Gene-chip studies of adipogenesis-regulated microRNAs in mouse primary adipocytes and human obesity

<p>Abstract</p> <p>Background</p> <p>Adipose tissue abundance relies partly on the factors that regulate adipogenesis, i.e. proliferation and differentiation of adipocytes. While components of the transcriptional program that initiates adipogenesis is well-known, the importance of microRNAs in adipogenesis is less well studied. We thus set out to investigate whether miRNAs would be actively modulated during adipogenesis and obesity.</p> <p>Methods</p> <p>Several models exist to study adipogenesis <it>in vitro</it>, of which the cell line 3T3-L1 is the most well known, albeit not the most physiologically appropriate. Thus, as an alternative, we produced EXIQON microarray of brown and white <it>primary </it>murine adipocytes (prior to and following differentiation) to yield global profiles of miRNAs.</p> <p>Results</p> <p>We found 65 miRNAs regulated during <it>in vitro </it>adipogenesis in primary adipocytes. We evaluated the similarity of our responses to those found in non-primary cell models, through literature data-mining. When comparing primary adipocyte profiles, with those of cell lines reported in the literature, we found a high degree of difference in 'adipogenesis' regulated miRNAs suggesting that the model systems may not be accurately representing adipogenesis. The expression of 10 adipogenesis-regulated miRNAs were studied using real-time qPCR and then we selected 5 miRNAs, that showed robust expression, were profiled in subcutaneous adipose tissue obtained from 20 humans with a range of body mass indices (BMI, range = 21-48, and all samples have U133+2 Affymetrix profiles provided). Of the miRNAs tested, mir-21 was robustly expressed in human adipose tissue and positively correlated with BMI (R2 = 0.49, p < 0.001).</p> <p>Conclusion</p> <p>In conclusion, we provide a preliminary analysis of miRNAs associated with primary cell <it>in vitro </it>adipogenesis and demonstrate that the inflammation-associated miRNA, mir-21 is up-regulated in subcutaneous adipose tissue in human obesity. Further, we provide a novel transcriptomics database of EXIQON and Affymetrix adipocyte profiles to facilitate data mining.</p

The health outcomes and physical activity in preschoolers (HOPP) study: rationale and design

<p>Abstract</p> <p>Background</p> <p>The early years are the period of growth for which we know the least about the impact of physical activity. In contrast, we know that more than 90 % of school-aged Canadian children, for example, are not meeting physical activity recommendations. Such an activity crisis is a major contributor to recent trends in childhood obesity, to which preschoolers are not immune. The World Health Organization estimated that more than 42 million children under the age of 5 years were overweight world-wide in 2010. If an activity crisis exists during the preschool years, we should also be concerned about its broader impact on health. Unfortunately, the relationship between physical activity and health during the early years is poorly understood. The goal of the Health Outcomes and Physical activity in Preschoolers (HOPP) study is to describe how the prevalence and patterns of physical activity in preschoolers are associated with indices of health.</p> <p>Methods</p> <p>The HOPP study is a prospective cohort study. We aim to recruit 400 3- to 5-year-old children (equal number of boys and girls) and test them once per year for 3 years. Each annual assessment involves 2 laboratory visits and 7 consecutive days of physical activity monitoring with protocols developed in our pilot work. At visit 1, we assess body composition, aerobic fitness, short-term muscle power, motor skills, and have the parents complete a series of questionnaires related to their child’s physical activity, health-related quality of life and general behaviour. Over 7 consecutive days each child wears an accelerometer on his/her waist to objectively monitor physical activity. The accelerometer is programmed to record movement every 3 s, which is needed to accurately capture the intensity of physical activity. At visit 2, we assess vascular structure and function using ultrasound. To assess the associations between physical activity and health outcomes, our primary analysis will involve mixed-effects models for longitudinal analyses.</p> <p>Discussion</p> <p>The HOPP study addresses a significant gap in health research and our findings will hold the potential to shape public health policy for active living during the early years.</p

An image analysis toolbox for high-throughput C. elegans assays

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.National Institutes of Health (U.S.) (U54 EB005149

Better Palliative Care for people with a Dementia: Summary of InterdisciplinaryWorkshop Highlighting Current Gaps and Recommendations for Future Research

Background: Dementia is the most common neurological disorder worldwide and is a life-limiting condition, but very often is not recognised as such. People with dementia, and their carers, have been shown to have palliative care needs equal in extent to those of cancer patients. However, many people with advanced dementia are not routinely being assessed to determine their palliative care needs, and it is not clear why this is so. Main body: An interdisciplinary workshop on "Palliative Care in Neurodegeneration, with a focus on Dementia", was held in Cork, Ireland, in May 2016. The key aim of this workshop was to discuss the evidence base for palliative care for people with dementia, to identify 'gaps' for clinical research, and to make recommendations for interdisciplinary research practice. To lead the discussion throughout the day a multidisciplinary panel of expert speakers were brought together, including both researchers and clinicians from across Ireland and the UK. Targeted invitations were sent to attendees ensuring all key stakeholders were present to contribute to discussions. In total, 49 experts representing 17 different academic and practice settings, attended. Key topics for discussion were pre-selected based on previously identified research priorities (e.g. James Lind Alliance) and stakeholder input. Key discussion topics included: i. Advance Care Planning for people with Dementia; ii. Personhood in End-of-life Dementia care; iii. Topics in the care of advanced dementia at home. These topics were used as a starting point, and the ethos of the workshop was that the attendees could stimulate discussion and debate in any relevant area, not just the key topics, summarised under iv. Other priorities. Conclusions: The care experienced by people with dementia and their families has the potential to be improved; palliative care frameworks may have much to offer in this endeavour. However, a solid evidence base is required to translate palliative care into practice in the context of dementia. This paper presents suggested research priorities as a starting point to build this evidence base. An interdisciplinary approach to research and priority setting is essential to develop actionable knowledge in this area

The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins.

BACKGROUND: Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform\u27s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. METHODOLOGY/PRINCIPAL FINDINGS: Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. CONCLUSIONS/SIGNIFICANCE: Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However, prohibiting phosphorylation of candidate 14-3-3-binding sites does not impact localization of the fusogen. Hypodermal membrane fusion activity persists when 14-3-3 expression levels are reduced

CUL-2^LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis

Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (CDC45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus laevis egg extracts, we show that the E3 ligase CUL-2(LRR-1) associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2(LRR1) as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2(LRR-1), but is then removed by a mitotic pathway that requires the CDC-48 cofactor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically

Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

<p>Abstract</p> <p>Background</p> <p>VEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice) were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein.</p> <p>Methods</p> <p>Tissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffe's <it>F</it>-test for multiple comparisons.</p> <p>Results</p> <p>Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory), cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM.</p> <p>Conclusion</p> <p>These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near-term, including cervical remodeling.</p

Membrane Invaginations Reveal Cortical Sites that Pull on Mitotic Spindles in One-Cell C. elegans Embryos

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell

The Germinal Center Kinase GCK-1 Is a Negative Regulator of MAP Kinase Activation and Apoptosis in the C. elegans Germline

The germinal center kinases (GCK) constitute a large, highly conserved family of proteins that has been implicated in a wide variety of cellular processes including cell growth and proliferation, polarity, migration, and stress responses. Although diverse, these functions have been attributed to an evolutionarily conserved role for GCKs in the activation of ERK, JNK, and p38 MAP kinase pathways. In addition, multiple GCKs from different species promote apoptotic cell death. In contrast to these paradigms, we found that a C. elegans GCK, GCK-1, functions to inhibit MAP kinase activation and apoptosis in the C. elegans germline. In the absence of GCK-1, a specific MAP kinase isoform is ectopically activated and oocytes undergo abnormal development. Moreover, GCK-1- deficient animals display a significant increase in germ cell death. Our results suggest that individual germinal center kinases act in mechanistically distinct ways and that these functions are likely to depend on organ- and developmental-specific contexts