Sirt1 Regulates DNA Methylation and Differentiation Potential of Embryonic Stem Cells by Antagonizing Dnmt3l.

Embryonic stem cell (ESC) abnormalities in genome methylation hamper the utility of their therapeutic derivatives; however, the underlying mechanisms are unknown. Here, we show that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, Sirt1, selectively prevents abnormal DNA methylation of some developmental genes in murine ESCs by antagonizing Dnmt3l. Transcriptome and DNA methylome analyses demonstrated that Sirt1-null (Sirt1-/-) ESCs repress expression of a subset of imprinted and germline genes concomitant with increased DNA methylation of regulatory elements. Dnmt3l was highly expressed in Sirt1-/- ESCs, and knockdown partially rescued abnormal DNA methylation of the Sirt1 target genes. The Sirt1 protein suppressed transcription of Dnmt3l and physically interacted with the Dnmt3l protein, deacetylating and destabilizing Dnmt3l protein. Sirt1 deficiency delayed neurogenesis and spermatogenesis. These differentiation delays were significantly or partially abolished by reintroduction of Sirt1 cDNA or Dnmt3l knockdown. This study sheds light on mechanisms that restrain DNA methylation of developmentally vital genes operating in ESCs.

Time-dependent Gene Profiling Indicates the Presence of Different Phases for Ischemia/Reperfusion Injury in Retina.

Ischemia/reperfusion (IR) injury has been associated with several retinal pathologies, and a few genes/gene products have been linked to IR injury. However, the big picture of temporal changes, regarding the affected gene networks, pathways, and processes remains to be determined. The purpose of the present study was to investigate initial, intermediate, and later stages to characterize the etiology of IR injury in terms of the pathways affected over time. Analyses indicated that at the initial stage, 0-hour reperfusion following the ischemic period, the ischemia-associated genes were related to changes in metabolism. In contrast, at the 24-hour time point, the signature events in reperfusion injury include enhanced inflammatory and immune responses as well as cell death indicating that this would be a critical period for the development of any interventional therapeutic strategies. Genes in the signal transduction pathways, particularly transmitter receptors, are downregulated at this time. Activation of the complement system pathway clearly plays an important role in the later stages of reperfusion injury. Together, these results demonstrate that the etiology of injury related to IR is characterized by the appearance of specific patterns of gene expression at any given time point during retinal IR injury. These results indicate that evaluation of treatment strategies with respect to time is very critical.

Identifying mRNA, microRNA and protein profiles of melanoma exosomes.

BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

Microvascular mural cell functionality of human embryonic stem cell-derived mesenchymal cells.

Microvascular mural or perivascular cells are required for the stabilization and maturation of the remodeling vasculature. However, much less is known about their biology and function compared to large vessel smooth muscle cells. We have developed lines of multipotent mesenchymal cells from human embryonic stem cells (hES-MC); we hypothesize that these can function as perivascular mural cells. Here we show that the derived cells do not form teratomas in SCID mice and independently derived lines show similar patterns of gene expression by microarray analysis. When exposed to platelet-derived growth factor-BB, the platelet-derived growth factor receptor ß is activated and hES-MC migrate in response to a gradient. We also show that in a serum-free medium, transforming growth factor ß1 (TGFß1) induces robust expression of multiple contractile proteins (α smooth muscle actin, smooth muscle myosin heavy chain, smooth muscle 22α, and calponin). TGFß1 signaling is mediated through the TGFßR1/Alk5 pathway as demonstrated by inhibition of α smooth muscle actin expression by treatment of the Alk5-specific inhibitor SB525334 and stable retroviral expression of the Alk5 dominant negative (K232R). Coculture of human umbilical vein endothelial cell (HUVEC) with hES-MC maintains network integrity compared to HUVEC alone in three-dimensional collagen I-fibronectin by paracrine signaling. Using high-resolution laser confocal microscopy, we show that hES-MC also make direct contact with HUVEC. This demonstrates that hESC-derived mesenchymal cells possess the molecular machinery expected in a perivascular progenitor cells and can play a functional role in stabilizing EC networks in in vitro three-dimensional culture.

Perioperative anticoagulation and antiplatelet therapy in renal transplant: is there an increase in bleeding complication?

BACKGROUND: Renal transplant recipients may have comorbidities requiring anticoagulation or antiplatelet therapy. While the effects of warfarin may be neutralized with plasma infusion, those of aspirin and clopidogrel are not easily reversible and may be associated with an increased risk of bleeding. We conducted this study to evaluate the risk of bleeding complications in patients receiving perioperative anticoagulation or antiplatelet therapy. METHODS: Medical records of patients who underwent renal transplantation from July 1, 2005 to April 30, 2009 were retrospectively reviewed. Patients receiving perioperative anticoagulation or antiplatelet therapy were identified. The incidence of reoperation, transfusion utilization and decrease in serum hemoglobin from pre-operative value (ΔHgb) were compared to those on no therapy. RESULTS: Of the 327 patients identified, 105 received pre-operative anticoagulation or antiplatelet therapy, 28 received therapy post-operatively, while 213 patients received no therapy. The incidence of reoperation, transfusion utilization and ΔHgb were not significantly increased with pre-operative anticoagulation or antiplatelet therapy. With post-operative heparin infusion, the incidence of reoperation and transfusion utilization were significantly increased (p values < 0.001). Patients with activated partial thromboplastin times (aPTT) >80 s experienced significant bleeding complications. CONCLUSION: A supratherapeutic aPTT with post-operative heparin infusion was associated with the greatest risk of bleeding complication.

Estimating key parameters in periodic breast cancer screening-application to the Canadian National Breast Screening Study data.

PROBLEM STATEMENT: Breast cancer screening in women of younger age has been controversial. The screening sensitivities, transition probabilities and sojourn time distributions are estimated for females aged 40-49 years and 50-59 years separately, using the Canadian National Breast Screening Study (CNBSS) data. The purpose is to estimate the lead time distribution and the probability of not detecting the cancer early. APPROACH: Within the 40-49-year-old and 50-59-year-old cohorts separately, the age-independent statistical model was applied. Bayesian estimators along with 95% highest probability density (HPD) credible intervals (CI) were calculated. Bayesian hypothesis testing was used to compare the parameter estimates of the two cohorts. The lead time density was also estimated for both the 40-49 and 50-59-year-old cohorts. RESULTS: The screening sensitivity, transition probability of the disease, and mean sojourn time were all found to increase with age. For the 40-49-year-old and 50-59-year-old cohorts, the posterior mean sensitivities were 0.70 (95% HPD-CI: 0.46, 0.93) and 0.77 (0.61, 0.93), respectively. The posterior mean transition probabilities were 0.0023 (0.0018, 0.0027) and 0.0031 (0.0024, 0.0038), while the posterior mean sojourn times were 2.55 (1.56, 4.26) years and 3.15 (2.12, 4.96) years. Bayes factors for the ratio of posterior probabilities that the respective parameter was larger vs. smaller in the 50-59-year-old cohort were estimated to be 2.09, 40.8 and 3.0 for the sensitivity, transition probability, and mean sojourn time, respectively. All three Bayes factors were larger than two, indicating greater than 2:1 odds in favor of the hypothesis that each of these parameters was greater in the 50-59-year-old cohort. The estimated mean lead times were 0.83 years and 0.96 years if the two cohorts were screened annually. CONCLUSIONS: The increase in sensitivity corresponds to an increase in the mean sojourn time. Breast cancer in younger women is more difficult to detect by screening tests and is more aggressive than breast cancer in older women. Women aged 50-59 tend to benefit more from screening compared with women aged 40-49.