indices. Statistical analysis will be conducted on an intention-to-treat basis. This study has been granted ethical approval by the University of Central Lancashire, Health Research Ethics Committee (ref: HEALTH 0016) and formally registered as a trial. Dissemination of the study findings from this investigation will be through publication in a leading peer-reviewed journal.Peer reviewe

The Role of Special Instructions in the Successive Performance of Different Tasks on the Star Discrimeter

The problem was to determine the facilitating and/or interfering effects of knowledge concerning the kinds of change that might be made in shifting from a perceptual-motor task that has just been learned to a subsequent interpolated task. The experimental design called for original learning (OL), interpolated learning (IL), and relearning (RL) phases of practice, with the period for informative instructions coming between the OL and IL phases. Task A was used for OL and RL and either Task B or Task J for IL. Familiarity with the principal features of the tasks will help in explaining the problem

Population pharmacokinetics of orally administered mefloquine in healthy volunteers and patients with uncomplicated Plasmodium falciparum malaria

Background The determination of dosing regimens for the treatment of malaria is largely empirical and thus a better understanding of the pharmacokinetic/pharmacodynamic properties of antimalarial agents is required to assess the adequacy of current treatment regimens and identify sources of suboptimal dosing that could select for drug-resistant parasites. Mefloquine is a widely used antimalarial, commonly given in combination with artesunate. Patients and methods Mefloquine pharmacokinetics was assessed in 24 healthy adults and 43 patients with Plasmodium falciparum malaria administered mefloquine in combination with artesunate. Population pharmacokinetic modelling was conducted using NONMEM. Results A two-compartment model with a single transit compartment and first-order elimination from the central compartment most adequately described mefloquine concentration-time data. The model incorporated population parameter variability for clearance (CL/F), central volume of distribution (VC/F) and absorption rate constant (KA) and identified, in addition to body weight, malaria infection as a covariate for VC/F (but not CL/F). Monte Carlo simulations predict that falciparum malaria infection is associated with a shorter elimination half-life (407 versus 566 h) and T>MIC (766 versus 893 h). Conclusions This is the first known population pharmacokinetic study to show falciparum malaria to influence mefloquine disposition. Protein binding, anaemia and other factors may contribute to differences between healthy individuals and patients. As VC/F is related to the earlier portion of the concentration-time profiles, which occurs during acute malaria, and CL/F is more related to the terminal phase during convalescence after treatment, this may explain why malaria was found to be a covariate for VC/F but not CL/

Inadequate reporting of research ethics review and informed consent in cluster randomized trials : review of random sample of published trials

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