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We asked this question: in normal humans,is either a normal dietary intake or normal serum concentration of phosphorus a determinant of the serum concentration of 1,25(OH)2D? In seven normal men whose dietary phosphorus was decreased from 2,300 to 625 mg/d,each intake for 8-9 d,under strictly controlled,normal metabolic conditions,we measured serum concentrations of 1,25(OH)2D daily,and concentrations of phosphorus hourly throughout a 24-h period,before and after restriction. Decreasing dietary phosphorus induced: (a) a 58% increase in serum levels of 1,25(OH)2D; (b) a 35% decrease in serum levels of phosphorus measured in the afternoon; (c) a 12% decrease in the 24-h mean serum level of phosphorus; but,(d) no decrease in morning fasting levels of phosphorus. Serum concentrations of 1,25(OH)2D varied inversely and significantly with 24-h mean concentrations of phosphorus (r = -0.77,P less than 0.001). When these data are combined with those of our prior study in which dietary phosphorus was varied over an extreme range,the relationship between serum levels of 1,25(OH)2D and 24-h mean serum levels of phosphorus is even stronger (r = -0.90,P less than 0.001). In the aggregate,the results demonstrate that in normal men,dietary phosphorus throughout a normal range and beyond,can finely regulate the renal production and serum concentration of 1,25(OH)2D,and provide evidence that this regulation is mediated by fine modulation of the serum concentration of phosphorus. View Publication

BACKGROUND Neuritic degeneration is an important early pathological step in neurodegeneration. AIM The purpose of this study was to explore the mechanisms connecting neuritic degeneration to the functional and morphological remodeling of endoplasmic reticulum (ER) and mitochondria. METHODS Here,we set up neuritic degeneration models by neurite cutting-induced neural degeneration in human-induced pluripotent stem cell-derived neurons. RESULTS We found that neuritic ER becomes fragmented and forms complexes with mitochondria,which induces IP3R-dependent mitochondrial Ca(2+) elevation and dysfunction during neuritic degeneration. Furthermore,mitochondrial membrane potential is required for ER fragmentation and mitochondrial Ca(2+) elevation during neuritic degeneration. Mechanically,tightening of the ER-mitochondria associations by expression of a short synthetic linker" and ER Ca(2+) releasing together could promote mitochondrial Ca(2+) elevation� View Publication

AIM To evaluate the tumor localization and efficacy pH-responsive expansile nanoparticles (eNPs) as a drug delivery system for pancreatic peritoneal carcinomatosis (PPC) modeled in nude rats. METHODS & MATERIALS A Panc-1-cancer stem cell xeno1graft model of PPC was validated in vitro and in vivo. Tumor localization was tracked via in situ imaging of fluorescent eNPs. Survival of animals treated with paclitaxel-loaded eNPs (PTX-eNPs) was evaluated in vivo. RESULTS The Panc-1-cancer stem cell xenograft model recapitulates significant features of PPC. Rhodamine-labeled eNPs demonstrate tumor-specific,dose- and time-dependent localization to macro- and microscopic tumors following intraperitoneal injection. PTX-eNPs are as effective as free PTX in treating established PPC; but,PTX-eNPs result in fewer side effects. CONCLUSION eNPs are a promising tool for the detection and treatment of PPC. View Publication

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury,although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation,respectively,attenuating the activity of the C3/C5 convertases and,consequently,avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study,we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly,FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture,resembling immature MoDCs,lower expression of the maturation marker CD83 and the costimulatory molecules CD40,CD80,and CD86,decreased production of key proinflammatory Th1-cytokines (IL-12,TNF-α,IFN-γ,IL-6,and IL-8),and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover,FH-treated MoDCs show low Ag uptake and,when challenged with LPS,display reduced CCR7 expression and chemotactic migration,impaired CD4(+) T cell alloproliferation,inhibition of IFN-γ secretion by the allostimulated T cells,and,conversely,induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus,this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity,transplantation,and autoimmunity. View Publication

Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase,MLL2 (KMT2B),during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here,we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically,by utilizing dimeric CRISPR RNA-guided nucleases,RFNs (commercially known as the NextGEN™ CRISPR),in combination with an excision-only piggyBac™ transposase,we demonstrate how to generate a point mutation of threonine-542,a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection,and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless donor-excision" approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations� View Publication

BACKGROUND Patient-derived induced pluripotent stem cells (iPSCs) are an innovative source as an in vitro model for neurological diseases. Recent studies have demonstrated the differentiation of brain microvascular endothelial cells (BMECs) from various stem cell sources,including iPSC lines. However,the impact of the culturing conditions used to maintain such stem cell pluripotency on their ability to differentiate into BMECs remains undocumented. In this study,we investigated the effect of different sources of Matrigel and stem cell maintenance medium on BMEC differentiation efficiency. METHODS The IMR90-c4 iPSC line was maintained on mTeSR1 or in essential-8 (E-8) medium on growth factor-reduced (GFR) Matrigel from three different manufacturers. Cells were differentiated into BMECs following published protocols. The phenotype of BMEC monolayers was assessed by immunocytochemistry. Barrier function was assessed by transendothelial electrical resistance (TEER) and permeability to sodium fluorescein,whereas the presence of drug efflux pumps was assessed by uptake assay using fluorescent substrates. RESULTS Stem cell maintenance medium had little effect on the yield and barrier phenotype of IMR90-derived BMECs. The source of GFR-Matrigel used for the differentiation process significantly impacted the ability of IMR90-derived BMECs to form tight monolayers,as measured by TEER and fluorescein permeability. However,the Matrigel source had minimal effect on BMEC phenotype and drug efflux pump activity. CONCLUSION This study supports the ability to differentiate BMECs from iPSCs grown in mTeSR1 or E-8 medium and also suggests that the origin of GFR-Matrigel has a marked inpact on BMEC barrier properties. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy,while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study,we generated TAP1- and TAPBP-deficient hESC lines,respectively,using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types,but maintained normal pluripotency,karyotypes,and differentiation ability. Thus,our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future. View Publication

CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement,these requirements are met by an increased glycolysis,due,at least in part,to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia,we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably,Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore,TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics,irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen,Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition,augmented proliferation,and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover,Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus,Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. View Publication

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid,we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3,Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells,whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. View Publication

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure,self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells,but its role in hESCs remains unclear. Here,we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly,knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly,FOXM1 depletion sensitized hESCs to oxidative stress. Moreover,genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1,which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together,our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs. View Publication

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism,genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs,the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells,which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation,metabolism,genetic network,and response to infection or other external stimuli. View Publication