Special Issue Introduction: Youth at Risk

Guest editors MeLisa Creamer, Anna Wilkinson, Deanna Hoelscher and Steven Kelder introduce Volume 8, Issue 2 of the Journal of Applied Research on Children

Development and characterisation of a cytokine supplemented serum free medium formulation for bone marrow derived mesenchymal stromal cells

Eng D ThesisBackground: The discovery and subsequent characterisation of adult human-derived stem cells appears poised to help revolutionise the fields of regenerative medicine and tissue engineering, offering clinicians the opportunity to develop fully functional replacement tissues, alongside holding significant promise as next generation gene or protein delivery vehicles. Despite their apparent potential however, current stem cell-based therapeutics typically rely on delivering massive doses of cells to sites of injury in order to help ensure adequate cell survival in the highly detrimental microenvironments presented by damaged and degrading biological material. In order to produce the cell numbers required for these types of treatments, relatively modest donor cell populations are subjected to extended periods of in vitro expansion, within highly regulated GMP culture conditions. One crucially important aspect of this manufacturing framework is the requirement for xeno-free expansion systems, including the use of serum-free culture medium. Unfortunately, whilst a number of functional serum-free media formulations are commercially available at the present time, their proprietary nature makes them both highly expensive and wholly unsuitable for use in academic research. Aims: The goal of this project is to begin development of a characterised cytokinesupplemented serum-free medium formulation using a design of experiments-based methodology. Methods: Primary bone marrow-derived mesenchymal stromal cells (BM-MSCs) were isolated, characterised and used to screen a series of selected cytokines and growth supplements for their ability to successfully support cell proliferation and continued survival in the absence of serum. Cells cultured in the resultant serum-free formulation were then compared to those grown in conventional medium in regard to genetic, metabolic and morphological factors. In addition, the impact of batch-to-batch variability on BM-MSC growth and metabolic activity was assessed as a means of determining the potential impact of raw material variation on cell quality and any related manufacturing processes. Finally, a number of different extracellular matrix proteins were also screened for the purpose of mediating cell-surface interactions in serum-free conditions. ii Results: We successfully identified a cytokine supplemented medium preparation capable of supporting the proliferation of BM-MSCs during serum-free culture. Evaluation of cells expanded in this medium provided evidence of altered secretory and genetic characteristics leading to shifted therapeutic potential. Furthermore, the identification of a combination of different extracellular matrix proteins able to enhance cell adhesion in the absence of serum served to provide the beginnings of a complete serum-free formulation. In addition, FBS batch variability was shown to have significant effects on cell proliferation and gene expression, including a number of genetic markers linked to differentiation potential and lineage specificity. Conclusions: We offer a new serum-free medium formulation for use in the expansion of primary BM-MSCs, alongside providing evidence of the impact of raw material variability on the therapeutically relevant characteristics of these cells

Boundary Layer Flow In Streamwise Concave Corners

Laminar boundary-layer theory has been correctly developed for streamwise corners using singular perturbation techniques. Theoretical predictions, however, do not compare well with experimental measurements due to the observed breakdown of flow stability and similarity. Both of these properties appear as basic assumptions of the boundary-layer theory, thereby providing needed simplification as well as imposing limitations upon the mathematical model.;Any theoretical investigation of similarity breakdown would require knowledge of the similar solution not only as an initial condition, but also for insight into how to proceed. Examination of the cross-flow behavior, which affects the onset of similarity breakdown, may suggest ways of formulating the problem without abandoning entirely the simplifying assumption of similarity.;The formulation of boundary-layer equations is reviewed beginning with time-independent Navier-Stokes equations. Tensor analysis is used so that the resulting equations are generally applicable to any similar flow configuration. A non-orthogonal Cartesian coordinate system is chosen to deal with streamwise concave corners (i.e. with corner angles less than 180(DEGREES)). Coordinate and flow-variable transforms are then used to define bounded quantities.;The computational procedures for obtaining the boundary conditions and solving the main equations are described briefly, noting some pitfalls that would hinder numerical computation. Results characterizing the mainstream flow and the secondary cross-flow are displayed and discussed for corners with angles of 30(DEGREES), 60(DEGREES), 90(DEGREES), 120(DEGREES) and 150(DEGREES). Ways to proceed with further investigation, while keeping the mathematical model simple, are then suggested

Uric acid-mediated modulation of the transcriptional regulator HucR from Deinococcus radiodurans

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. This study presents a biochemical characterization of a novel MarR homolog, HucR (hypothetical uricase regulator), from the DNA damage-resistant eubacterium, Deinococcus radiodurans. Circular dichroism spectroscopy suggests that HucR has ~47% alpha-helix and 10% beta-strand conformation at 25 deg C, and undergoes a transition to a disordered state with Tm = 51.1 ± 0.0 deg C. HucR binds as a homodimer with high sequence-specificity to a single site in its promoter region (hucO) with an apparent Kd = 0.29 ± 0.02 nM. DNaseI and hydroxyl-radical footprinting indicate HucR binding site sizes of ~24 bp and 21 bp, respectively. The binding site contains a pseudopalindromic sequence comprised of 8 bp inverted repeats separated by 2 bp that overlaps predicted promoter elements for hucR and a putative uricase (dr1160). Specific phenolic weak acids, notably uric acid, antagonize HucR-hucO complex formation. In vivo, uric acid increases transcript levels of hucR and dr1160, ~1.6-fold, and stimulates uricase activity 1.5-fold. HucR-hucO complex formation involves protein conformational changes and a decrease in the helical twist of the DNA duplex. Intrinsic fluorescence measurements show that uric acid induces HucR conformational changes, and its apparent Kd = 11.6 ± 3.7 micromolar and Hill coefficient of 0.7 ± 0.1 suggest negative cooperativity. An amino acid substitution in the predicted HucR wing (HucR-R118A) reduces DNA-binding affinity ~5-fold (Kd = 1.60 ± 0.14 nM), whereas a substitution in the predicted recognition helix (HucR-S104A) does not significantly alter DNA-binding affinity (Kd = 0.23 ± 0.03 nM). Each mutation decreases complex stability on the gel, but does not affect sequence-specificity. Intrinsic fluorescence spectra suggest altered conformations of the HucR-variants and altered mechanisms of DNA association. The mutations at HucR positions 118 and 104 also alter a predicted weak ligand-binding site, as indicated by minor changes in uric acid affinities for HucR-R118A and HucR-S104A (Kd = 9.7 ± 3.2 micromolar and 7.4 ± 0.5 micromolar, respectively) and modest attenuations of protein-hucO complex formation in response to uric acid

Negative cooperativity of uric acid binding to the transcriptional regulator HucR from Deinococcus radiodurans

Members of the MarR family of winged helix transcriptional regulators have been shown to regulate multidrug and oxidative stress response, pathogenesis, and catabolism of aromatic compounds. Many respond to anionic lipophilic compounds in their capacity to bind DNA, and the co-crystal structure of MarR bound to salicylate revealed two ligand-binding pockets, SAL-A and SAL-B. The MarR homolog, HucR, from Deinococcus radiodurans has been shown to repress expression of a predicted uricase, and DNA-binding by HucR is antagonized by uric acid, the substrate of uricase. We provide a biochemical investigation of DNA-binding and uric acid-binding by HucR. Equilibrium analytical ultracentrifugation indicates that HucR exists as a dimer. Intrinsic fluorescence spectra suggest that the association of the HucR dimer with its cognate DNA involves conformational flexibility in the globular interior and/or dimerization domain of the protein, and near-UV circular dichroism spectra indicate a concomitant change in the helical twist of the DNA duplex. DNA-binding affinity, measured by electrophoretic mobility-shift assays, for HucR mutants bearing single amino acid substitutions suggests the importance of the β-hairpin wing in DNA binding. Analysis of intrinsic fluorescence spectra demonstrates that uric acid induces conformational changes in HucR and binds with an apparent Kd=11.6(±3.7) μM and a Hill coefficient of 0.7±0.1, indicating negative cooperativity. Fluorescence and DNA-binding properties of the HucR variants indicate that SAL-A is a low-affinity, uric acid-binding site and that negative cooperativity exists between homologous, high-affinity sites. The conservation of residues comprising site SAL-A suggests that it is a low-affinity, ligand-binding site in MarR homologs. Mechanistic considerations suggest that HucR is regulated by uric acid to maintain optimal cellular levels of this scavenger of free radicals in response to oxidative stress and DNA damage. © 2005 Elsevier Ltd. All rights reserved

Differential DNA binding and protection by dimeric and dodecameric forms of the ferritin homolog Dps from Deinococcus radiodurans

Bacterial iron storage proteins such as ferritin serve as intracellular iron reserves. Members of the DNA protection during starvation (Dps) family of proteins are structurally related to ferritins, and their function is to protect the genome from iron-induced free radical damage. Some members of the Dps family bind DNA and are thought to do so only as fully assembled dodecamers. We present the cloning and characterization of a Dps homolog encoded by the radiation-resistant eubacterium Deinococcus radiodurans and show that DNA binding does not require its assembly into a dodecamer. D. radiodurans Dps-1, the product of gene DR2263, adopts a stably folded conformation, as demonstrated by circular dichroism spectroscopy, and undergoes a transition to a disordered state with a melting temperature of 69.2(±0.1)°C. While a dimeric form of Dps-1 is observed under low-salt conditions, a dodecameric assembly is highly favored at higher concentrations of salt. Both oligomeric forms of Dps-1 exhibit ferroxidase activity, and Fe(II) oxidation/mineralization is seen for dodecameric Dps-1. Notably, addition of Ca2+ (to millimolar concentrations) to dodecameric Dps-1 can result in the reduction of bound Fe(III). Dimeric Dps-1 protects DNA from both hydroxyl radical cleavage and from DNase I-mediated cleavage; however, dodecameric Dps-1 is unable to provide efficient protection against hydroxyl radical-mediated DNA cleavage. While dodecameric Dps-1 does bind DNA, resulting in formation of large aggregates, cooperative DNA binding by dimeric Dps-1 leads to formation of protein-DNA complexes of finite stoichiometry. © 2005 Elsevier Ltd. All rights reserved

HucR, a novel uric acid-responsive member of the MarR family of transcriptional regulators from Deinococcus radiodurans

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. We describe the characterization of HucR, a novel MarR homolog from Deinococcus radiodurans that demonstrates phenolic sensing capabilities. HucR binds as a homodimer to a single site within its promoter/operator region with Kd = 0.29 ± 0.02 nM. The HucR binding site contains a pseudopalindromic sequence, composed of 8-bp half-sites separated by 2 bp. The location of the HucR binding site in the intergenic region between hucR and a putative uricase suggests a mechanism of simultaneous co-repression of these two genes. The substrate of uricase, uric acid, is an efficient antagonist of DNA binding, reducing HucR-DNA complex formation to 50% at 0.26 mM ligand, compared with 5.2 and 46 mM for the aromatic compounds salicylate and acetylsalicylate, respectively. Enhanced levels in vivo of hucR and uricase transcript and increased uricase activity under conditions of excess uric acid further indicate a novel regulatory mechanism of aromatic catabolism in D. radiodurans. Since uric acid is a scavenger of reactive oxygen species, we hypothesize that HucR is a participant in the intrinsic resistance of D. radiodurans to high levels of oxidative stress