Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

As previously shown, higher levels of NOTCH1 and increased NF-kappa B signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow ( BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells ( CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency ( than expected by chance) of NF-kappa B-binding sites (BS), including potentially novel NF-kappa B targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappa B, and other important TFs on more primitive HSC sets.

Consequences for the Bovine Embryo of Being Derived from a Spermatozoon Subjected to Post-Ejaculatory Aging and Heat Shock: Development to the Blastocyst Stage and Sex Ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the. embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.

Differential expression of HIF-1 alpha in CD44(+)CD24(-/) (low) breast ductal carcinomas

Background: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1 alpha expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44(+)CD24(-/low) immunophenotype. Methods: Immunohistochemical analysis of CD44(+)CD24(-/low) expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results: In univariate analysis, CD44(+)CD24(-/low) expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44(+)CD24(-/low) immunophenotype showed relationship with HIF-1 alpha (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44(+)CD24(-/low) positive breast tumors.

Gene silencing during development of in vitro-produced female bovine embryos

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mu M ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to ""inactivation memory"" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and > 95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (> 90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Self-bound models of compact stars and recent mass-radius measurements

The exact composition of a specific class of compact stars, historically referred to as ""neutron stars,'' is still quite unknown. Possibilities ranging from hadronic to quark degrees of freedom, including self-bound versions of the latter, have been proposed. We specifically address the suitability of strange star models (including pairing interactions) in this work, in the light of new measurements available for four compact stars. The analysis shows that these data might be explained by such an exotic equation of state, actually selecting a small window in parameter space, but still new precise measurements and also further theoretical developments are needed to settle the subject.

Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea

Background: Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy. Methods: Organs of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGF alpha). Immunofluorescence assays were conducted for phenotype characterization. Results: The TGF alpha group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells. Conclusions: Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.

Morphological and growth alterations on early development stages of Iridaea cordata (Rhodophyta) under different intensities of UVB radiation

This study describes the effects of different intensities of UVB radiation on growth and morphology of early development stages of Iridaea cordata in germlings, young gametophytes originated in the laboratory and young fronds collected in the Magellan Strait, Chile. The experiments were carried out during four weeks in controlled conditions of temperature and photoperiod and the results were compared with a control treatment (without UVB). All UVB irradiation treatments caused bleaching and decrease in growth rates of germlings. Additionally, initial upright fronds were not observed in any of the UVB treatments, where as those cultivated in UVB absence developed erect ones in the second week of culture. The young gametophytes exhibited morphological alteration (small number and size of basal ramifications, curling of tips, bleaching and necrosis) and decrease in growth when exposed to UVB radiation. Young fronds collected from the field showed mainly morphological alterations (curling of frond). Morphological alterations in young gametophytes and young fronds of I. cordata could be interpreted as a defense against UVB by reducing the area exposed to radiation. However, high level of UVB radiation can produce irreparable damage, such as necrosis, observed in young gametophytes originated in the laboratory. Finally, the UVB effects on early developmental stages of I. cordata depend on the UVB irradiance and time of exposition.

Development of a DNA-dosimeter system for monitoring the effects of solar-ultraviolet radiation

Solar radiation sustains and affects all life forms on Earth. In recent years, the increase in environmental levels of solar-UV radiation due to depletion of the stratospheric ozone layer, as a result of anthropogenic emission of destructive chemicals, has highlighted serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions, where the intensity of solar radiation is higher. To better understand the impact of the harmful effects of solar-UV radiation on the DNA molecule, we developed a reliable biological monitoring system based on the exposure of plasmid DNA to artificial UV lamps and sunlight. The determination and quanti. cation of different types of UV photoproducts were performed through the use of specific DNA repair enzymes and antibodies. As expected, a significant number of CPDs and 6-4PPs was observed when the DNA-dosimeter system was exposed to increasing doses of UVB radiation. Moreover, CPDs could also be clearly detected in plasmid DNA when this system was exposed to either UVA or directly to sunlight. Interestingly, although less abundant, 6-4PPs and oxidative DNA damage were also generated after exposure to both UVA and sunlight. These results confirm the genotoxic potential of sunlight, reveal that UVA may also produce CPDs and 6-4PPs directly in naked DNA and demonstrate the applicability of a DNA-dosimeter system for monitoring the biological effects of solar-UV radiation.

Multistability and Self-Similarity in the Parameter-Space of a Vibro-Impact System

The dynamics of a dissipative vibro-impact system called impact-pair is investigated. This system is similar to Fermi-Ulam accelerator model and consists of an oscillating one-dimensional box containing a point mass moving freely between successive inelastic collisions with the rigid walls of the box. In our numerical simulations, we observed multistable regimes, for which the corresponding basins of attraction present a quite complicated structure with smooth boundary. In addition, we characterize the system in a two-dimensional parameter space by using the largest Lyapunov exponents, identifying self-similar periodic sets. Copyright (C) 2009 Silvio L.T. de Souza et al.

Accurate prediction of the Si/SiO(2) interface band offset using the self-consistent ab initio DFT/LDA-1/2 method

We use the density functional theory/local-density approximation (DFT/LDA)-1/2 method [L. G. Ferreira , Phys. Rev. B 78, 125116 (2008)], which attempts to fix the electron self-energy deficiency of DFT/LDA by half-ionizing the whole Bloch band of the crystal, to calculate the band offsets of two Si/SiO(2) interface models. Our results are similar to those obtained with a ""state-of-the-art"" GW approach [R. Shaltaf , Phys. Rev. Lett. 100, 186401 (2008)], with the advantage of being as computationally inexpensive as the usual DFT/LDA. Our band gap and band offset predictions are in excellent agreement with experiments.

Dispersion of electron g-factor with optical transition energy in (In,Ga)As/GaAs self-assembled quantum dots

The electron spin precession about an external magnetic field was studied by Faraday rotation on an inhomogeneous ensemble of singly charged, self-assembled (In,Ga)As/GaAs quantum dots. From the data the dependence of electron g-factor on optical transition energy was derived. A comparison with literature reports shows that the electron g-factors are quite similar for quantum dots with very different geometrical parameters, and their change with transition energy is almost identical. (C) 2011 American Institute of Physics. [doi:10.1063/1.3588413]

Chaotic advection in blood flow

In this paper we argue that the effects of irregular chaotic motion of particles transported by blood can play a major role in the development of serious circulatory diseases. Vessel wall irregularities modify the flow field, changing in a nontrivial way the transport and activation of biochemically active particles. We argue that blood particle transport is often chaotic in realistic physiological conditions. We also argue that this chaotic behavior of the flow has crucial consequences for the dynamics of important processes in the blood, such as the activation of platelets which are involved in the thrombus formation.

Gene Expression Noise in Spatial Patterning: hunchback Promoter Structure Affects Noise Amplitude and Distribution in Drosophila Segmentation

Positional information in developing embryos is specified by spatial gradients of transcriptional regulators. One of the classic systems for studying this is the activation of the hunchback (hb) gene in early fruit fly (Drosophila) segmentation by the maternally-derived gradient of the Bicoid (Bcd) protein. Gene regulation is subject to intrinsic noise which can produce variable expression. This variability must be constrained in the highly reproducible and coordinated events of development. We identify means by which noise is controlled during gene expression by characterizing the dependence of hb mRNA and protein output noise on hb promoter structure and transcriptional dynamics. We use a stochastic model of the hb promoter in which the number and strength of Bcd and Hb (self-regulatory) binding sites can be varied. Model parameters are fit to data from WT embryos, the self-regulation mutant hb(14F), and lacZ reporter constructs using different portions of the hb promoter. We have corroborated model noise predictions experimentally. The results indicate that WT (self-regulatory) Hb output noise is predominantly dependent on the transcription and translation dynamics of its own expression, rather than on Bcd fluctuations. The constructs and mutant, which lack self-regulation, indicate that the multiple Bcd binding sites in the hb promoter (and their strengths) also play a role in buffering noise. The model is robust to the variation in Bcd binding site number across a number of fly species. This study identifies particular ways in which promoter structure and regulatory dynamics reduce hb output noise. Insofar as many of these are common features of genes (e. g. multiple regulatory sites, cooperativity, self-feedback), the current results contribute to the general understanding of the reproducibility and determinacy of spatial patterning in early development.