Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

Vitronectin decreases microvascular endothelial cell apoptosis

Angiogenesis after tissue injury occurs in a matrix environment consisting of fibrin, fibronectin, and vitronectin as the major extracellular matrix (ECM) constituents. ECM-integrin interactions is critical for angiogenesis and failure to bind a ligand to certain integrin receptors (α_vβ₃ or α_vβ₅) inhibits angiogenesis. The ligand that binds to α_vβ₃ or α_vβ₅ integrin receptors during microvascular angiogenesis has not been identified. Our hypothesis is that provisional matrix molecules provide the environmental context cues to microvascular endothelial cells and promote angiogenesis by decreased programmed cell death. Using cultured human microvascular endothelial cells, we show that vitronectin, in comparison to growth on alternative provisional matrix molecules (fibronectin, fibrinogen plus thrombin), collagen I, and basement membrane molecules (collagen IV), significantly reduces microvascular endothelial cell death in vitro. This reduction was observed using morphologic criteria, TdT-mediated dUTP nick end labeling (TUNEL) assay, histone release into the cytoplasm, and thymidine release into the supernatant. Though our data confirm that vitronectin may bind to more than one integrin receptor to reduce MEC apoptosis, binding to the α_v component appears to be the critical integrin subcomponent for reducing apoptosis. J. Cell. Physiol. 175:149–155, 1998. © 1998 Wiley-Liss, Inc.     

Variation of reaction dynamics for OH hydrogen abstraction from glycine between ab initio levels of theory

The variation in reaction dynamics of OH hydrogen abstraction from glycine between HF, MP2, CCSD(T), M05-2X, BHandHLYP, and B3LYP levels was demonstrated. The abstraction mode shows distinct patterns between these five levels and determines the barrier height, and the spin density transfer between OH radical and glycine. These differences are mainly resulted from the spin density distribution and geometry of the alpha carbon during the abstraction. The captodative effect which is commonly believed as one of the major factors to stabilize the caron-centered radical can only be observed in DFT levels but not in HF and MP2 levels. Difference in the abstraction energy were found in these calculation levels, by using the result of CCSD(T) as reference, B3LYP, BHandHLYP, and M05-2X underestimated the reaction barrier about 5.1, 0.1, and 2.4 kcal mol^-1, while HF and MP2 overestimated 19.1 kcal mol^-1 and 1.6 kcal mol^-1, respectively. These differences can be characterized by the vibration mode of imaginary frequency of transition states, which indicates the topology around transition states and determines reaction barrier height. In this model system, BHandHLYP provides the best prediction of the energy barrier among those tested methods.     

The first two-dimensional reference map of the fission yeast, Schizosaccharomyces pombe proteins

Cytosolic proteins of Schizosaccharomyces pombe were separated by two-dimensional (2-D) gel electrophoresis, to construct the first 2-D reference map. In the pI range 4-7, more than 500 spots were detected by silver staining, and 70 different proteins corresponding to 111 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry, where necessary. In the pI range 6-9, approximately 330 spots were detected, and 31 proteins corresponding to 38 spots were identified by mass spectrometry. More than 50% of the identified proteins were involved in amino acid, carbohydrate or nucleotide metabolism, and energy production. A second large group of identified proteins comprises heat shock and other stress related proteins and chaperones.     

Application of RAPD and SCAR markers for purity testing of F1 hybrid seed in chili pepper (Capsicum annuum)

A simpler and better method for purity testing of hybrid pepper seed was developed. The simplest method for extracting genomic DNA, the NaOH method, was chosen. Two RAPD markers identifying male and female parents were also developed, and the PCR products of male- and female-specific RAPD markers were cloned and sequenced. From these sequences, new longer primers were constructed for conversion into SCAR markers. In blind tests the RAPD and SCAR markers were able to reliably detect contaminating exotic seeds. These PCR-based markers are therefore directly applicable for purity testing by seed companies. In addition, the PCR products of the SCAR markers could be identified by direct staining methods such as ethidium bromide and pellet painting without electrophoresis.     

Structural basis for the changes in redox potential in the nitrogenase Phe135Trp Fe protein with MgADP Bound

The crystal structure of the Azotobacter vinelandii nitrogenase Fe protein with phenylalanine at position 135 substituted by tryptophan has been determined in MgADP-bound form by X-ray diffraction methods. Amino acid substitution studies have suggested that the phenylalanine at position 135 located near the [4Fe-4S] cluster contributes to both the midpoint potential and nucleotide-induced changes of the [4Fe-4S] cluster. Substitution of tryptophan for phenylalanine at position 135 resulted in a significant positive shift in the midpoint potential in both the isolated and nucleotide-bound states. The factors thought to control the midpoint potential of the [FeS] cluster include solvent accessibility, dipolar environment, and structural strain. The structure derived in the present work provides an explanation for the more positive midpoint potential observed in the nucleotide-bound state, and suggests important insights into the contributions of the nucleotide interaction to the conformational states that are the keys to nitrogenase catalysis. The presence of MgADP in Phe135Trp Fe protein reveals the mechanism of the long-range communication from the nucleotide-binding site that controls its affinity for the MoFe protein component.     

Gene-transferring efficiencies of novel diamino cationic lipids with varied hydrocarbon chains

Utilizing three biocompatible components, a series of novel cationic lipids has been chemically synthesized and tested for their gene-transferring capabilities in 293 transformed kidney cells and B16BL6 mouse melanoma cells. The synthesized cationic lipids consisting of a core of lysine and aspartic acid with hydrocarbon chains of varied length were assigned the acronyms DLKD (O,O'-dilauryl N-lysylaspartate), DMKD (O,O'-dimyristyl N-lysylaspartate), DPKD (O,O'-dipalmityl N-lysylaspartate), and DSKD (O,O'-distearyl N-lysylaspartate). The gene-transferring capabilities of these cationic lipids were found to be dependent on the hydrocarbon chain length. Under similar experimental conditions, the order of gene transfection efficiency was DMKD > DLKD > DPKD > DSKD. Addition of cholesterol or dioleoyl phosphatidylethanolamine (DOPE) as a colipid did not change this order. Colipid addition affected the transfection efficiency positively or negatively depending on the length of the cationic lipid acyl chain. On the whole, the length of the hydrophobic carbon chain was a major factor governing the gene-transferring capabilities of this series of cationic lipids. The observed differences in transfection efficiency may be due to differing binding affinities to DNA molecules as well as differences in the surface charge potential of the liposome-DNA complexes (lipoplexes) in the aqueous environment.     

Insulin suppresses PDK-4 expression in skeletal muscle independently of plasma FFA

Starvation and experimental diabetes induce a stable increase in pyruvate dehydrogenase kinase (PDK) activity in skeletal muscle, which is largely due to a selective upregulation of PDK-4 expression. Increased free fatty acid (FFA) level has been suggested to be responsible for the upregulation. Because these metabolic states are also characterized by insulin deficiency, the present study was designed to examine whether insulin has a significant effect to regulate PDK mRNA expression in rat skeletal muscle. In study 1, overnight-fasted rats received an infusion of saline or insulin for 5 h (n = 6 each). During the insulin infusion, plasma glucose was clamped at basal levels (euglycemic hyperinsulinemic clamp). A third group (n = 6) received Intralipid infusion during the clamp to prevent a fall in plasma FFA. PDK-2 mRNA level in gastrocnemius muscle was not altered by insulin or FFA (i.e., Intralipid infusion). In contrast, PDK-4 mRNA level was decreased 72% by insulin (P < 0.05), and Intralipid infusion prevented only 20% of the decrease. PDK-4 protein level was decreased approximately 20% by insulin (P < 0.05), but this effect was not altered by Intralipid infusion. In study 2, overnight-fasted rats were refed or received an infusion of saline or nicotinic acid (NA, 30 micromol/h) for 5 h (n = 5 each). During the refeeding and NA infusion, plasma FFA levels were similarly (i.e., 60-70% vs. saline control) lowered. Muscle PDK-4 mRNA level decreased 77% after the refeeding (P < 0.05) but not after the NA infusion. In conclusion, the present data indicate that insulin had a profound effect to suppress PDK-4 expression in skeletal muscle and that, contrary to previous suggestions, circulating FFA had little impact on PDK-4 mRNA expression, at least within 5 h.