Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells

Rationale The human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation. Methods and results Here, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2\u27-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network- like structures in the presence of VEGF. Conclusion Chromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tubeforming cells in a 2D matrix in VEGF-dependent manner

Session 1F: Modeling CHARGE Syndrome in Human Fibroblast Cells via CRISPR/Cas9

Mutations in chromodomain helicase DNA binding protein 7 (CHD7) gene located in human chromosome 8 have been linked to “CHARGE” syndrome, a cluster of disease seen in a number children, for which there is no pharmacotherapy. As there is no cellular or animal model system to study the function of CHD7, the goal of this investigation is to mimic CHARGE disease in a dish by the use of CRISPR/Cas9 to edit the CHD7 gene in human fibroblast cells. Accordingly, I have generated lentivirus particles encoding Cas9 and CHD7 guide(g)RNA in all-in-one vector, and transduced fibroblast cells. CHD7 mutation will be confirmed by qRT-PCR and DNA sequencing; microscopy, Western blot analyses, and the function of mutant protein will be analyzed. Thereafter, the fibroblast cells that harbor a specific mutation in CHD7 gene will be used to screen drugs using a chemical compound library. Through this assay, a molecule that acts on mutant CHD7 protein as an agonist or antagonist could be found, thereby one could fantasize a therapeutic approach to alleviate CHARGE syndrome. There are limitations to cell-based assays, therefore, animal experiments will be needed to address the toxicity and efficacy of any chemical that might be discovered

Recognizing scientific excellence in the biology of cell adhesion

The prestigious 2005 Japan Prize for Cell Biology has been awarded to Dr. Masatoshi Takeichi, Director of RIKEN Developmental Biology, Kobe, Japan, and Dr. Erkki Ruoslahti, Distinguished Professor, The Burnham Institute, La Jolla, USA for their "Fundamental contribution in elucidating the molecular mechanisms of cell adhesion". The award is scheduled to be presented to the scientists in ceremonies in Tokyo on April 20, 2005 as part of a week-long celebration of "Japan Prize Week"

Signaling through Raf-1 in the Neovasculature and Target Validation by Nanoparticles

A recent study demonstrated that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) activate Raf-1 kinase in an experimental neovasculature system. The study showed that bFGF and VEGF activate p21-activated protein kinase-1 (PAK-1) and Src kinase, respectively. PAK-1 and Src kinases phosphorylate specific serine and tyrosine residues within the activation loop of Raf-1 kinase. Their findings further suggest that phosphorylation at these sites protects endothelial cells from apoptosis induced by both intrinsic and extrinsic factors. The tumor neovasculature provides specific molecular markers or "zip codes". This group of investigators has previously shown that nanosphere-aided targeting of the neovasculature with mutant Raf-1 causes regression of the tumor vasculature. Thus, nanoparticles coated with "zip code"-specific homing biomolecules may be useful for delivering anti-angiogenic molecules that can induce tumor regression

Kennedy Space Center Spaceport Analysis

Until the Shuttle Atlantis' final landing on July 21, 2011, Kennedy Space Center (KSC) served as NASA's main spaceport, which is a launch and landing facility for rockets and spacecraft that are attempting to enter orbit. Many of the facilities at KSC were created to assist the Shuttle Program. One of the most important and used facilities is the Shuttle Landing Facility (SLF), This was the main landing area for the return of the shuttle after her mission in space. However, the SLF has also been used for a number of other projects including straight-line testing by Gibbs Racing, weather data collection by NOAA, and an airfield for the KSC helicopters. This runway is three miles long with control tower at midfield and a fire department located at the end in care of an emergency. This facility, which was part of the great space race, will continue to be used for historical events as Kennedy begins to commercialize its facilities. KSC continues to be an important spaceport to the government, and it will transform into an important spaceport for the commercial industry as well. During my internship at KSC's Center Planning and Development Directorate, I had the opportunity to be a part of the negotiation team working on the agreement for Space Florida to control the Shuttle Landing Facility. This gave me the opportunity to learn about all the changes that are occurring here at Kennedy Space Center. Through various meetings, I discovered the Master Plan and its focus is to transform the existing facilities that were primarily used for the Shuttle Program, to support government operations and commercial flights in the future. This. idea is also in a new strategic business plan and completion of a space industry market analysis. All of these different documentations were brought to my attention and I. saw how they came together in the discussions of transitioning the SLF to a commercial operator, Space Florida. After attending meetings and partaking in discussions for the SLF Agreement, I formed the idea of a Spaceport Analysis as my over internship project. As previously stated, I had the opportunity to sit in on the market analysis meetings and read through the analysis itself. I suggested the creation of a Strengths Weaknesses Opportunities Threats (SWOT) analysis, which allows an individual to see an overview of the company's strengths and weaknesses alongside any industry opportunities and threats. After discussions with the lead writer of the new strategic business plan and getting approval, I took the action upon myself and created the Kennedy Space Center SWOT Analysis

Steer stress response as affected by genotype and transportation

Bovine cytochrome P450 3A28 is responsible for metabolizing ergot alkaloids that cattle ingest when feeding on endophyte-infested tall fescue grass. The objective of this research was to determine associations among genotype, transportation, and stress responses. Angus crossbred steers (n = 47) were genotyped (CC, CG, or GG) for a single-nucleotide polymorphism (C994G) in cytochrome P450 3A28. Genotypes were determined by polymerase chain reaction (PCR) amplification followed by restriction enzyme (Alu1) digestion. Steers were backgrounded on a mixedcultivar tall fescue pasture. Following the stocker phase, steers were transported to the feedlot for finishing. Stress responses were determined 27 h prior to, and 6 and 20 h after transport. Plasma concentrations of prolactin and cortisol, and white blood cell expression of prolactin, cytochrome P450, tumor necrosis factor-alpha, and short form prolactin receptor were our indicators of stress. Both time and genotypic effects were determined. Time (P \u3c 0.05) relative to transportation was associated with expression of all four genes tested. In addition, plasma concentrations of cortisol and prolactin, as well as their ratio were affected (P \u3c 0.05) by time. In contrast, neither genotype nor the interaction between genotype and time affected (P \u3e 0.1) our stress indicators. In previous studies, C994G genotype has been associated with cattle productivity; however, those effects were not observed in this study

Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF- and VEGF-induced capillary morphogenesis of endothelial cells

BACKGROUND: Angiogenesis, or the remodeling of existing vasculature serves as a lifeline to nourish developing embryos and starved tissues, and to accelerate wound healing, diabetic retinopathy, and tumor progression. Recent studies indicate that angiogenesis requires growth factor activity as well as cell adhesion events mediated by α(5)β(1 )and α(v)β(3 )integrins. We previously demonstrated that human lipid phosphate phosphohydrolase-3 (LPP3) acts as a cell-associated ligand for α(5)β(1 )and α(v)β(3 )integrins. Here, we test the hypothesis that an anti-LPP3 antibody can inhibit basic fibroblast growth factor (bFGF)-and vascular endothelial growth factor (VEGF)-induced capillary morphogenesis of endothelial cells (ECs). RESULTS: We report that bFGF and VEGF up-regulate LPP3 protein expression in ECs. Immunoprecipitation analyses show that LPP3 is a cell surface protein and undergoes N-glycosylation. Fluorescent activated cell sorting (FACS) data suggest that anti-LPP3-RGD detects native neoepitope on the surface of activated ECs. Moreover, we demonstrate LPP3 protein expression in tumor endothelium alongside VEGF. The embedding of ECs into three-dimensional type I collagen in the presence of bFGF and VEGF induce capillary formation. Importantly, we show that the addition of an anti-LPP3 antibody specifically and significantly blocks bFGF- and VEGF-induced capillary morphogenesis of ECs. CONCLUSION: These data suggest that activated ECs as well as tumor endothelium express LPP3 protein. In an in vitro assay, the anti-LPP3-RGD specifically blocks bFGF and VEGF induced capillary morphogenesis of ECs. Our results, therefore, suggest a role for LPP3 in angiogenesis

Tracking sexual dimorphism in Telfairia occidentalis Hooker f. (Cucurbitaceae) with morphological and molecular markers

Morphological (foliar features) and molecular (DNA fingerprinting) markers were used to characterize sexual dimorphism in Telfairia occidentalis Hook. f. (Cucurbitaceae). A total of thirty strains comprising fifteen males and fifteen females from market-sourced fruits bred to the third generation were used forthe investigation. The amplified fragment length polymorphism (AFLP) technique was combined with silver staining to determine the level of DNA polymorphism in the strains. Results showed that the females were generally larger in size than the males in leaf morphological features, while DNAfingerprints revealed the existence of polymorphism in the plant. However, this genetic variability did not clearly correspond with presence of sexual dimorphism in the species. The implications of these results in understanding the genetics, breeding and conservation of the plant’s germplasm arediscussed

Malware propagation in urban D2D networks

We introduce and analyze models for the propagation of malware in pure D2D networks given via stationary Cox--Gilbert graphs. Here, the devices form a Poisson point process with random intensity measure λ, Λ where Λ is stationary and given, for example, by the edge-length measure of a realization of a Poisson--Voronoi tessellation that represents an urban street system. We assume that, at initial time, a typical device at the center of the network carries a malware and starts to infect neighboring devices after random waiting times. Here we focus on Markovian models, where the waiting times are exponential random variables, and non-Markovian models, where the waiting times feature strictly positive minimal and finite maximal waiting times. We present numerical results for the speed of propagation depending on the system parameters. In a second step, we introduce and analyze a counter measure for the malware propagation given by special devices called white knights, which have the ability, once attacked, to eliminate the malware from infected devices and turn them into white knights. Based on simulations, we isolate parameter regimes in which the malware survives or is eliminated, both in the Markovian and non-Markovian setting