Tying the knot: the cystine signature and molecular-recognition processes of the vascular endothelial growth factor family of angiogenic cytokines

The cystine-knot motif, made up of three intertwined disulfide bridges, is a unique feature of several toxins, cyclotides and growth factors, and occurs in a variety of species, including fungi, insects, molluscs and mammals. Growth factor molecules containing the cystine-knot motif serve as ligands for a diverse range of receptors and play an important role in extracellular signalling. This superfamily of polypeptides comprises several homodimeric and heterodimeric molecules that are central characters in both health and disease. Amongst these molecules are a group of proteins that belong to the vascular endothelial growth factor (VEGF) subfamily. The members of this family are known angiogenic factors that regulate processes leading to blood vessel formation in physiological and pathological conditions. The focus of the present review is on the structural characteristics of proteins that belong to the VEGF family and on signal-transduction pathways that become initiated via the VEGF receptors.     

Elevational gradients in bird diversity in the Eastern Himalaya: an evaluation of distribution patterns and their underlying mechanisms

Background

Understanding diversity patterns and the mechanisms underlying those patterns along elevational gradients is critically important for conservation efforts in montane ecosystems, especially those that are biodiversity hotspots. Despite recent advances, consensus on the underlying causes, or even the relative influence of a suite of factors on elevational diversity patterns has remained elusive.

Methods and Principal Findings

We examined patterns of species richness, density and range size distribution of birds, and the suite of biotic and abiotic factors (primary productivity, habitat variables, climatic factors and geometric constraints) that governs diversity along a 4500-m elevational gradient in the Eastern Himalayan region, a biodiversity hotspot within the world''s tallest mountains. We used point count methods for sampling birds and quadrats for estimating vegetation at 22 sites along the elevational gradient. We found that species richness increased to approximately 2000 m, then declined. We found no evidence that geometric constraints influenced this pattern, whereas actual evapotranspiration (a surrogate for primary productivity) and various habitat variables (plant species richness, shrub density and basal area of trees) accounted for most of the variation in bird species richness. We also observed that ranges of most bird species were narrow along the elevation gradient. We find little evidence to support Rapoport''s rule for the birds of Sikkim region of the Himalaya.

Conclusions and Significance

This study in the Eastern Himalaya indicates that species richness of birds is highest at intermediate elevations along one of the most extensive elevational gradients ever examined. Additionally, primary productivity and factors associated with habitat accounted for most of the variation in avian species richness. The diversity peak at intermediate elevations and the narrow elevational ranges of most species suggest important conservation implications: not only should mid-elevation areas be conserved, but the entire gradient requires equal conservation attention.     

PEG-albumin supraplasma expansion is due to increased vessel wall shear stress induced by blood viscosity shear thinning

We studied the extreme hemodilution to a hematocrit of 11% induced by three plasma expanders: polyethylene glycol (PEG)-conjugated albumin (PEG-Alb), 6% 70-kDa dextran, and 6% 500-kDa dextran. The experimental component of our study relied on microelectrodes and cardiac output to measure both the rheological properties of plasma-expander blood mixtures and nitric oxide (NO) bioavailability in vessel walls. The modeling component consisted of an analysis of the distribution of wall shear stress (WSS) in the microvessels. Our experiments demonstrated that plasma expansion with PEG-Alb caused a state of supraperfusion with cardiac output 40% above baseline, significantly increased NO vessel wall bioavailability, and lowered peripheral vascular resistance. We attributed this behavior to the shear thinning nature of blood and PEG-Alb mixtures. To substantiate this hypothesis, we developed a mathematical model of non-Newtonian blood flow in a vessel. Our model used the Quemada rheological constitutive relationship to express blood viscosity in terms of both hematocrit and shear rate. The model revealed that the net effect of the hemodilution induced by relatively low-viscosity shear thinning PEG-Alb plasma expanders is to reduce overall blood viscosity and to increase the WSS, thus intensifying endothelial NO production. These changes act synergistically, significantly increasing cardiac output and perfusion due to lowered overall peripheral vascular resistance.     

Diagnosis of Parkinson's disease based on disease-specific autoantibody profiles in human sera

Parkinson's disease (PD), hallmarked by a variety of motor disorders and neurological decline, is the second most common neurodegenerative disease worldwide. Currently, no diagnostic test exists to identify sufferers, and physicians must rely on a combination of subjective physical and neurological assessments to make a diagnosis. The discovery of definitive blood-borne biomarkers would be a major step towards early and reliable diagnosis. Despite attention devoted to this search, such biomarkers have remained elusive. In the present study, we used human protein microarrays to reveal serum autoantibodies that are differentially expressed among PD and control subjects. The diagnostic significance of each of these autoantibodies was evaluated, resulting in the selection of 10 autoantibody biomarkers that can effectively differentiate PD sera from control sera with a sensitivity of 93.1% and specificity of 100%. PD sera were also distinguishable from sera obtained from Alzheimer's disease, breast cancer, and multiple sclerosis patients with accuracies of 86.0%, 96.6%, and 100%, respectively. Results demonstrate that serum autoantibodies can be used as highly specific and accurate biomarkers for PD diagnosis throughout the course of the disease.     

Optimization of Cardiovascular Stent against Restenosis: Factorial Design-Based Statistical Analysis of Polymer Coating Conditions

The objective of this study was to optimize the physicodynamic conditions of polymeric system as a coating substrate for drug eluting stents against restenosis. As Nitric Oxide (NO) has multifunctional activities, such as regulating blood flow and pressure, and influencing thrombus formation, a continuous and spatiotemporal delivery of NO loaded in the polymer based nanoparticles could be a viable option to reduce and prevent restenosis. To identify the most suitable carrier for S-Nitrosoglutathione (GSNO), a NO prodrug, stents were coated with various polymers, such as poly (lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG) and polycaprolactone (PCL), using solvent evaporation technique. Full factorial design was used to evaluate the effects of the formulation variables in polymer-based stent coatings on the GSNO release rate and weight loss rate. The least square regression model was used for data analysis in the optimization process. The polymer-coated stents were further assessed with Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy analysis (FTIR), Scanning electron microscopy (SEM) images and platelet adhesion studies. Stents coated with PCL matrix displayed more sustained and controlled drug release profiles than those coated with PLGA and PEG. Stents coated with PCL matrix showed the least platelet adhesion rate. Subsequently, stents coated with PCL matrix were subjected to the further optimization processes for improvement of surface morphology and enhancement of the drug release duration. The results of this study demonstrated that PCL matrix containing GSNO is a promising system for stent surface coating against restenosis.     

A Small Molecule Agonist of EphA2 Receptor Tyrosine Kinase Inhibits Tumor Cell Migration In Vitro and Prostate Cancer Metastasis In Vivo

During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo.     

Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation

In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM-MSC) or adipose tissue (AT-MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM-MSC with bovine AC, (ii) BM-MSC from HLA-A2+ with AC from HLA-A2- donors, or (iii) human green fluorescent protein transduced BM-MSC with AC. Human BM-MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT-PCR. Coculture of NC or AC (25%) with BM-MSC or AT-MSC (75%) in pellets resulted in up to 1.6-fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM-MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2-fold) if cocultured with BM-MSC and maintained a differentiated phenotype. Chondro-induction in MSC-chondrocyte coculture is a robust process mediated by two concomitant effects: MSC-induced chondrocyte proliferation and chondrocyte-enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair.     

Molecular Basis for Involvement of CYP1B1 in MYOC Upregulation and Its Potential Implication in Glaucoma Pathogenesis

CYP1B1 has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in CYP1B1 have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with Myocilin (MYOC). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce MYOC expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate MYOC expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to MYOC promoter. Hence any mutation in CYP1B1 that reduces its 17β estradiol metabolizing activity might lead to MYOC upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the MYOC promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in MYOC promoter and subsequent upregulation in MYOC level in HTM cells on 17β estradiol treatment. Interestingly, CYP1B1 mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.     

Characterizing the Radioresponse of Pluripotent and Multipotent Human Stem Cells

The potential capability of stem cells to restore functionality to diseased or aged tissues has prompted a surge of research, but much work remains to elucidate the response of these cells to genotoxic agents. To more fully understand the impact of irradiation on different stem cell types, the present study has analyzed the radioresponse of human pluripotent and multipotent stem cells. Human embryonic stem (ES) cells, human induced pluripotent (iPS) cells, and iPS-derived human neural stem cells (iPS-hNSCs) cells were irradiated and analyzed for cell survival parameters, differentiation, DNA damage and repair and oxidative stress at various times after exposure. While irradiation led to dose-dependent reductions in survival, the fraction of surviving cells exhibited dose-dependent increases in metabolic activity. Irradiation did not preclude germ layer commitment of ES cells, but did promote neuronal differentiation. ES cells subjected to irradiation exhibited early apoptosis and inhibition of cell cycle progression, but otherwise showed normal repair of DNA double-strand breaks. Cells surviving irradiation also showed acute and persistent increases in reactive oxygen and nitrogen species that were significant at nearly all post-irradiation times analyzed. We suggest that stem cells alter their redox homeostasis to adapt to adverse conditions and that radiation-induced oxidative stress plays a role in regulating the function and fate of stem cells within tissues compromised by radiation injury.     

Toward electrocorticographic control of a dexterous upper limb prosthesis: building brain-machine interfaces

One of the most exciting and compelling areas of research and development is building brain machine interfaces (BMIs) for controlling prosthetic limbs. Prosthetic limb technology is advancing rapidly, and the modular prosthetic limb (MPL) of the Johns Hopkins University/ Applied Physics Laboratory (JHU/APL) permits actuation with 17 degrees of freedom in 26 articulating joints. There are many signals from the brain that can be leveraged, including the spiking rates of neurons in the cortex, electrocorticographic (ECoG) signals from the surface of the cortex, and electroencephalographic (EEG) signals from the scalp. Unlike microelectrodes that record spikes, ECoG does not penetrate the cortex and has a higher spatial specificity, signal-to-noise ratio, and bandwidth than EEG signals. We have implemented an ECoG-based system for controlling the MPL in the Johns Hopkins Hospital Epilepsy Monitoring Unit, where patients are implanted with ECoG electrode grids for clinical seizure mapping and asked to perform various recorded finger or grasp movements. We have shown that low-frequency local motor potentials (LMPs) and ECoG power in the high gamma frequency (70,150 Hz) range correlate well with grasping parameters, and they stand out as good candidate features for closed-loop control of the MPL.