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PubMed2004

Gene expression characteristic in human auricular cartilage tissue engineering.

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.

Actins↗

PubMed2003

Tissue engineering the kidney.

The means by which kidney function can be replaced in humans include dialysis and renal allotransplantation. Dialytic therapies are lifesaving, but often poorly tolerated. Transplantation of human kidneys is limited by the availability of donor organs. During the past decades, a number of different approaches have been applied toward tissue engineering the kidney as a means to replace renal function. The goals of one or another of them included the recapitulation of renal filtration, reabsorptive and secretory functions, and replacement of endocrine/metabolic activities. This review will delineate the progress to date recorded for five approaches: (1) integration of new nephrons into the kidney; (2) growing new kidneys in situ; (3) use of stem cells; (4) generation of histocompatible tissues using nuclear transplantation; and (5) bioengineering of an artificial kidney. All five approaches utilize cellular therapy. The first four employ transplantation as well, and the fifth uses dialysis.

Animals↗

PubMed2005

Microfabricated grooved substrates as platforms for bioartificial liver reactors.

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.

3T3 Cells↗

PubMed1996

Gene-modified tumor cells as cellular vaccine.

The identification and characterization of many tumor antigens and the parallel explosion of knowledge of the cellular and molecular mechanisms of antigen recognition by the immune system have given renewed hopes that immunogenetherapy could be a promising modality to treat certain tumors. Many different novel strategies have been developed to derive genetically modified tumor cells and use them as cellular vaccines to induce useful antitumor immunity in a variety of animal tumor models. This review discusses induction of tumor immunity by injecting tumor cells that are genetically engineered to secrete various cytokines and to express major histocompatibility complex molecules and/or costimulatory molecules. While there has been a great success in inducing excellent antitumor immunity in a variety of tumor models, there are some difficulties and limitations in the application of these gene-modified tumor cells for the treatment of preexisting tumors. A number of improvements and modifications are already underway to overcome some of these problems.

Animals↗

PubMed2005

Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles.

Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.

Adaptation, Physiological↗

PubMed2005

Use of constraint-based modeling for the prediction and validation of antimicrobial targets.

The overall process of antimicrobial drug discovery and development seems simple, to cure infectious disease by identifying suitable antibiotic drugs. However, this goal has been difficult to fulfill in recent years. Despite the promise of the high-throughput innovations sparked by the genomics revolution, discovery, and development of new antibiotics has lagged in recent years exacerbating the already serious problem of evolution of antibiotic resistance. Therefore, both new antimicrobials are desperately needed as are improvements to speed up or improve nearly all steps in the process of discovering novel antibiotics and bringing these to clinical use. Another product of the genomic revolution is the modeling of metabolism using computational methodologies. Genomic-scale networks of metabolic reactions based on stoichiometry, thermodynamics and other physico-chemical constraints that emulate microbial metabolism have been developed into valuable research tools in metabolic engineering and other fields. This constraint-based modeling is predictive in identifying critical reactions, metabolites, and genes in metabolism. This is extremely useful in determining and rationalizing cellular metabolic requirements. In turn, these methods can be used to predict potential metabolic targets for antimicrobial research especially if used to increase the confidence in prioritization of metabolic targets. The many different capacities of constraint-based modeling also enable prediction of cellular response to specific inhibitors such as antibiotics and this may, ultimately find a role in drug discovery and development. Herein, we describe the principles of metabolic modeling and how they might initially be applied to antimicrobial research.

Anti-Bacterial Agents↗

PubMed2007

In vivo tumor targeting and radionuclide imaging with self-assembled nanoparticles: mechanisms, key factors, and their implications.

The development of more selective delivery systems for cancer diagnosis and chemotherapy is one of the most important goals of current anticancer research. The purpose of this study is to evaluate various self-assembled nanoparticles as candidates to shuttle radionuclide and/or drugs into tumors and to investigate the mechanisms underlying the tumor targeting with self-assembled nanoparticles. By combining different hydrophobic moieties and hydrophilic polymer backbones, various self-assembled nanoparticles were prepared, and their in vivo distributions in tumor-bearing mice were studied by radionuclide imaging. One type of nanoparticles (fluorescein isothiocyanate-conjugated glycol chitosan (FGC) nanoparticles) exhibited highly selective tumoral localization. Scintigraphic images obtained 1 day after the intravenous injection of FGC nanoparticles clearly delineated the tumor against adjacent tissues. The mechanisms underlying the tumor targeting with self-assembled nanoparticles were investigated in terms of the physicochemical properties of nanoparticles and tumor microenvironments. FGC nanoparticles were preferentially localized in perivascular regions, implying their extravasation to tumors through the hyperpermeable tumor vasculature. The magnitude and pattern of tumoral distribution of self-assembled nanoparticles were influenced by several key factors--(i) in vivo colloidal stability: nanoparticles should maintain their intact nanostructures in vivo for a long period of time, (ii) particle size, (iii) intracellular uptake of nanoparticle: fast cellular uptake greatly facilitates the tumor targeting, (iv) tumor angiogenesis: pathological angiogenesis permits access of nanoparticles to tumors. We believe that this work can provide insight for the engineering of nanoparticles and be extended to cancer therapy and diagnosis, so as to deliver multiple therapeutic agents and imaging probes at high local concentrations.

Animals↗

PubMed2003

Advances in flux balance analysis.

Biology is going through a paradigm shift from reductionist to holistic, systems-based approaches. The complete genome sequence for a number of organisms is available and the analysis of genome sequence data is proving very useful. Thus, genome sequencing projects and bioinformatic analyses are leading to a complete 'parts catalog' of the molecular components in many organisms. The next challenge will be to reconstruct and simulate overall cellular functions based on the extensive reductionist information. Recent advances have been made in the area of flux balance analysis, a mathematical modeling approach often utilized by metabolic engineers to quantitatively simulate microbial metabolism.

Computational Biology↗

PubMed2006

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology.

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.

Biotechnology↗

PubMed2004

Distinct cell-to-fiber junctions are critical for the establishment of cardiotypical phenotype in a 3D bioartificial environment.

The first step toward improving the cell-matrix interactions that occur in bioartificial myocardial tissue is an understanding of the ultrastructural links between cells and host fibers. Here, we identify a distinct type of junction that helps the cells to find anchorage in the three-dimensional environment, and we evaluate the phenotype of the resulting tissue. Neonatal rat cardiomyocytes were seeded in two different collagen scaffolds after pre-hydration of the scaffold. Conventional and electron microscopy were used to analyze the tissue microstructure. Viability was assessed by life/dead assay and physical properties of the resulting tissue were evaluated. The resulting tissue displayed high cellular viability, spontaneous contractions over 12 weeks, and responded to passive stretch similar to native rat myocardium. Contractile force responded physiologically to calcium (Ca), adrenaline, and stretch administration. Ultrastructural studies revealed a cell-to-fiber junction, as well as a background matrix configuration, which has not been described before in this context. The cells aligned along collagen fibers and engaged in complex intercalations. The cell-to-fiber affinity is essential for the phenotypical performance of bioartificial myocardial tissue equivalents. Moreover, given the appropriate porosity of the scaffold, pre-hydration promotes migration and affinity of cells to host structures.

Animals↗

PubMedDate not supplied

Molecular level investigations of the inter- and intramolecular interactions of pH-responsive artificial triblock proteins.

Intelligent materials that can undergo physical gelation in response to environmental stimuli have potential impacts in the bioengineering and biomedical fields where the entrapment of cellular or molecular species is desired. Here, we utilize atomic force microscopy (AFM) to perform molecular level investigations of designer artificial proteins that undergo physical gelation. These are engineered as triblock copolymers with independent interchain binding and solvent retention functions, namely, two terminal leucine zipper-like peptide sequences and a central alanylglycine rich sequence, respectively. AFM force measurements between probes and surfaces functionalized with molecules of this triblock protein revealed adhesive interactions that increased in average force and frequency as the pH was lowered from pH 11.2 to 7.4 to 4.5, reflecting an increase in the numbers of interacting molecular strands. In bulk solution, lowering the pH results in a viscous liquid to gel transition. The modular design of the triblock protein was also exploited for single molecule force spectroscopy investigations, which revealed altered intramolecular interactions in response to changes in pH. An increased understanding of the inter- and intramolecular forces involved in biomolecule driven gelation processes is not only of great fundamental interest in the study of the biomolecular systems involved but may also prove key in enabling the rational design of new generations of intelligent hydrogel systems.

Amino Acid Sequence↗

PubMed2005

Mobile genetic elements: the agents of open source evolution.

Horizontal genomics is a new field in prokaryotic biology that is focused on the analysis of DNA sequences in prokaryotic chromosomes that seem to have originated from other prokaryotes or eukaryotes. However, it is equally important to understand the agents that effect DNA movement: plasmids, bacteriophages and transposons. Although these agents occur in all prokaryotes, comprehensive genomics of the prokaryotic mobile gene pool or 'mobilome' lags behind other genomics initiatives owing to challenges that are distinct from cellular chromosomal analysis. Recent work shows promise of improved mobile genetic element (MGE) genomics and consequent opportunities to take advantage - and avoid the dangers - of these 'natural genetic engineers'. This review describes MGEs, their properties that are important in horizontal gene transfer, and current opportunities to advance MGE genomics.

Conjugation, Genetic↗

PubMed2002

Intracellular ribozyme applications.

The exquisite target selectivity of trans -acting ribozymes has fostered their use as potential therapeutic agents and tools for down-regulating cellular transcripts. In living cells, free diffusion of RNAs is extremely limited, if it exists at all. Thus, getting ribozymes to base-pair with their cognate targets requires co-localizing the ribozyme transcript with the target RNA. In addition, not all sites along a given target RNA are equally accessible to ribozyme base pairing. Cellular proteins greatly influence the trafficking and structure of RNA, and therefore making ribozymes work effectively in cells a significant challenge. This article addresses the problems of getting engineered ribozymes to effectively pair with and cleave targets in cells. The work described here illuminates methods for target-site selection on native mRNAs, methods for ribozyme expression, and strategies for obtaining a discrete intracellular localization of ribozymes.

Blotting, Northern↗

PubMed2002

Evaluation of cellular affinity and compatibility to biodegradable polyesters and Type-II collagen-modified scaffolds using immortalized rat chondrocytes.

Immortalized rat chondrocytes (IRCs) were employed to evaluate the cytocompatibility of different biodegradable polyester scaffolds for chondrocyte seeding and cartilage tissue engineering in vitro due to the limitation of using freshly harvested chondroctyes. Cells were seeded onto the films and the porous substrates as well as into the three-dimensional scaffolds made of the biodegradable polyesters including poly(l-lactide) (PLLA) and two poly(lactide-co-glycolide)s (PLGAs). The materials were characterized by water contact angle, electron spectroscopy for chemical analysis (ESCA), and microscopy. PLGA50/50, one of the PLGAs, had the largest cell numbers at 24 h and 96 h (close to the tissue culture polystyrene control), possibly due to its lower contact angle, higher oxygen/carbon (O/C) atomic ratio, and larger degradation rate. When the surface was further modified by cross-linked Type-II collagen, cell population was significantly enhanced (two- to fourfold). The adhesion and proliferation behavior of IRCs on different materials was parallel to that of rabbit chondrocytes, but was more reproducible in general. IRCs are thus suitable for evaluation of different polymer scaffolds. Despite the favorable cytocompatibility of PLGA50/50, blending with a small portion of PLLA is required for easy fabrication and collagen modification. Scaffolds made of blended materials by freeze-drying procedure with the surface modified by cross-linked Type-II collagen were demonstrated as the ideal templates for chondrocyte seeding in our study.

Animals↗

PubMed2003

Modeling regulatory networks at Virginia Tech.

The life of a cell is governed by the physicochemical properties of a complex network of interacting macromolecules (primarily genes and proteins). Hence, a full scientific understanding of and rational engineering approach to cell physiology require accurate mathematical models of the spatial and temporal dynamics of these macromolecular assemblies, especially the networks involved in integrating signals and regulating cellular responses. The Virginia Tech Consortium is involved in three specific goals of DARPA's computational biology program (Bio-COMP): to create effective software tools for modeling gene-protein-metabolite networks, to employ these tools in creating a new generation of realistic models, and to test and refine these models by well-conceived experimental studies. The special emphasis of this group is to understand the mechanisms of cell cycle control in eukaryotes (yeast cells and frog eggs). The software tools developed at Virginia Tech are designed to meet general requirements of modeling regulatory networks and are collected in a problem-solving environment called JigCell.

Animals↗

PubMed2000

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Cell Differentiation↗

PubMed2005

Mitochondrial calcium signalling in cell death.

The development of targeted probes (based on the molecular engineering of luminescent or fluorescent proteins) has allowed the specific measurement of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This approach gave novel information on different aspects of cellular Ca2+ homeostasis. Regarding mitochondria, it was possible to demonstrate that, upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the matrix. We will discuss the basic characteristics of this process, its role in modulating physiological and pathological events, such as the regulation of aerobic metabolism and the induction of cell death, and new insight into the regulatory mechanisms operating in vivo.

Animals↗

PubMed1993

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Alleles↗