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Gil V.,Cellular Bioengineering | Gil V.,University of Barcelona | Gil V.,CIBER ISCIII | Del Rio J.A.,Cellular Bioengineering | And 2 more authors.
Nature Protocols | Year: 2012

This protocol uses rat tail-derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat tail-derived type I collagen or, alternatively, by commercial Matrigel. The neural tissue is placed in the hydrogel with other brain tissue pieces or cell aggregates genetically modified to secrete a particular molecule that can generate a gradient inside the hydrogel. The present method is uncomplicated and generally reproducible, and only a few specific details need to be considered during its preparation. Moreover, the degree and behavior of axonal growth or neural migration can be observed directly using phase-contrast, fluorescence microscopy or immunocytochemical methods. This protocol can be carried out in 4 weeks. © 2012 Nature America, Inc. All rights reserved.

Nagy K.J.,U.S. National Cancer Institute | Nagy K.J.,University of Delaware | Giano M.C.,U.S. National Cancer Institute | Giano M.C.,University of Delaware | And 3 more authors.
Journal of the American Chemical Society | Year: 2011

Chirality can be used as a design tool to control the mechanical rigidity of hydrogels formed from self-assembling peptides. Hydrogels prepared from enantiomeric mixtures of self-assembling β-hairpins show nonadditive, synergistic, enhancement in material rigidity compared to gels prepared from either pure enantiomer, with the racemic hydrogel showing the greatest effect. CD spectroscopy, TEM, and AFM indicate that this enhancement is defined by nanoscale interactions between enantiomers in the self-assembled state. © 2011 American Chemical Society.

Kainerstorfer J.M.,Eunice Kennedy Shriver National Institute of Child Health and Human Development | Smith P.D.,Cellular Bioengineering | Gandjbakhche A.H.,Eunice Kennedy Shriver National Institute of Child Health and Human Development
IEEE Journal on Selected Topics in Quantum Electronics | Year: 2012

Imaging of large tissue areas (>5 cm 2) is often desired in clinical settings, e.g., of tissue oxygenation. Intrinsic contrasts such as absorption and scattering changes have the potential to enhance diagnostics and help monitor diseased tissue. An overview of existing noncontact multispectral diffuse reflectance imaging modalities for tissue characterization based on intrinsic optical contrast due to absorption and scattering properties of tissue is given here. Also, an overview of instrumentation advances, modeling approaches, applications, and ongoing work is described, primarily in, but not limited to, skin imaging, demonstrating the benefits and limitations of wide-field diffuse reflectance imaging. © 2012 IEEE.

Cendra M.d.M.,Cellular Bioengineering | Juarez A.,Cellular Bioengineering | Juarez A.,University of Barcelona | Torrents E.,Cellular Bioengineering
PLoS ONE | Year: 2012

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed. © 2012 Cendra et al.

Torrents E.,University of Stockholm | Torrents E.,Cellular Bioengineering | Sjoberg B.-M.,University of Stockholm
Biological Chemistry | Year: 2010

Bacillus anthracis is a severe mammalian pathogen. The deoxyribonucleotides necessary for DNA replication and repair are provided via the ribonucleotide reductase (RNR) enzyme. RNR is also important for spore germination and cell proliferation upon infection. We show that the expression of B. anthracis class Ib RNR responds to the environment that the pathogen encounters upon infection. We also show that several anti-proliferative agents (radical scavengers) specifically inhibit the B. anthracis RNR. Owing to the importance of RNR in the pathogenic infection process, our results highlight a promising potential to inhibit the growth of B. anthracis early during infection. © 2010 by Walter de Gruyter Berlin New York.

Guan Y.,Cellular Bioengineering | Kisaalita W.,Cellular Bioengineering
Colloids and Surfaces B: Biointerfaces | Year: 2011

The purpose of this study was to investigate the effect of microstructured material surface on cell adhesion and locomotion in real-time. ArF excimer laser direct-writing ablation was used to fabricate microwell patterns with precise control of size and spacing on glass. The influence of the ablation process parameters (laser fluence, pulse number and repetition rate) on the micromachining quality (depth, width, aspect ratio and edge effects) of the microwells was established. Human fibroblast cells, as an example of anchorage-dependent cells, were seeded onto the microstructured glass substrate and time-lapse microscopy was used to study cell adhesion and locomotion. The interaction with microstructured materials resulted in fibroblast cell repulsion and the cells exhibited a higher locomotion speed (75.77 ± 3.36 μm/h) on the structures in comparison with plane glass control (54.01 ± 15.53 μm/h). Further studies are needed to firmly establish the potential of microstructuring, for example, in elongating the life spans of implantable devices. © 2010 Elsevier B.V.

Lai Y.,Cellular Bioengineering | Cheng K.,Cellular Bioengineering | Kisaalita W.,Cellular Bioengineering
PLoS ONE | Year: 2012

It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells' functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns. © 2012 Lai et al.

Wang L.,Cellular Bioengineering | Kisaalita W.S.,Cellular Bioengineering
Journal of Biomedical Materials Research - Part B Applied Biomaterials | Year: 2010

Micropatterns were fabricated in nanofibrous poly-L-lactic acid (PLLA) films by laser micromachining and the resulting scaffolds were characterized with respect to architecture, thermal, mechanical, and mass transport properties. Also, human neural stem cells were successfully cultured in these micropatterned nanofibrous scaffolds (MNFSs). The scaffolds were incorporated in high-density well plates (e.g., 96-well plates), creating a platform for high-throughput screening of drugs with physiologically more relevant networked neural cultures. Through mathematical modeling of the transport of model stimulants, the feasibility of stimulating neural networks cultured in MNFSs was demonstrated. More work is needed to establish biological network activity-MNFS architecture relationships. © 2010 Wiley Periodicals, Inc.

Zhao H.,Cellular Bioengineering | Schuck P.,Cellular Bioengineering
Acta Crystallographica Section D: Biological Crystallography | Year: 2015

Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design. © 2015.

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