Betaine and Trimethylamine-N-Oxide as Predictors of Cardiovascular Outcomes Show Different Patterns in Diabetes Mellitus: An Observational Study

Background

Betaine is a major osmolyte, also important in methyl group metabolism. Concentrations of betaine, its metabolite dimethylglycine and analog trimethylamine-N-oxide (TMAO) in blood are cardiovascular risk markers. Diabetes disturbs betaine: does diabetes alter associations between betaine-related measures and cardiovascular risk?

Methods

Plasma samples were collected from 475 subjects four months after discharge following an acute coronary admission. Death (n = 81), secondary acute MI (n = 87), admission for heart failure (n = 85), unstable angina (n = 72) and all cardiovascular events (n = 283) were recorded (median follow-up: 1804 days).

Results

High and low metabolite concentrations were defined as top or bottom quintile of the total cohort. In subjects with diabetes (n = 79), high plasma betaine was associated with increased frequencies of events; significantly for heart failure, hazard ratio 3.1 (1.2–8.2) and all cardiovascular events, HR 2.8 (1.4–5.5). In subjects without diabetes (n = 396), low plasma betaine was associated with events; significantly for secondary myocardial infarction, HR 2.1 (1.2–3.6), unstable angina, HR 2.3 (1.3–4.0), and all cardiovascular events, HR 1.4 (1.0–1.9). In diabetes, high TMAO was a marker of all outcomes, HR 2.7 (1.1–7.1) for death, 4.0 (1.6–9.8) for myocardial infarction, 4.6 (2.0–10.7) for heart failure, 9.1 (2.8–29.7) for unstable angina and 2.0 (1.1–3.6) for all cardiovascular events. In subjects without diabetes TMAO was only significant for death, HR 2.7 (1.6–4.8) and heart failure, HR 1.9 (1.1–3.4). Adding the estimated glomerular filtration rate to Cox regression models tended to increase the apparent risks associated with low betaine.

Conclusions

Elevated plasma betaine concentration is a marker of cardiovascular risk in diabetes; conversely low plasma betaine concentrations indicate increased risk in the absence of diabetes. We speculate that the difference reflects control of osmolyte retention in tissues. Elevated plasma TMAO is a strong risk marker in diabetes.     

Railway Embankments as New Habitat for Pollinators in an Agricultural Landscape

Pollinating insect populations, essential for maintaining wild plant diversity and agricultural productivity, rely on (semi)natural habitats. An increasing human population is encroaching upon and deteriorating pollinator habitats. Thus the population persistence of pollinating insects and their associated ecosystem services may depend upon on man-made novel habitats; however, their importance for ecosystem services is barely understood. We tested if man-made infrastructure (railway embankments) in an agricultural landscape establishes novel habitats that support large populations of pollinators (bees, butterflies, hoverflies) when compared to typical habitats for these insects, i.e., semi-natural grasslands. We also identified key environmental factors affecting the species richness and abundance of pollinators on embankments. Species richness and abundance of bees and butterflies were higher for railway embankments than for grasslands. The occurrence of bare (non-vegetated) ground on embankments positively affected bee species richness and abundance, but negatively affected butterfly populations. Species richness and abundance of butterflies positively depended on species richness of native plants on embankments, whereas bee species richness was positively affected by species richness of non-native flowering plants. The density of shrubs on embankments negatively affected the number of bee species and their abundance. Bee and hoverfly species richness were positively related to wood cover in a landscape surrounding embankments. This is the first study showing that railway embankments constitute valuable habitat for the conservation of pollinators in farmland. Specific conservation strategies involving embankments should focus on preventing habitat deterioration due to encroachment of dense shrubs and maintaining grassland vegetation with patches of bare ground.     

Diagnostic Overshadowing and Other Challenges Involved in the Diagnostic Process of Patients with Mental Illness Who Present in Emergency Departments with Physical Symptoms – A Qualitative Study

We conducted a qualitative study in the Emergency Departments (EDs) of four hospitals in order to investigate the perceived scope and causes of ‘diagnostic overshadowing’ – the misattribution of physical symptoms to mental illness – and other challenges involved in the diagnostic process of people with mental illness who present in EDs with physical symptoms. Eighteen doctors and twenty-one nurses working in EDs and psychiatric liaisons teams in four general hospitals in the UK were interviewed. Interviewees were asked about cases in which mental illness interfered with diagnosis of physical problems and about other aspects of the diagnostic process. Interviews were transcribed and analysed thematically. Interviewees reported various scenarios in which mental illness or factors related to it led to misdiagnosis or delayed treatment with various degrees of seriousness. Direct factors which may lead to misattribution in this regard are complex presentations or aspects related to poor communication or challenging behaviour of the patient. Background factors are the crowded nature of the ED environment, time pressures and targets and stigmatising attitudes held by a minority of staff. The existence of psychiatric liaison team covering the ED twenty-four hours a day, seven days a week, can help reduce the risk of misdiagnosis of people with mental illness who present with physical symptoms. However, procedures used by emergency and psychiatric liaison staff require fuller operationalization to reduce disagreement over where responsibilities lie.     

MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome.     

Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

Myxoma Virus Expressing a Fusion Protein of Interleukin-15 (IL15) and IL15 Receptor Alpha Has Enhanced Antitumor Activity

Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1^-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8⁺ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.     

Using Eye Movements to Evaluate the Cognitive Processes Involved in Text Comprehension

The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Measuring eye movements during reading is one of the most precise methods for measuring moment-by-moment (online) processing demands during text comprehension. Cognitive processing demands are reflected by several aspects of eye movement behavior, such as fixation duration, number of fixations, and number of regressions (returning to prior parts of a text). Important properties of eye tracking equipment that researchers need to consider are described, including how frequently the eye position is measured (sampling rate), accuracy of determining eye position, how much head movement is allowed, and ease of use. Also described are properties of stimuli that influence eye movements that need to be controlled in studies of text comprehension, such as the position, frequency, and length of target words. Procedural recommendations related to preparing the participant, setting up and calibrating the equipment, and running a study are given. Representative results are presented to illustrate how data can be evaluated. Although the methodology is described in terms of reading comprehension, much of the information presented can be applied to any study in which participants read verbal stimuli.     

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.     

Regional differences in short stature in England between 2006 and 2019: A cross-sectional analysis from the National Child Measurement Programme

BackgroundShort stature, defined as height for age more than 2 standard deviations (SDs) below the population median, is an important indicator of child health. Short stature (often termed stunting) has been widely researched in low- and middle-income countries (LMICs), but less is known about the extent and burden in high-income settings. We aimed to map the prevalence of short stature in children aged 4–5 years in England between 2006 and 2019.Methods and findingsWe used data from the National Child Measurement Programme (NCMP) for the school years 2006–2007 to 2018–2019. All children attending state-maintained primary schools in England are invited to participate in the NCMP, and heights from a total of 7,062,071 children aged 4–5 years were analysed. We assessed short stature, defined as a height-for-age standard deviation score (SDS) below −2 using the United Kingdom WHO references, by sex, index of multiple deprivation (IMD), ethnicity, and region. Geographic clustering of short stature was analysed using spatial analysis in SaTScan. The prevalence of short stature in England was 1.93% (95% confidence interval (CI) 1.92–1.94). Ethnicity adjusted spatial analyses showed geographic heterogeneity of short stature, with high prevalence clusters more likely in the North and Midlands, leading to 4-fold variation between local authorities (LAs) with highest and lowest prevalence of short stature. Short stature was linearly associated with IMD, with almost 2-fold higher prevalence in the most compared with least deprived decile (2.56% (2.53–2.59) vs. 1.38% (1.35–1.41)). There was ethnic heterogeneity: Short stature prevalence was lowest in Black children (0.64% (0.61–0.67)) and highest in Indian children (2.52% (2.45–2.60)) and children in other ethnic categories (2.57% (2.51–2.64)). Girls were more likely to have short stature than boys (2.09% (2.07–2.10) vs. 1.77% (1.76–1.78), respectively). Short stature prevalence declined over time, from 2.03% (2.01–2.05) in 2006–2010 to 1.82% (1.80–1.84) in 2016–2019. Short stature declined at all levels of area deprivation, with faster declines in more deprived areas, but disparities by IMD quintile were persistent. This study was conducted cross-sectionally at an area level, and, therefore, we cannot make any inferences about the individual causes of short stature.ConclusionsIn this study, we observed a clear social gradient and striking regional variation in short stature across England, including a North–South divide. These findings provide impetus for further investigation into potential socioeconomic influences on height and the factors underlying regional variation.

Joanna Orr and coauthors investigate regional differences in short stature among children in England, between 2006-2019, using a cross-sectional analysis of the National Child Measurement Program data.