SEARCH · PubMed Health

Results for “reverse engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources

PubMed2003

Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee.

This study assessed the feasibility of a devitalized knee as a scaffold for an engineered chimeric joint. Embryonic chick knees (19 days old), devitalized by lyophilization or multiple freeze-thaw cycles, were tested as scaffolds for repopulation with bovine articular chondrocytes (bACs). bACs were seeded into porous three-dimensional collagen sponges and were cultured for 1 day before fabrication of chimeric constructs. A pair of cell-seeded sponges was inserted into the joint space to contact preshaved articular surfaces. In some constructs, a sterile membrane of expanded polytetrafluoroethylene (ePTFE) was inserted between the collagen sponges. Histologic analysis showed that at 1 week, sponges with bACs were adherent to the shaved articular surfaces of the joint with accumulation of metachromatic extracellular matrix. Penetration of bACs and neomatrix into the devitalized matrix appeared to begin in preexistent epiphyseal canals and was observed to some extent in all specimens. Membranes of ePTFE maintained a joint space at 2 and 3 weeks, whereas there was fusion across the two sponges in many specimens lacking the membrane. Gene expression analysis demonstrated that lyophilization, but not multiple freeze-thaw cycles, completely devitalized the chick knees. These studies identified several design parameters crucial for successful engineering of a chimeric joint.

Animals↗

PubMed2000

[Development of influenza vaccines against newly emerging A/H5N1 virus].

Emergence of highly virulent influenza A/H5N1 viruses in Hong Kong in 1997 posed a threat of pandemic and brought an urgent need to develop a suitable seed virus for vaccine production. The virulence of the H5N1 viruses to chicken embryos should hamper the efficient production of the vaccine. In addition, potential virulence to humans raised safety issue in manufacturing vaccine. Toward vaccine development, one approach is to use an avirulent avian influenza virus antigenically similar to the virulent ones as a surrogate vaccine strain. The other approach is based on the attenuation of pathogenicity of virulent H5N1 virus by genetic engineering of the hemagglutinin gene and selection of a gene constellation. The reverse genetics technique can make the latter approach possible. Candidate strains suitable for vaccine production could be prepared by using either approach.

Animals↗

PubMed1995

Retrovirus-mediated transduction of an engineered intron-containing purine nucleoside phosphorylase gene.

We constructed and tested several retroviral vectors containing abbreviated purine nucleoside phosphorylase (PNP) genes in the reverse orientation, a strategy compatible with transduction of intron-containing genes. We observed two types of deletions in these vectors after one round of replication: (i) Deletions flanked by direct repeats with one copy of the repeat retained in the provirus, presumably resulting from reverse transcriptase slippage during (-) strand DNA synthesis. (ii) Deletions due to fortuitous splice sites in the PNP complementary strand. Two splice donor sites and three splice acceptor sites were identified in a 3.0-kb PNP minigene. We found that the splice donor sites (but not the splice acceptor sites) could be predicted by sequence analysis of the PNP complementary strand. To increase the frequency of intact PNP gene transduction, we introduced sequence modifications: The putative PNP polyadenylation signal and a truncated 117-bp 3' flank were recovered from a rearranged provirus and inserted in place of a 1.2-kb genomic 3' flank. Sequences associated with deletions were eliminated from the PNP 5' untranslated region, and two fortuitous splice donor signals in the complementary strand were inactivated. A retroviral vector LN-PMG11, containing the engineered 2.9-kb PNP minigene in the reverse orientation, was transduced intact in 23% (5/22) of clones after one round of replication and in 87% (20/23) of clones after a second round of replication from two primary virus producer clones. Directed mutagenesis of sequences preventing intact retroviral transduction thus provided a 2.9-kb PNP gene that was transduced intact and expressed at a high level.

Base Sequence↗

PubMed1996

Airway reactivity in welders: a controlled prospective cohort study.

In a 3-year survey, respiratory symptoms, spirometry, and methacholine reactivity were measured annually in welders (n = 51) and non-welder controls subjects (n = 54) to determine whether welding-related symptoms are associated with accelerated decline in lung function or changes in airway reactivity. In the cross-workshift study, maximal midexpiratory flow rate declined reversibly during a welding day, whereas 1-second forced expiratory volume and forced-vital capacity were unchanged. In the longitudinal study, the welders had significantly more reversible work-related symptoms of cough, phlegm, wheeze, and chest tightness than the non-welder shipyard control subjects. In this group of actively working welders, across-workshift changes in midflow and reversible symptoms were related to the welding occupation, but evidence for chronic irreversible effects on spirometry or airway reactivity was not seen over the 3 years of observation. The short period of observation was not optimal for detecting a chronic effect on lung function. Work practices and engineering controls may be successfully preventing irreversible respiratory effects, but not mild reversible effects, in this group of welders.

Adult↗

PubMed2002

Molecular approaches for the evaluation of immune responses to zona pellucida (ZP) and development of second-generation ZP vaccines.

It has long been established that there are major variations in both the immunogenicity and antigenicity of native zona pellucida (ZP) proteins. These differences appear to be more pronounced with respect to genetically engineered ZP proteins, which do not have native post-translational modifications (for example glycosylation and sulphation). As the number of animal species that are now included in population management programmes using native porcine zona pellucida (PZP) proteins expands, it is increasingly important to carry out studies to evaluate the immune response variations among different species as well as the individual variation within a species. In an attempt to compare these complex immune responses, we have evaluated antibodies from numerous species immunized with native, genetically engineered ZP and synthetic ZP peptides. Such an immunocontraceptive method could have great potential. These studies are critical not only for the development of predictable immune responses that result in permanent sterilization versus reversible contraceptive effects, but also for predicting which vaccinogens (native ZP protein versus genetically engineered ZP proteins) might have detrimental effects on animal and human populations.

Animals↗

PubMed2002

Biological response of chondrocytes to hydrogels.

Primary bovine chondrocytes were encapsulated in alginate and alginate combined with cartilage matrix extract, Cartrigel, for the purpose of cartilage tissue engineering. The cell constructs were incubated in vitro and gene expression of cartilage-specific extracellular matrix molecules was quantitated and localized with in situ hybridization with a decrease in expression observed in the alginate-Cartrigel constructs. Further understanding of cell response to scaffolds will allow rational design and development of hydrogels for cartilage tissue engineering.

Alginates↗

PubMed2006

Gene therapy and tissue engineering in orthopaedic surgery.

Despite setbacks in other fields, gene therapy in orthopaedic surgery continues to serve as the basis for novel treatments of various musculoskeletal disorders. Even in the brief time since the last review of scientific progress in this area, another orthopaedic-related disease has joined the ranks of those studied in gene therapy clinical trials. Armed with new techniques and new reagents, and committed to the increased use of tissue engineering, physicians and scientists continue to work together to accelerate tissue repair and reverse the course of chronic debilitating diseases.

Genetic Therapy↗

PubMed1996

Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.

Animals↗

PubMed2004

An engineered EBV vector expressing human factor VIII and von Willebrand factor in cultured B-cells.

BACKGROUND: Hemophilia A is a congenital disorder caused by a deficiency of the blood-clotting factor VIII (FVIII) and is an attractive candidate for gene therapy. Most of the studies have only explored the potential of hepatocytes and muscle cells as the targets for gene transfer. Attempts to transfer the genes into hematopoietic cells have so far been mostly unsuccessful due to inefficiency of most viral vectors to transduce these cells and the supposed inability of the cells to express FVIII. METHODS: We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry either the full-length FVIII cDNA or its B-domain-deleted form; a modified version of the vector that carries B-domain-deleted FVIII along with the von Willebrand factor (vWF) cDNA or the reporter gene DsRed2 was also used. All these vectors have been safety modified with viral thymidine kinase cDNA to transduce human B-cells in culture. RESULTS: Low-level expression of FVIII in the order of 5-8 ng FVIIIC/ml were observed in the cells stably transduced with full-length FVIII, while cells with the B-domain-deleted version expressed 8-16 ng FVIIIC/ml. Expression of vWF and B-domain-deleted FVIII resulted in a moderate expression of 18-30 ng FVIIIC/ml. Long-term expression for 12-16 weeks was observed in these cells regardless of selection pressure. CONCLUSIONS: These results support the development of an episomal engineered EBV vector for treatment of hemophilia A using the hematopoietic cells as a target for providing immediate secretion of functionally active product in the circulating bloodstream.

B-Lymphocytes↗

PubMed1997

Structure and conformation of helical nucleic acids: analysis program (SCHNAaP).

We present a new versatile program, SCHNAaP, for the analysis of double-helical nucleic acid structures. The program uses mathematically rigorous and fully reversible procedures for calculating the structural parameters: the Cambridge University Engineering Department Helix computation Scheme (CEHS) is used to determine the local helical parameters and an analogous procedure is used to determine the global helical parameters. These parameters form a complete set that conforms to the "Cambridge Accord" on definitions and nomenclature of nucleic acid structure parameters. In addition to the two standard Watson-Crick base-pairs, the program handles mismatched base-pairs and chemically modified bases. An analysis of the sugar-phosphate backbone conformation is included. Standardized base-stacking diagrams of each dinucleotide step with reference to the mid-step triad are generated. Structures are classified as one of the four polymorphic families, A/B, Z, W or R, although W- and R-DNA (two types of hypothetical structure) have yet to be observed experimentally.

Base Composition↗

PubMed1988

Neurobiological approach to computing devices.

According to the old metaphor of classical cybernetics the brain can be considered as a computer. Newer theoretical endeavours reverse the question and ask: what could neurobiology offer to engineers of near-future generation computer systems? Three not completely disjoint abstract functions of the nervous system, namely pattern formation, pattern recognition and action, can be treated in a unified conceptual framework. Storage and retrieval mechanisms of information are connected to fault-tolerant, adaptive parallel structures. "Learning" and "plastic behaviour" are interpreted in terms of the theory of non-linear dynamic systems. As neural development and plasticity can be approached by deterministic models superimposed by random influence, noise might also have a positive role to play during the operation of technical computing devices. Molecular computation is discussed in relation to eventual hardware realization of "neurobiology-based" computers.

Cybernetics↗

PubMed2006

Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for periodontal tissue engineering.

The current rapid progression in tissue engineering and local gene delivery system has enhanced our applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.

Adenoviridae↗

PubMed2000

The Escherichia coli lacZ reversion mutagenicity assay.

The Escherichia coli lacZ reversion assay, based on the set of episomal lacZ alleles engineered by Miller et al., provides an attractive system for studies of mutagenesis and mutational specificity. Each strain in the lacZ set reverts by a specific base substitution or frameshift event. Revertants are selected by growth on lactose minimal medium. In this review, I describe the development of the assay and its subsequent modifications and improvements. Examples of its application are presented and detailed protocols for the implementation of the assay are given.

Escherichia coli↗

PubMed2003

A temperature responsive biopolymer for mercury remediation.

Tunable biopolymers based on elastin-like polypeptides (ELP) were engineered for the selective removal of mercury. ELP undergoes a reversible thermal precipitation within a wide range of temperatures and was exploited to enable easy recovery of the sequestered mercury. A bacterial metalloregulatory protein, MerR, which binds mercury with an unusually high affinity and selectivity, was fused to the ELP to provide the highly selective nature of the biopolymers. Selective binding of mercury was demonstrated at an expected ratio of 0.5 mercury/biopolymer, and minimal binding of competing heavy metals (cadmium, nickel, and zinc), even at 100-fold excess, was observed. The sequestered mercury was extracted easily, enabling continuous reuse of the biopolymers. In repeating cycles, mercury concentration was reduced to ppb levels, satisfying even drinking water limits. Utility of the biopolymers with mercury-contaminated Lake Elsinore water was demonstrated with no decrease in efficiency. The nanoscale biopolymers reported here using metalloregulatory proteins represent a "green" technology for environmentally benign mercury removal. As nature offers a wide selection of specific metalloregulatory proteins, this technology offers promising solutions to remediation of other important pollutants such as arsenic or chromium.

Adsorption↗

PubMed2026

DNA-guided CRISPR-Cas12 for cellular RNA targeting.

Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70-95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12-ΨDNA-RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.

Journal Article↗

PubMed2004

Independent downstream gene expression profiles in the presence of estrogen receptor alpha or beta.

The two known forms of estrogen receptor (ER), alpha and beta, exhibit differences in structure, affinity for certain ligands, and tissue distribution, suggesting differential roles. It is of interest from several perspectives to determine whether the two receptors elicit similar or differing responses within the same cell type in the presence of the same ligand. To evaluate roles of ER, we have examined responses to estrogen in a rat embryonic fibroblast cell line model, normally naive to ER, engineered to stably express ERalpha or ERbeta. Rat1+ERalpha, Rat1+ERbeta, and precursor Rat1 cell lines were treated with estradiol-17beta (E(2); 1 nM) or an ethanol vehicle for 24 h. Total RNA was extracted, and cDNA generated and subjected to suppression subtractive hybridization (SSH), followed by differential screening using dot blot hybridization. In the presence of ERalpha, products were identified that represent classic responses to E(2), including markers for cell proliferation. In the presence of ERbeta, an alternate transcription profile was observed, including upregulation of pro-alpha-2(I) collagen. These data support a model in which ERalpha and ERbeta regulate unique subsets of downstream genes within a given cell type.

Animals↗

PubMed2006

Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.

Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

Basic Helix-Loop-Helix Proteins↗

PubMed2003

A reversibly switching surface.

We report the design of surfaces that exhibit dynamic changes in interfacial properties, such as wettability, in response to an electrical potential. The change in wetting behavior was caused by surface-confined, single-layered molecules undergoing conformational transitions between a hydrophilic and a moderately hydrophobic state. Reversible conformational transitions were confirmed at a molecular level with the use of sum-frequency generation spectroscopy and at a macroscopic level with the use of contact angle measurements. This type of surface design enables amplification of molecular-level conformational transitions to macroscopic changes in surface properties without altering the chemical identity of the surface. Such reversibly switching surfaces may open previously unknown opportunities in interfacial engineering.

Adsorption↗