A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Silica polymer bonding of stressed silica grains: An early growth of intergranular tensile strength

© 2015 Elsevier Ltd. All rights reserved.Laboratory tests on microscale are reported in which millimeter-sized amorphous silica cubes were kept highly compressed in a liquid environment of de-ionized water solutions with different silica ion concentrations for up to four weeks. Such an arrangement simulates an early evolution of bonds between two sand grains stressed in situ. In-house designed Grain Indenter-Puller apparatus allowed measuring strength of such contacts after 3-4 weeks. Observations reported for the first time confirm a long-existing hypothesis that a stressed contact with microcracks generates silica polymers, forming a bonding structure between the grains on a timescale in the order of a few weeks. Such structure exhibits intergranular tensile force at failure of 1-1.5 mN when aged in solutions containing silica ion concentrations of 200-to 500-ppm. The magnitude of such intergranular force is 2-3 times greater than that of water capillary force between the same grains.

TEACHING AMERICAN CIVIL WAR MUSIC IDSTORY WITH MODERN EDITIONS OF PERIOD MUSIC FOR FULL CONCERT BAND

This dissertation explores a method of teaching the history of Civil War music and musicians through modern full-band editions of original brass band music. In the study of music history the period of the Civil War is rarely discussed, or at best, mentioned only if a student takes a specific course on the history of bands and happens to look deeply into the background of some of the early band pioneers such as Patrick Gilmore, who served in the Union Army as a bandmaster. The history of the musicians, bands, and music performed during the Civil War deserves study to provide a way for students and audiences to learn this history. This project includes lesson plans that can be used with the arrangements of the period music as well as select published music that is also representative of the period. Included with the historical information are four arrangements of original brass band music now scored for full concert band. Each arrangement includes a section scored for brass only with optional brass band parts. Historical information is provided on the Civil War period bands and how each side used them, on the composers of the music, and also on the individual compositions. The historical information can be used to supplement the lesson plans to teach the history, as well as for program notes for audiences. The research involved locating information on both Union and Confederate bands available in books, other dissertations, articles, and interviews with Civil War music historians. The original brass band music is scored for full band. This method will allow teachers and conductors to highlight this period of wind band history and to share it with both students and audiences. Included with this project are photos and video footage taken during a visit with the 1st Brigade Band of Watertown, Wisconsin, an historical organization dedicated to recreating the music and performances of an actual Civil War era band.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

Development of numerical techniques for near-field aeroacoustic computations

This work is concerned with the development of a numerical scheme capable of producing accurate simulations of sound propagation in the presence of a mean flow field. The method is based on the concept of variable decomposition, which leads to two separate sets of equations. These equations are the linearised Euler equations and the Reynolds-averaged Navier–Stokes equations. This paper concentrates on the development of numerical schemes for the linearised Euler equations that leads to a computational aeroacoustics (CAA) code. The resulting CAA code is a non-diffusive, time- and space-staggered finite volume code for the acoustic perturbation, and it is validated against analytic results for pure 1D sound propagation and 2D benchmark problems involving sound scattering from a cylindrical obstacle. Predictions are also given for the case of prescribed source sound propagation in a laminar boundary layer as an illustration of the effects of mean convection. Copyright © 1999 John Wiley & Sons, Ltd.

Monotone scheme and boundary conditions for finite volume simulation of magnetohydrodynamic internal flows at high Hartmann number

A monotone scheme for finite volume simulation of magnetohydrodynamic internal flows at high Hartmann number is presented. The numerical stability is analysed with respect to the electromagnetic force. Standard central finite differences applied to finite volumes can only be numerically stable if the vector products involved in this force are computed with a scheme using a fully staggered grid. The electromagnetic quantities (electric currents and electric potential) must be shifted by half the grid size from the mechanical ones (velocity and pressure). An integral treatment of the boundary layers is used in conjunction with boundary conditions for electrically conducting walls. The simulations are performed with inhomogeneous electrical conductivities of the walls and reach high Hartmann numbers in three-dimensional simulations, even though a non-adaptive grid is used.

A semi-Lagrangian finite volume method for Newtonian contraction flows

A new finite volume method for solving the incompressible Navier--Stokes equations is presented. The main features of this method are the location of the velocity components and pressure on different staggered grids and a semi-Lagrangian method for the treatment of convection. An interpolation procedure based on area-weighting is used for the convection part of the computation. The method is applied to flow through a constricted channel, and results are obtained for Reynolds numbers, based on half the flow rate, up to 1000. The behavior of the vortex in the salient corner is investigated qualitatively and quantitatively, and excellent agreement is found with the numerical results of Dennis and Smith [Proc. Roy. Soc. London A, 372 (1980), pp. 393-414] and the asymptotic theory of Smith [J. Fluid Mech., 90 (1979), pp. 725-754].

The use of evacuation modelling techniques in the design of very large transport aircraft and blended wing body aircraft

Very Large Transport Aircraft (VLTA) pose considerable challenges to designers, operators and certification authorities. Questions concerning seating arrangement, nature and design of recreational space, the number, design and location of internal staircases, the number of cabin crew required and the nature of the cabin crew emergency procedures are just some of the issues that need to be addressed. Other more radical concepts such as blended wing body (BWB) design, involving one or two decks with possibly four or more aisles offer even greater challenges. Can the largest exits currently available cope with passenger flow arising from four or five aisles? Do we need to consider new concepts in exit design? Should the main aisles be made wider to accommodate more passengers? In this paper we demonstrate how computer based evacuation models can be used to investigate these issues through examination of staircase evacuation procedures for VLTA and aisle/exit configuration for BWB cabin layouts.

Numerical simulation of flow-induced cavity noise in self-sustained oscillations

The generation and near-field radiation of aerodynamic sound from a low-speed unsteady flow over a two-dimensional automobile door cavity is simulated by using a source-extraction-based coupling method. In the coupling procedure, the unsteady cavity flow field is first computed solving the Reynolds averaged Navier–Stokes (RANS) equations. The radiated sound is then calculated by using a set of acoustic perturbation equations with acoustic source terms which are extracted from the time-dependent solutions of the unsteady flow. The aerodynamic and its resulting acoustic field are computed for the Reynolds number of 53,266 based on the base length of the cavity. The free stream flow velocity is taken to be 50.9m/s. As first stage of the numerical investigation of flow-induced cavity noise, laminar flow is assumed. The CFD solver is based on a cell-centered finite volume method. A dispersion-relation-preserving (DRP), optimized, fourth-order finite difference scheme with fully staggered-grid implementation is used in the acoustic solver

Numerical investigation of a source extraction technique based on an acoustic correction method

An aerodynamic sound source extraction from a general flow field is applied to a number of model problems and to a problem of engineering interest. The extraction technique is based on a variable decomposition, which results to an acoustic correction method, of each of the flow variables into a dominant flow component and a perturbation component. The dominant flow component is obtained with a general-purpose Computational Fluid Dynamics (CFD) code which uses a cell-centred finite volume method to solve the Reynolds-averaged Navier–Stokes equations. The perturbations are calculated from a set of acoustic perturbation equations with source terms extracted from unsteady CFD solutions at each time step via the use of a staggered dispersion-relation-preserving (DRP) finite-difference scheme. Numerical experiments include (1) propagation of a 1-D acoustic pulse without mean flow, (2) propagation of a 2-D acoustic pulse with/without mean flow, (3) reflection of an acoustic pulse from a flat plate with mean flow, and (4) flow-induced noise generated by the an unsteady laminar flow past a 2-D cavity. The computational results demonstrate the accuracy for model problems and illustrate the feasibility for more complex aeroacoustic problems of the source extraction technique.

New genus and two new species of the family Ethmolaimidae (Nematoda: Chromadorida), found in two different cold-seep environments

This study describes a new genus Dystomanema gen. nov. with two new species, D. cadizensis sp. nov. and D. brandtae sp. nov. within the family Ethmolaimidae, subfamily Neotonchinae, based on specimens from two low-activity cold-seep environments at distant geographical locations. The new genus was first identified in samples from the Darwin mud volcano (1100 m depth) in the Gulf of Cadiz and later on also found in samples from a low-activity seep in the Larsen B embayment (820m depth) off the eastern Antarctic Peninsula. Until now, the family Ethmolaimidae contained nine genera: Ethmolaimus and Paraethmolaimus in the subfamily Ethmolaiminae, and Comesa, Filitonchoides, Filitonchus, Gomphionchus, Gomphionema, Nannolaimus, and Neothonchus in the subfamily Neotonchinae. The most important family characteristics are: an annulated cuticle bearing transverse rows of dots, cephalic sensilla arrangement of 6+6+4, a spiral amphid, an oesophagus with muscular posterior bulb, paired gonads and males with cup-shaped precloacal supplements. The new genus resembles Comesa and Neotonchus, but is typified by a ventrally displaced oral opening with three very small teeth that are easily overlooked. D. cadizensis gen. nov. sp. nov. is characterized by the 1401-2123 mu m long body; cuticle transversally striated with fine punctation; head conical; low lips; amphid spiralled 3 turns, oral opening ventrally displaced, male with outstretched testes; spicules of equal size; gubernaculum plate-like and ten to twelve conspicuous cup-shaped precloacal supplements with external longitudinal articulated flange. D. brandtae gen. nov. sp. nov. can be distinguished by the 2438-3280 mu m long body; cuticle transversally striated with fine punctuation; head conical; low lips; amphid spiraled 3+ turns; oral opening ventrally displaced; male with anterior testes outstretched and posterior one smaller and reflexed; spicules of equal size; gubernaculum plate-like and twenty conspicuous cup-shaped precloacal supplements with external longitudinal articulated flange. Notes on the ecology and habitat of the new genus are provided in light of its discovery in cold-seep environments.

Disentangling the impacts of heat wave magnitude, duration and timing on the structure and diversity of sessile marine assemblages

Extreme climatic events, including heat waves (HWs) and severe storms, influence the structure of marine and terrestrial ecosystems. Despite growing consensus that anthropogenic climate change will increase the frequency, duration and magnitude of extreme events, current understanding of their impact on communities and ecosystems is limited. Here, we used sessile invertebrates on settlement panels as model assemblages to examine the influence of HW magnitude, duration and timing on marine biodiversity patterns. Settlement panels were deployed in a marina in southwest UK for ≥5 weeks, to allow sufficient time for colonisation and development of sessile fauna, before being subjected to simulated HWs in a mesocosm facility. Replicate panel assemblages were held at ambient sea temperature (∼17 °C), or +3 °C or +5 °C for a period of 1 or 2 weeks, before being returned to the marina for a recovery phase of 2–3 weeks. The 10-week experiment was repeated 3 times, staggered throughout summer, to examine the influence of HW timing on community impacts. Contrary to our expectations, the warming events had no clear, consistent impacts on the abundance of species or the structure of sessile assemblages. With the exception of 1 high-magnitude long-duration HW event, warming did not alter not assemblage structure, favour non-native species, nor lead to changes in richness, abundance or biomass of sessile faunal assemblages. The observed lack of effect may have been caused by a combination of (1) the use of relatively low magnitude, realistic heat wave treatments compared to previous studies (2), the greater resilience of mature adult sessile fauna compared to recruits and juveniles, and (3) the high thermal tolerance of the model organisms (i.e., temperate fouling species, principally bryozoans and ascidians). Our study demonstrates the importance of using realistic treatments when manipulating climate change variables, and also suggests that biogeographical context may influence community-level responses to short-term warming events, which are predicted to increase in severity in the future.

La Mancha Triassic and Lower Lias Stratigraphy, a well log interpretation

La estratigrafía del intervalo sedimentario correspondiente al Keuper, Triásico, y al Lías basal ha atraído históricamente mucho menos interés que los sedimentos adyacentes del Muschelkalk y Buntsandstein, y del Lías Inferior y Medio. Se ha confeccionado una correlación de 12 sondeos profundos del área de La Mancha para mostrar la distribución de facies de estas formaciones, así como su ordenación en secuencias sedimentarias. Los datos de pozo muestran la existencia de secuencias evaporíticas complejas y multiepisódicas en la unidad inferior K1, de carácter más salino, y en la unidad superior K4-K5, relativamente más anhidrítica. El episodio clástico correspondiente a las unidades K2-K3 está extendido en toda la zona, con varias áreas preferenciales de acumulación de arenas. La unidad K6, la más alta del Keuper, constituye un excelente nivel guía. La sedimentación continúa en el Lías basal con otra potente unidad evaporítica, la Zona de Anhidrita, que muestra un episodio salino muy signifi cativo en la parte central de la correlación. Es de resaltar la buena correlación que existe entre los afl oramientos y las sondeos para estas formaciones.

Sequence and Structure Based Protein Folding Studies With Implications

As the expression of the genetic blueprint, proteins are at the heart of all biological systems. The ever increasing set of available protein structures has taught us that diversity is the hallmark of their architecture, a fundamental characteristic that enables them to perform the vast array of functionality upon which all of life depends. This diversity, however, is central to one of the most challenging problems in molecular biology: how does a folding polypeptide chain navigate its way through all of the myriad of possible conformations to find its own particular biologically active form? With few overarching structural principles to draw upon that can be applied to all protein architecture, the search for a solution to the protein folding problem has yet to produce an algorithm that can explain and duplicate this fundamental biological process. In this thesis, we take a two-pronged approach for investigating the protein folding process. Our initial statistical studies of the distributions of hydrophobic and hydrophilic residues within α-helices and β-sheets suggest (i) that hydrophobicity plays a critical role in helix and sheet formation; and (ii) that the nucleation of these motifs may result in largely unidirectional growth. Most tellingly, from an examination of the amino acids found in the smallest β-sheets, we do not find any evidence of a β-nucleating code in the primary protein sequence. Complementing these statistical analyses, we have analyzed the structural environments of several ever-widening aspects of protein topology. Our examination of the gaps between strands in the smallest β-sheets reveals a common organizational principle underlying β-formation involving strands separated by large sequential gaps: with very few exceptions, these large gaps fold into single, compact structural modules, bringing the β-strands that are otherwise far apart in the sequence close together in space. We conclude, therefore, that β-nucleation in the smallest sheets results from the co-location of two strands that are either local in sequence, or local in space following prior folding events. A second study of larger β-sheets both corroborates and extends these findings: virtually all large sequential gaps between pairs of β-strands organize themselves into an hierarchical arrangement, creating a bread-crumb model of go-and-come-back structural organization that ultimately juxtaposes two strands of a parental β-structure that are far apart in the sequence in close spatial proximity. In a final study, we have formalized this go-and-come-back notion into the concept of anti-parallel double-strandedness (DS), and measure this property across protein architecture in general. With over 90% of all residues in a large, non-redundant set of protein structures classified as DS, we conclude that DS is a unifying structural principle that underpins all globular proteins. We postulate, moreover, that this one simple principle, anti-parallel double-strandedness, unites protein structure, protein folding and protein evolution.

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT-PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.