Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: A case study with the metastatic breast cancer cell line MDA-MB-231

Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (−50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism

Physical activity as a treatment for depression: the TREAD randomised trial protocol

Depression is one of the most common reasons for consulting a General Practitioner (GP) within the UK. Whilst antidepressants have been shown to be clinically effective, many patients and healthcare professionals would like to access other forms of treatment as an alternative or adjunct to drug therapy for depression. A recent systematic review presented some evidence that physical activity could offer one such option, although further investigation is needed to test its effectiveness within the context of the National Health Service.The aim of this paper is to describe the protocol for a randomised, controlled trial (RCT) designed to evaluate an intervention developed to increase physical activity as a treatment for depression within primary care

In vivo cholinergic basal forebrain atrophy predicts cognitive decline in de novo Parkinson’s disease

Cognitive impairments are a prevalent and disabling non-motor complication of Parkinson’s disease, but with variable expression and progression. The onset of serious cognitive decline occurs alongside substantial cholinergic denervation, but imprecision of previously available techniques for in vivo measurement of cholinergic degeneration limit their use as predictive cognitive biomarkers. However, recent developments in stereotactic mapping of the cholinergic basal forebrain have been found useful for predicting cognitive decline in prodromal stages of Alzheimer’s disease. These methods have not yet been applied to longitudinal Parkinson’s disease data. In a large sample of people with de novo Parkinson’s disease (n = 168), retrieved from the Parkinson’s Progressive Markers Initiative database, we measured cholinergic basal forebrain volumes, using morphometric analysis of T1-weighted images in combination with a detailed stereotactic atlas of the cholinergic basal forebrain nuclei. Using a binary classification procedure, we defined patients with reduced basal forebrain volumes (relative to age) at baseline, based on volumes measured in a normative sample (n = 76). Additionally, relationships between the basal forebrain volumes at baseline, risk of later cognitive decline, and scores on up to 5 years of annual cognitive assessments were assessed with regression, survival analysis and linear mixed modelling. In patients, smaller volumes in a region corresponding to the nucleus basalis of Meynert were associated with greater change in global cognitive, but not motor scores after 2 years. Using the binary classification procedure, patients classified as having smaller than expected volumes of the nucleus basalis of Meynert had ∼3.5-fold greater risk of being categorized as mildly cognitively impaired over a period of up to 5 years of follow-up (hazard ratio = 3.51). Finally, linear mixed modelling analysis of domain-specific cognitive scores revealed that patients classified as having smaller than expected nucleus basalis volumes showed more severe and rapid decline over up to 5 years on tests of memory and semantic fluency, but not on tests of executive function. Thus, we provide the first evidence that volumetric measurement of the nucleus basalis of Meynert can predict early cognitive decline. Our methods therefore provide the opportunity for multiple-modality biomarker models to include a cholinergic biomarker, which is currently lacking for the prediction of cognitive deterioration in Parkinson’s disease. Additionally, finding dissociated relationships between nucleus basalis status and domain-specific cognitive decline has implications for understanding the neural basis of heterogeneity of Parkinson’s disease-related cognitive decline

‘Unpenned’: a collection of responses to fairy tales, together with a critical commentary

The creative component of the thesis comprises a collection of prose poems, poetry and short stories, all of which draw on and re-present the landscape of fairy tales. Some narratives are visited several times, from the viewpoint of different characters or time frames. The prose moves closer to poetry as the collection progresses, and also becomes more surrealist in nature. The liminality of the spaces described becomes more pronounced. Whereas the earlier pieces tend to be character-driven and intertextual, the later ones move further away from the original tales, and focus more sharply on topographical features and on forming a creative relationship to them. The critical commentary takes the form of a guidebook, the chapters of which focus on different aspects of fairy tale settings: forests, castles, cottages and so on. After an introductory chapter establishing premise and methodology, the commentary examines the ways in which settings are presented in the creative work, placing this in the context of fairy tales (and imaginative responses to them), and of the critical material surrounding them. I also interweave, throughout, an exploration of my own creative process, touching on automatic writing, surrealism, postmodernism and metanarrative. Theoretical frameworks are employed where they seem most relevant, particularly those pertaining to psychoanalytic thinkers such as Klein. The commentary concludes with a chapter on Agency and Style in which I further examine artistic choices I have made and the rationale underpinning them

Experts and novices: Examining academic email requests to faculty and developmental change during study abroad

This longitudinal study seeks to contribute to a shortage of email investigations examining expert (L1) and novice (L2) English practices and tracking L2 developmental change during a UK study abroad period. Using a corpus of 315 authentic request emails, distinct features of Chinese ESL and British students’ email practices were examined, in addition to changes in Chinese ESL practices between the beginning and end of the ten-month period abroad. Findings firstly indicated that choice of request strategies, internal modification, and request perspective showed much variation between the two groups due to different approaches to projecting politeness. Secondly, exposure to the L2 and engagement in email writing had minimal impact on pragmatic performance over the academic year

Openings and Closing in institutionally-situated email requests

The study abroad experience presents opportunities to enrich linguistic and cultural knowledge, but learners also face challenges negotiating within and between language systems. This chapter describes one such interlanguage challenge: managing online pragmatic interactions in upward email requests. Openings and closings sequences in emails of two academic discourse communities are examined: (L1) experts (n=162) and (L2) novices (n=159) from China. The request data are analysed for evidence of variance between the groups and to what extent a ten-month sojourn changes novice email practice. Results revealed that novices and experts adopt markedly different strategies for doing interpersonal work. Experts tend to take a less formal, egalitarian stance when composing emails while novices opt for increased levels of formality in structure and style. Most novice mismanagement of openings and closings can be traced back to either L1 influences or, more commonly, an overreliance on formal letter writing techniques

Maternal vitamin D status during pregnancy and body composition and cardiovascular risk markers in Indian children: the Mysore Parthenon Study

Background: Metabolic consequences of vitamin D deficiency have become a recent research focus. Maternal vitamin D status is thought to influence musculoskeletal health in children, but its relation with offspring metabolic risk is not known. Objective: We aimed to examine the association between maternal vitamin D status and anthropometric variables, body composition, and cardiovascular risk markers in Indian children. Design: Serum 25-hydroxyvitamin D [25(OH)D] concentrations were measured at 28–32 wk gestation in 568 women who delivered at Holdsworth Memorial Hospital, Mysore, India. Anthropometric variables, glucose and insulin concentrations, blood pressure, and fasting lipid concentrations were measured in the offspring at 5 and 9.5 y of age. Muscle-grip strength was measured by using a hand-held dynamometer at age 9.5 y. Arm-muscle area was calculated as a measure of muscle mass. Fasting insulin resistance was calculated by using the homeostasis model assessment equation. Results: Sixty-seven percent of women had vitamin D deficiency [serum 25(OH)D concentration &lt;50 nmol/L]. At ages 5 and 9.5 y, children born to vitamin D–deficient mothers had smaller arm-muscle area in comparison with children born to mothers without deficiency (P &lt; 0.05). There was no difference in grip strength between offspring of women with and without vitamin D deficiency. At 9.5 y, children of vitamin D–deficient mothers had higher fasting insulin resistance than did children of nondeficient women (P = 0.04). There were no associations between maternal vitamin D status and other offspring risk factors at either age. Conclusion: Intrauterine exposure to low 25(OH)D concentrations is associated with less muscle mass and higher insulin resistance in children. <br/

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease