Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.     

Dihydropyridines and atypical MDR: a novel perspective of designing general reversal agents for both typical and atypical MDR

Multidrug resistance (MDR) is defined as resistance of tumor cells to the cytotoxic action of multiple structurally dissimilar and functionally divergent drugs commonly used in chemotherapy. 1,4-Dihydropyridines (DHP(s)) are one of the major classes of Ca2+ channel blockers, and it has been shown that these agents are a new class of drug resistance reversers in cancer treatment. Analysis of various investigations on MDR reversing effects of DHPs shows that they can be a potential class for designing compounds simultaneously effective on both typical and atypical MDR. Also, it is possible to include some considerations on essential structural features for MDR reversing and further decreasing of Ca2+ channel blocking activity as an adverse effect.     

LIGR, a protease-activated receptor-2-derived peptide, enhances skin pigmentation without inducing inflammatory processes

The protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR-2 with Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), a known PAR-2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu-Ile-Gly-Arg-NH(2) (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno-deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho-GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR-2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR-2 signaling pathways, including [Ca(2+)] mobilization, cAMP induction, the induction of cyclooxgenase-2 (COX-2) expression and the secretion of prostaglandin E2, interleukin-6 and -8. PAR-2 siRNA inhibited LIGR-induced phagocytosis, indicating that LIGR signals via PAR-2. Our data suggest that LIGR is a more specific regulator of PAR-2-induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned-like skin color, without enhancing inflammatory processes, and without the need of UV exposure.     

Actin Phylogeny and Intron Distribution in Bangiophyte Red Algae(Rhodoplantae)

The molecular phylogeny of red algal actin genes, with emphasis on the paraphyletic “Bangiophyceae,” was examined and compared to the rhodophyte SSU rDNA phylogeny. Nineteen new genomic actin sequences and seven SSU rDNA sequences were obtained and subjected to diverse phylogenetic analyses (maximum likelihood, distance/neighbor-joining, maximum parsimony, Bayesian analyses, and, with respect to protein sequences, also quartet puzzling). The actin trees confirmed most of the major clades found in the SSU rDNA phylogenies, although with a lower resolution. An actin gene duplication in the florideophycean lineage is reported, presumably related to an increased complexity of sexual reproduction. In addition, the distribution and characteristics of spliceosomal introns found in some of the actin sequences were examined. Introns were found in almost all florideophycean actin genes, whereas only two bangiophyte sequences contained introns. One intron in the florideophycean actin genes was also found in metazoan, and, shifted by one or two nucleotides, in a glaucocystophyte, a cryptophyte, and two fungal actin genes, and thus may be an ancient intron.[Reviewing Editor: Dr. Yves Van de Peer]     

Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.     

Can there be a truly systematic and comprehensive theory of race?

ABSTRACT

In this ambitious new book, Mustafa Emirbayer and Matthew Desmond contend that there has never been a truly comprehensive and systematic theory of race. They go on to argue that ‘Much of our best work no longer tells us how to understand or reconstruct racial dynamics but simply gives us concrete proof of their continuing significance’ (3). To what extent does The Racial Order theoretically advance existing theorizing of race? An important contribution – and a central plank in the book – is the way in which a wide variety of cultural and social phenomena is discussed and interwoven into the analysis. The authors draw most heavily on Bourdieu, Dewey, and Durkheim, in their elaboration of the racial order.     

Logarithmic and Power Law Input-Output Relations in Sensory Systems with Fold-Change Detection

Two central biophysical laws describe sensory responses to input signals. One is a logarithmic relationship between input and output, and the other is a power law relationship. These laws are sometimes called the Weber-Fechner law and the Stevens power law, respectively. The two laws are found in a wide variety of human sensory systems including hearing, vision, taste, and weight perception; they also occur in the responses of cells to stimuli. However the mechanistic origin of these laws is not fully understood. To address this, we consider a class of biological circuits exhibiting a property called fold-change detection (FCD). In these circuits the response dynamics depend only on the relative change in input signal and not its absolute level, a property which applies to many physiological and cellular sensory systems. We show analytically that by changing a single parameter in the FCD circuits, both logarithmic and power-law relationships emerge; these laws are modified versions of the Weber-Fechner and Stevens laws. The parameter that determines which law is found is the steepness (effective Hill coefficient) of the effect of the internal variable on the output. This finding applies to major circuit architectures found in biological systems, including the incoherent feed-forward loop and nonlinear integral feedback loops. Therefore, if one measures the response to different fold changes in input signal and observes a logarithmic or power law, the present theory can be used to rule out certain FCD mechanisms, and to predict their cooperativity parameter. We demonstrate this approach using data from eukaryotic chemotaxis signaling.     

Production of a recombinant alkane hydroxylase (AlkB2) from <Emphasis Type="Italic">Alcanivorax</Emphasis><Emphasis Type="Italic">borkumensis</Emphasis>

Alcanivorax borkumensis is an oil-degrading marine bacterium. Its genome contains genes coding for three cytochrome P450s and two integral membrane alkane hydroxylases (AlkB1 & AlkB2), all assumed to perform hydroxylation of different linear or branched alkanes. Although, the sequence of alkB2 has been determined, the molecular characterization and the substrate specificity of AlkB2 require more precise investigation. In this study, AlkB2 from A. borkumensis SK2 was expressed in Escherichia coli to examine the functionality of AlkB2 as a hydroxylating enzyme. Furthermore, the activity of the enzyme in the presence of the accessory proteins, rubredoxin (RubA) and rubredoxin reductase (RubB), produced in E. coli BL21(DE3)plysS cells, was determined. Recombinant AlkB2 is produced in an active form and rubredoxin is the intermediate electron donor to AlkB2 and can replace AlkG function, when NADH is the prime electron donor.     

Long- but not short-term heat acclimation produces an apoptosis-resistant cardiac phenotype: a lesson from heat stress and ischemic/reperfusion insults

Long-term heat acclimation (AC, 30d/34°C) is a phenotypic adaptation leading to increased thermotolerance during heat stress (HS, 2 h 41°C). AC also renders protection against ischemic/reperfusion (I/R, 30′ global ischemia/40′ reperfusion) insult via cross-tolerance mechanisms. In contrast to the protected AC phenotype, the onset of acclimation (34°C, AC2d) is characterized by cellular perturbations, suggesting increased susceptibility to HS and I/R insults. In this investigation, we tested the hypothesis that apoptosis resistance is part of the AC repertoire and that, at the initial phase of acclimation (AC2d), cytoprotection is impaired. TUNEL staining and caspase 3 levels in HS and I/R insulted hearts affirmed this hypothesis. To examine the role of the mitochondria in life/death decision in AC2d and 30d AC settings vs. control hearts, we studied the Bcl-2 apoptotic cascade and found increased levels of the anti-apoptotic Bcl-X_L and decreased levels of the pro-apoptotic death promoter Bad in hearts from AC2d and AC animals. In these groups, cytochrome c (cyt c) was elevated in the mitochondria and remained unchanged in the cytosol. This adaptation was insufficient to negate apoptosis in AC2d rats. At this early acclimation phase (and in controls), increased caspase 8 activity confirmed activation of the extrinsic (Fas ligand) apoptosis pathway. In conclusion, the elevated Bcl-X_L/Bad ratio and decreased cyt c leakage to the cytosol are insufficient to protect the heart and interactions with additional cytoprotective pathways involved in acclimation (elevated HSP70, ROS, and sarcolemmal adaptations to abolish extrinsic apoptosis pathways) are required to induce the apoptosis-resistant AC phenotype.