The role of DA1 in organ size control in Arabidopsis thaliana

Despite the sizes of organs and organisms being a key defining feature, very little is known about how the final sizes of organs is determined. Recent progress has highlighted the important role of the DA1 peptidase as a negative regulator of organ size in Arabidopsis thaliana. Previous studies have proposed that DA1, and the E3 ligase BIG BROTHER (a protein known to regulate DA1 activity), work synergistically to regulate the duration of cell proliferation. In the prulresent study, we take a multidisciplinary approach to further our understanding of the biological activities of DA1 and BB. Protoplast transient expression analyses were used to explore potential new substrates for DA1 peptidase activity, and to work towards identifying a conserved target site for DA1mediated cleavage. Using confocal microscopy and bespoke segmentation software, I embarked on a global analysis of leaf cellular phenotypes in the da1-1, bb, and da1-1bb mutants throughout early development. This allowed a holistic comparison to wild type of parameters such as total cell number, and cell area, density, and circularity. In addition, scanning electron microscopy was used to examine cells in mature leaves of wild type, and da1-1, bb, and da1-1bb mutants, revealing novel insights into the control of final organ size in these mutants relative to wild type. Finally, innovative live cell imaging has, for the first time, allowed cell divisions to be observed in plants carrying the da1-1, bb, and da1-1bb mutations. My observations and interpretation establish new insights into how DA1 and BB control growth by controlling the arrest of cell proliferation, and the population-level rate of cell proliferation. The approaches I have developed show the promise of quantitative cell imaging for understanding organ growth, and establish a framework for precisely comparing the effects of different mutations on organ growth

A scanning electron microscope technique for identifying the mineralogy of dust in ice cores

Dust particles in an ice core from East Rongbuk Glacier on the northern slope of Qomolangma (Mount Everest; 28deg 01\u27 N, 58deg 00\u27 E; 6518m a.s.l.), central Himalaya, have been identified as mica using a combination of scanning electron microscope-based techniques and energy-dispersive X-ray spectroscopy to identify the elements present, and electron backscatter diffraction to identify the crystal type. This technique for identifying individual crystalline dust particles in samples of glacial ice could be especially useful in the future for identifying water-soluble crystals in ice, for studying the strain history (glaciotectonics) of basal ice or in studies of icemica composites used as analogs of quartz-mica rocks

The -765G>C Cyclooxygenase-2 Promoter Polymorphism is associated with Type 2 Diabetes Mellitus, Low High-density Lipoprotein and Manifest Angina

Background & Aims: Cycloxygenase-2 (COX-2) catalyses the rate limiting step of prostaglandin biosynthesis. Despite previous studies, it is still unclear whether COX-2 is beneficial or detrimental to cardiovascular risk. The aim of this study was to examine the -765G>C (rs20417) PTGS2 promoter gene variant, which encodes COX-2, in relation to markers of cardiovascular risk in a sample of well-characterised subjects with diabetes mellitus. Methods & Results: We observed that the CC genotype was more prevalent in Type 2 diabetes mellitus compared to Type 1 (84.2 vs 15.8%; p≤0.05), and was significantly associated with clinically manifest angina (GG vs GC vs CC: 14.3% vs 15.6% vs 28.0%; p=0.009) and lower HDL-cholesterol levels (GG vs GC vs CC: 1.3mmol/L vs 1.4mmol/L vs 1.2mmol/L; p=0.032). This is in line with previous studies showing that -765G>C genotype variant alters Sp1 binding, resulting in decreased COX-2 activity which is associated with atherosclerosis. Conclusion: We conclude that the CC genotype may contribute to a reduction of prostaglandin E2 mediated insulin secretion, predisposing those individuals to Type 2 diabetes mellitus. Further prospective work is warranted in order to examine the association between COX-2 and cardiovascular risk

Development and characterization of an in vitro system of the human retina using cultured cell lines

Background: Previously developed in vitro cultures of the human retina have been solo or dual cell cultures. We developed a triple-cell culture in vitro model utilizing a membrane system to produce a better representation of a functional and morphological human retina. Methods: Retinal microvascular endothelial cells (HRMVEC/ACBRI181, Cell systems), retinal pigment epithelium cells (RPE/ARPE-19, ATCC) and Müller glial cells (MIO-M1, UCL) were grown in a triple-culture. Our optimized triple-culture media contained a mix of specific endothelial medium and high glucose Dulbecco's Modified Eagle's medium (DMEM), where all three layers were viable for up to 5 days. Co-culture effect on morphological changes (cell staining) and gene expression of functional genes (pigment epithelial derived factor (PEDF) and vascular endothelial growth factor (VEGF)) were measured from RNA via real time PCR. Expression of tight junction protein 1 (TJP1) was measured in RNA isolated from ARPE-19s, to assess barrier stability. Results: The triple-culture promotes certain cell functionality through up-regulation of TJP1, increasing PEDF and decreasing VEGF expression highlighting its importance for the assessment of disease mechanisms distinct from a solo culture which would not allow the true effect of the native microenvironment to be elucidated. Conclusion: This model's novelty and reliability allows for the assessment of singular cellular function within the retinal microenvironment and overall assessment of retinal health, whilst eliminating the requirement of animal-based models

Acyl-ghrelin mediated lipid retention and inflammation in obesity-related Type 2 diabetes

Acyl-ghrelin has various peripheral effects including the potential role in mediating cellular lipid removal and macrophage polarization. Previous reports are contradictory as to how glycaemia and acyl-ghrelin mediates lipid retention and inflammation within individuals with Type 2 diabetes (T2D). Our aim was to explore acyl-ghrelin levels and ghrelin expression in relation to lipid and inflammatory markers within an ex vivo human model, biopsied visceral adipose tissue.Results indicated that acyl-ghrelin was associated with a decline in key lipid homeostasis genes ABCG1 and LXRβ expression. Within T2D there was also a down regulation of these genes which was independent of acyl-ghrelin levels. Circulatory pro-inflammatory markers (IL-6 and TNFα) had no association with ghrelin expression nor circulating acyl-ghrelin levels. Anti-inflammatory marker (IL-10) and total antioxidant status (TAOS%) were positively associated with ghrelin expression across samples from all groups combined (total sample cohort) and specifically within the obesity sample cohorts.Data supported the hypothesis that hyperglycaemia and acyl-ghrelin have a regulatory role in lipid retention. Furthermore, that both acyl- and desacyl-ghrelin is responsible for a protective inflammatory response; however this response is diminished in T2D

Can behaviour during immunisation be used to identify attachment patterns? A feasibility study

<b>Background</b> Infant attachment is a strong predictor of mental health, and current measures involve placing children into a stressful situation in order to observe how the child uses their primary caregiver to assuage their distress.<p></p> <b>Objectives</b> This study aimed to explore observational correlates of attachment patterns during immunisation.<p></p> <b>Participants and setting</b> 18 parent–child pairs were included in the study. They were all recruited through a single general medical practice.<p></p> <b>Methods</b> Infant immunisation videos were observed and coded for parenting behaviours as well as pain promoting and pain reducing strategies. Results were compared between different attachment groups, as measured with the Manchester Child Attachment Story Task. <p></p> <b>Results</b> Parents of securely attached children scored higher on positive Mellow Parenting Observational System behaviours, but not at a statistically significant level. Parents of securely attached children were also significantly more likely to engage in pain reducing behaviours (p <0.01) than parents of insecurely attached children.<p></p> <b>Conclusions</b> Robust composite measures for attachment informative behaviours in the immunisation situation should be developed and tested in a fully powered study

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/ CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins