104 research outputs found

    Bioactivation of 3D Cell-Imprinted Polydimethylsiloxane Surfaces by Bone Protein Nanocoating for Bone Tissue Engineering

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    Physical and chemical parameters that mimic the physiological niche of the human body have an influence on stem cell fate by creating directional signals to cells. Micro/nano cell-patterned polydimethylsiloxane (PDMS) substrates, due to their ability to mimic the physiological niche, have been widely used in surface modification. Integration of other factors such as the biochemical coating on the surface can achieve more similar microenvironmental conditions and promote stem cell differentiation to the target cell line. Herein, we investigated the effect of physical topography, chemical functionalization by acid bone lysate (ABL) nanocoating, and the combined functionalization of the bone proteins' nanocoated surface and the topographically modified surface. We prepared four distinguishing surfaces: plain PDMS, physically modified PDMS by 3D cell topography patterning, chemically modified PDMS with bone protein nanocoating, and chemically modified nano 3D cell-imprinted PDMS by bone proteins (ABL). Characterization of extracted ABL was carried out by Bradford staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, followed by the MTT assay for evaluation of cell viability on ABL-coated PDMS. Moreover, field emission scanning electron microscopy and profilometry were used for the determination of optimal coating thickness, and the appropriate coating concentration was identified and used in the study. The binding and retention of ABL to PDMS were confirmed by Fourier transform infrared spectroscopy and bicinchoninic acid assay. Sessile drop static water contact angle measurements on substrates showed that the combined chemical functionalization and nano 3D cell-imprinting on the PDMS surface improved surface wettability by 66% compared to plain PDMS. The results of ALP measurement, alizarin red S staining, immunofluorescence staining, and real-time PCR showed that the nano 3D cell-imprinted PDMS surface functionalized by extracted bone proteins, ABL, is able to guide the fate of adipose derived stem cellss toward osteogenic differentiation. Eventually, chemical modification of the cell-imprinted PDMS substrate by bone protein extraction not only improved the cell adhesion and proliferation but also contributed to the topographical effect itself and caused a significant synergistic influence on the process of osteogenic differentiation

    NEDD9 Stabilizes Focal Adhesions, Increases Binding to the Extra-Cellular Matrix and Differentially Effects 2D versus 3D Cell Migration

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    The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 −/− MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9−/− MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9−/− MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9−/−, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.NHMRC Project Grants 512551 and 63251

    NEDD9 Stabilizes Focal Adhesions, Increases Binding to the Extra-Cellular Matrix and Differentially Effects 2D versus 3D Cell Migration

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    The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 −/− MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9−/− MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9−/− MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9−/−, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.NHMRC Project Grants 512551 and 63251

    The effect of preoperative aspirin use on postoperative bleeding and perioperative myocardial infarction in patients undergoing coronary artery bypass surgery

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    Background: We tried to evaluate the clinical outcomes (mortality, postoperative bleeding and perioperative myocardial infarction) of patients who underwent first elective coronary artery bypass grafting and received aspirin during the preoperative period. Methods: The study was a prospective, randomized and single-blinded clinical trial. Two hundred patients were included and divided into two groups. One group received aspirin 80-160 mg, while in the other aspirin was stopped at least seven days before surgery. The primary end-points of the study were in-hospital mortality and hemorrhage-related complications (postoperative blood loss in the intensive care unit, re-exploration for bleeding and red blood cell and non-red blood cell requirements). The secondary end-point was perioperative myocardial infarction. Results: There were no differences in patient characteristics between the aspirin users and non-aspirin users. We found a significant difference between postoperative blood loss (608 ± ± 359.7 ml vs. 483 ± 251.5 ml, p = 0.005) and red blood cell product requirements (1.32 2+ ± 0.97 unit packed cell vs. 0.94 ± 1.02 unit packed cell, p = 0.008). There was no significant difference between the two groups regarding platelet requirement and the rate of in-hospital mortality and re-exploration for bleeding. Similarly, we found no significant difference in the incidence of definite and probable perioperative myocardial infarction (p = 0.24 and p = 0.56 respectively) or in-hospital mortality between the two groups. Conclusion: Preoperative aspirin administration increased postoperative bleeding and red blood cell requirements with no effect on mortality, re-exploration rate and perioperative myocardial infarction. We recommend withdrawal of aspirin seven days prior to surgery. Copyright © 2007 Via Medica

    The association between baseline persistent pain and weight change in patients attending a specialist weight management service

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    To quantify the influence of baseline pain levels on weight change at one-year follow-up in patients attending a National Health Service specialist weight management programme.We compared one-year follow-up weight (body mass) change between patient sub-groups of none-to-mild, moderate, and severe pain at baseline. A mean sub-group difference in weight change of ≥5kg was considered clinically relevant.Of the 141 complete cases, n = 43 (30.5%) reported none-to-mild pain, n = 44 (31.2%) reported moderate pain, and n = 54 (38.3%) reported severe pain. Covariate-adjusted mean weight loss (95%CI) was similar for those with none-to-mild (8.1kg (4.2 to 12.0kg)) and moderate pain (8.3kg (4.9 to 11.7kg). The mean weight loss of 3.0kg (-0.4 to 6.4kg) for the severe pain group was 5.1kg (-0.6 to 10.7, p = 0.08) lower than the none-to-mild pain group and 5.3kg (0.4 to 10.2kg, p = 0.03) lower than the moderate pain group.Patients with severe pain upon entry to a specialist weight management service in England achieve a smaller mean weight loss at one-year follow-up than those with none-to-moderate pain. The magnitude of the difference in mean weight loss was clinically relevant, highlighting the importance of addressing severe persistent pain in obese patients undertaking weight management programmes

    Genetic Analysis of Human Traits In Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

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    Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding

    A functional variant in the Stearoyl-CoA desaturase gene promoter enhances fatty acid desaturation in pork

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    There is growing public concern about reducing saturated fat intake. Stearoyl-CoA desaturase (SCD) is the lipogenic enzyme responsible for the biosynthesis of oleic acid (18:1) by desaturating stearic acid (18:0). Here we describe a total of 18 mutations in the promoter and 3′ non-coding region of the pig SCD gene and provide evidence that allele T at AY487830:g.2228T>C in the promoter region enhances fat desaturation (the ratio 18:1/18:0 in muscle increases from 3.78 to 4.43 in opposite homozygotes) without affecting fat content (18:0+18:1, intramuscular fat content, and backfat thickness). No mutations that could affect the functionality of the protein were found in the coding region. First, we proved in a purebred Duroc line that the C-T-A haplotype of the 3 single nucleotide polymorphisms (SNPs) (g.2108C>T; g.2228T>C; g.2281A>G) of the promoter region was additively associated to enhanced 18:1/18:0 both in muscle and subcutaneous fat, but not in liver. We show that this association was consistent over a 10-year period of overlapping generations and, in line with these results, that the C-T-A haplotype displayed greater SCD mRNA expression in muscle. The effect of this haplotype was validated both internally, by comparing opposite homozygote siblings, and externally, by using experimental Duroc-based crossbreds. Second, the g.2281A>G and the g.2108C>T SNPs were excluded as causative mutations using new and previously published data, restricting the causality to g.2228T>C SNP, the last source of genetic variation within the haplotype. This mutation is positioned in the core sequence of several putative transcription factor binding sites, so that there are several plausible mechanisms by which allele T enhances 18:1/18:0 and, consequently, the proportion of monounsaturated to saturated fat.This research was supported by grants from the Spanish Ministry of Science and Innovation (AGL2009-09779 and AGL2012-33529). RRF is recipient of a PhD scholarship from the Spanish Ministry of Science and Innovation (BES-2010-034607). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript
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