Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

<p>Abstract</p> <p>Background</p> <p>Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in <it>E. coli </it>had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in <it>E. coli</it>.</p> <p>Results</p> <p>An expression plasmid containing the open reading frame for tung tree (<it>Vernicia fordii</it>) DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in <it>E. coli </it>BL21(DE3). Immunoblotting showed that the recombinant DGAT1 (rDGAT1) was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification.</p> <p>Conclusions</p> <p>This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.</p

Transcriptional and functional profilling of human embryonic stem cell-derived cardiomyocytes

Human embryonic stemcells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stemcell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies. © 2008 Cao et al.published_or_final_versio

PGB pair production at LHC and ILC as a probe of the topcolor-assisted technicolor models

The topcolor-assisted technicolor (TC2) model predicts some light pseudo goldstone bosons (PGBs), which may be accessible at the LHC or ILC. In this work we study the pair productions of the charged or neutral PGBs at the LHC and ILC. For the productions at the LHC we consider the processes proceeding through gluon-gluon fusion and quark-antiquark annihilation, while for the productions at the ILC we consider both the electron-positron collision and the photon-photon collision. We find that in a large part of parameter space the production cross sections at both colliders can be quite large compared with the low standard model backgrounds. Therefore, in future experiments these productions may be detectable and allow for probing TC2 model.Comment: 26 pages, 16 figures. slight changes in the text; notations for curves changed; references adde

Structure-Function Analysis of Diacylglycerol Acyltransferase Sequences from 70 Organisms

<p>Abstract</p> <p>Background</p> <p>Diacylglycerol acyltransferase families (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants with value-added properties and provide clues for therapeutic intervention for obesity and related diseases. The objective of this analysis was to identify conserved sequence motifs and amino acid residues for better understanding of the structure-function relationship of these important enzymes.</p> <p>Results</p> <p>117 DGAT sequences from 70 organisms including plants, animals, fungi and human are obtained from database search using tung tree DGATs. Phylogenetic analysis separates these proteins into DGAT1 and DGAT2 subfamilies. These DGATs are integral membrane proteins with more than 40% of the total amino acid residues being hydrophobic. They have similar properties and amino acid composition except that DGAT1s are approximately 20 kDa larger than DGAT2s. DGAT1s and DGAT2s have 41 and 16 completely conserved amino acid residues, respectively, although only two of them are shared by all DGATs. These residues are distributed in 7 and 6 sequence blocks for DGAT1s and DGAT2s, respectively, and located at the carboxyl termini, suggesting the location of the catalytic domains. These conserved sequence blocks do not contain the putative neutral lipid-binding domain, mitochondrial targeting signal, or ER retrieval motif. The importance of conserved residues has been demonstrated by site-directed and natural mutants.</p> <p>Conclusions</p> <p>This study has identified conserved sequence motifs and amino acid residues in all 117 DGATs and the two subfamilies. None of the completely conserved residues in DGAT1s and DGAT2s is present in recently reported isoforms in the multiple sequences alignment, raising an important question how proteins with completely different amino acid sequences could perform the same biochemical reaction. The sequence analysis should facilitate studying the structure-function relationship of DGATs with the ultimate goal to identify critical amino acid residues for engineering superb enzymes in metabolic engineering and selecting enzyme inhibitors in therapeutic application for obesity and related diseases.</p

Social Determinants of Community Health Services Utilization among the Users in China: A 4-Year Cross-Sectional Study

Background To identify social factors determining the frequency of community health service (CHS) utilization among CHS users in China. Methods Nationwide cross-sectional surveys were conducted in 2008, 2009, 2010, and 2011. A total of 86,116 CHS visitors selected from 35 cities were interviewed. Descriptive analysis and multinomial logistic regression analysis were employed to analyze characteristics of CHS users, frequency of CHS utilization, and the socio-demographic and socio-economic factors influencing frequency of CHS utilization. Results Female and senior CHS clients were more likely to make 3–5 and ≥6 CHS visits (as opposed to 1–2 visits) than male and young clients, respectively. CHS clients with higher education were less frequent users than individuals with primary education or less in 2008 and 2009; in later surveys, CHS clients with higher education were the more frequent users. The association between frequent CHS visits and family income has changed significantly between 2008 and 2011. In 2011, income status did not have a discernible effect on the likelihood of making ≥6 CHS visits, and it only had a slight effect on making 3–5 CHS visits. Conclusion CHS may play an important role in providing primary health care to meet the demands of vulnerable populations in China. Over time, individuals with higher education are increasingly likely to make frequent CHS visits than individuals with primary school education or below. The gap in frequency of CHS utilization among different economic income groups decreased from 2008 to 2011

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.Oncogene advance online publication, 25 July 2016; doi:10.1038/onc.2016.260

LHC Predictions from a Tevatron Anomaly in the Top Quark Forward-Backward Asymmetry

We examine the implications of the recent CDF measurement of the top-quark forward-backward asymmetry, focusing on a scenario with a new color octet vector boson at 1-3 TeV. We study several models, as well as a general effective field theory, and determine the parameter space which provides the best simultaneous fit to the CDF asymmetry, the Tevatron top pair production cross section, and the exclusion regions from LHC dijet resonance and contact interaction searches. Flavor constraints on these models are more subtle and less severe than the literature indicates. We find a large region of allowed parameter space at high axigluon mass and a smaller region at low mass; we match the latter to an SU(3)xSU(3)/SU(3) coset model with a heavy vector-like fermion. Our scenario produces discoverable effects at the LHC with only 1-2 inverse femtobarns of luminosity at 7-8 TeV. Lastly, we point out that a Tevatron measurement of the b-quark forward-backward asymmetry would be very helpful in characterizing the physics underlying the top-quark asymmetry.Comment: 35 pages, 10 figures, 4 table

Top quark forward-backward asymmetry in R-parity violating supersymmetry

The interaction of bottom squark-mediated top quark pair production, occurring in the R-parity violating minimal supersymmetric standard model (MSSM), is proposed as an explanation of the anomalously large $t\bar{t}$ forward-backward asymmetry (FBA) observed at the Tevatron. We find that this model can give a good fit to top quark data, both the inclusive and invariant mass-dependent asymmetries, while remaining consistent (at the 2-$\sigma$ level) with the total and differential production cross-sections. The scenario is challenged by strong constraints from atomic parity violation (APV), but we point out an extra diagram for the effective down quark-Z vertex, involving the same coupling constant as required for the FBA, which tends to weaken the APV constraint, and which can nullify it for reasonable values of the top squark masses and mixing angle. Large contributions to flavor-changing neutral currents can be avoided if only the third generation of sparticles is light.Comment: 24 pages, 7 figures. v3: included LHC top production cross section data; model still consistent at 2 sigma leve

Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A

International audienceAims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A

Focus on vulnerable populations and promoting equity in health service utilization ––an analysis of visitor characteristics and service utilization of the Chinese community health service

Background Community health service in China is designed to provide a convenient and affordable primary health service for the city residents, and to promote health equity. Based on data from a large national study of 35 cities across China, we examined the characteristics of the patients and the utilization of community health institutions (CHIs), and assessed the role of community health service in promoting equity in health service utilization for community residents. Methods Multistage sampling method was applied to select 35 cities in China. Four CHIs were randomly chosen in every district of the 35 cities. A total of 88,482 visitors to the selected CHIs were investigated by using intercept survey method at the exit of the CHIs in 2008, 2009, 2010, and 2011. Descriptive analyses were used to analyze the main characteristics (gender, age, and income) of the CHI visitors, and the results were compared with that from the National Health Services Survey (NHSS, including CHIs and higher levels of hospitals). We also analyzed the service utilization and the satisfactions of the CHI visitors. Results The proportions of the children (2.4%) and the elderly (about 22.7%) were lower in our survey than those in NHSS (9.8% and 38.8% respectively). The proportion of the low-income group (26.4%) was apparently higher than that in NHSS (12.5%). The children group had the lowest satisfaction with the CHIs than other age groups. The satisfaction of the low-income visitors was slightly higher than that of the higher-income visitors. The utilization rate of public health services was low in CHIs. Conclusions The CHIs in China appears to fulfill the public health target of uptake by vulnerable populations, and may play an important role in promoting equity in health service utilization. However, services for children and the elderly should be strengthened