技术资料

Because of recent declining malaria transmission in Latin America,some authorities have recommended against chemoprophylaxis for most travelers to this region. However,the predominant parasite species in Latin America,Plasmodium vivax,can form hypnozoites sequestered in the liver,causing malaria relapses. Additionally,new evidence shows the potential severity of vivax infections,warranting continued consideration of prophylaxis for travel to Latin America. Individualized travel risk assessments are recommended and should consider travel locations,type,length,and season,as well as probability of itinerary changes. Travel recommendations might include no precautions,mosquito avoidance only,or mosquito avoidance and chemoprophylaxis. There are a range of good options for chemoprophylaxis in Latin America,including atovaquone-proguanil,doxycycline,mefloquine,and--in selected areas--chloroquine. Primaquine should be strongly considered for nonpregnant,G6PD-nondeficient patients traveling to vivax-endemic areas of Latin America,and it has the added benefit of being the only drug to protect against malaria relapses. View Publication

New drugs for preserving and restoring pancreatic β-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1,also known as BETA2); this transcription factor functions in neuronal and pancreatic β-cell differentiation and is essential for insulin gene transcription. Here,we probed effects of Isx on human cadaveric islets and MIN6 pancreatic β cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure,Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity,Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary,we have identified a small molecule with antidiabetic activity,providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes. View Publication

HIV-1 Tat-interacting protein of 110 kDa [Tip110; p110(nrb)/SART3/p110] is an RNA binding nuclear protein implicated in regulation of HIV-1 gene and host gene transcription,pre-mRNA splicing,and cancer immunology. Recently,we demonstrated a role for Tip110 in regulation of hematopoiesis. Here,we show that TIP110 is also expressed in human embryonic stem cells (hESCs) and expression was decreased with differentiation of these ESCs. TIP110 was found,through up- and down-modulation of expression of Tip110,to be important in maintaining pluripotent factor (NANOG,OCT4,and SOX2) expression in and pluripotency of hESCs,although the mechanisms involved and whether the Tip110 effects are direct remain to be determined. View Publication

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Recent studies have reported the anti-tumor effects of the AhR in breast cancer. In this study,we investigated the anti-tumor effect of AhR activation based on the cancer stem cell hypothesis. We show that AhR activation suppressed mammosphere formation of MCF-7 cells and decreased the proportion of cells with high ALDH-1 (aldehyde dehydrogenase 1) activity. In addition,we also demonstrate that AhR activation regulates self-renewal signaling by down-regulating Wnt/$$-catenin and Notch. View Publication

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines,from 38 laboratories worldwide,for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal,but there was a progressive tendency to acquire changes on prolonged culture,commonly affecting chromosomes 1,12,17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants,determined from the SNP arrays,also appeared sporadically. No common variants related to culture were observed on chromosomes 1,12 and 17,but a minimal amplicon in chromosome 20q11.21,including three genes expressed in human ES cells,ID1,BCL2L1 and HM13,occurred in textgreater20% of the lines. Of these genes,BCL2L1 is a strong candidate for driving culture adaptation of ES cells. View Publication

Considerable efforts have been made since the 1950s to better understand the cellular and molecular mechanisms of action of metformin,a potent antihyperglycaemic agent now recommended as the first-line oral therapy for T2D (Type 2 diabetes). The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production,mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I. In addition,the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase),a cellular metabolic sensor,providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. The demonstration that respiratory chain complex I,but not AMPK,is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice. Beyond its effect on glucose metabolism,metformin has been reported to restore ovarian function in PCOS (polycystic ovary syndrome),reduce fatty liver,and to lower microvascular and macrovascular complications associated with T2D. Its use has also recently been suggested as an adjuvant treatment for cancer or gestational diabetes and for the prevention in pre-diabetic populations. These emerging new therapeutic areas for metformin will be reviewed together with recent findings from pharmacogenetic studies linking genetic variations to drug response,a promising new step towards personalized medicine in the treatment of T2D. View Publication

(R)-Trichostatin A (TSA) is a Streptomyces product which causes the induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at the very low concentrations. We found that TSA caused an accumulation of acetylated histone species in a variety of mammalian cell lines. Pulse-labeling experiments indicated that TSA markedly prolonged the in vivo half-life of the labile acetyl groups on histones in mouse mammary gland tumor cells,FM3A. The partially purified histone deacetylase from wild-type FM3A cells was effectively inhibited by TSA in a noncompetitive manner with Ki = 3.4 nM. A newly isolated mutant cell line of FM3A resistant to TSA did not show the accumulation of the acetylated histones in the presence of a higher concentration of TSA. The histone deacetylase preparation from the mutant showed decreased sensitivity to TSA (Ki = 31 nM,noncompetitive). These results clearly indicate that TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme. View Publication

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic $\$-cells. In other systems,Musashi genes regulate cell fate via Notch signaling,which we recently showed regulates $\$-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms,as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of $\$-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1,while it decreased Ins1 and Ins2 expression,revealing a molecular link between ER stress and $\$-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1,stimulate proliferation,inhibit apoptosis and reduce insulin expression,whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together,these results demonstrate overlapping,but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and $\$-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory $\$-cell proliferation and the loss of $\$-cell gene expression seen in specific phases of the progression to type 2 diabetes. View Publication

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However,natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs),which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities,and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (˜50%) to hPSCs,which are importantly accompanied by a considerable increase in gene-targeting frequencies,up to 0.12%. While this level is likely sufficient for numerous applications,we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (textgreater1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus,this study demonstrates that under appropriate selective pressures,AAV vectors can be created to mediate efficient gene targeting in hPSCs,alone or in the presence of ZFN- mediated double-stranded DNA breaks. View Publication

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important,given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here,we present an in-depth quantitative mass spectrometry-based analysis of hESCs,two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless,a small group of 58 proteins,mainly related to metabolism,antigen processing and cell adhesion,was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap,highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. View Publication

Human embryonic stem cells (hESC) and hESC-derived cardiomyocytes (hESC-CM) hold great promise for the treatment of cardiovascular diseases. However the mechanobiological properties of hESC and hESC-CM remains elusive. In this paper,we examined the dynamic and static micromechanical properties of hESC and hESC-CM,by manipulating via optical tweezers at the single-cell level. Theoretical approaches were developed to model the dynamic and static mechanical responses of cells during optical stretching. Our experiments showed that the mechanical stiffness of differentiated hESC-CM increased after cardiac differentiation. Such stiffening could associate with increasingly organized myofibrillar assembly that underlines the functional characteristics of hESC-CM. In summary,our findings lay the ground work for using hESC-CMs as models to study mechanical and contractile defects in heart diseases. View Publication

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process,but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b,two known vitamin-C-dependent H3K36 demethylases,as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore,we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367,an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming. View Publication