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Biomedical subjects

Gang Bao

Publications and source records attributed to Gang Bao.

At least 19 recordsLinked to original sources
PubMed2025

In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene.

Homology-directed repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested Repair Drive, a platform technology for selectively expanding HDR-corrected hepatocytes in adult mice in vivo. Repair Drive involves transient conditioning of the liver by knocking down an essential gene, fumarylacetoacetate hydrolase (Fah), and delivering an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive increased the percentage of correctly targeted hepatocytes in healthy wild-type mice up to 25%, which resulted in a fivefold increased expression of a therapeutic transgene, human factor IX (FIX). Repair Drive was well tolerated and did not induce toxicity or tumorigenesis during a 1-year follow-up. This approach may broaden the range of liver diseases that can be treated with somatic genome editing.

Animals↗
PubMed2006

Nanostructured probes for RNA detection in living cells.

The ability to visualize in real-time the expression level and localization of specific RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we review the recent development of nanostructured oligonucleotide probes for living cell RNA detection, and discuss the biological and engineering issues and challenges of quantifying gene expression in vivo. In particular, we describe methods that use dual FRET (fluorescence resonance energy transfer) or single molecular beacons in combination with peptide-based or membrane-permeabilization-based delivery, to image the relative level, localization, and dynamics of RNA in live cells. Examples of detecting endogenous mRNAs, as well as imaging their subcellular localization and colocalization are given to illustrate the biological applications, and issues in molecular beacon design, probe delivery, and target accessibility are discussed. The nanostructured probes promise to open new and exciting opportunities in sensitive gene detection for a wide range of biological and medical applications.

Animals↗
PubMed2006

Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons.

Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 x 10(3.6) 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.

Animals↗
PubMed2005

DNA functionalized single-walled carbon nanotubes for electrochemical detection.

Single-walled carbon nanotubes (SWNTs) were effectively dispersed and functionalized by wrapping with single-stranded DNA (ssDNA). The ssDNA-SWNTs attach strongly on glass substrate and easily form a uniform film, making it possible for electrochemical analysis and sensing. The film was fabricated into a working electrode, which exhibited good electrochemical voltammetric properties, such as flat and wide potential window, well-defined quasi-reversible voltammetric responses, and quick electron transfer for a Fe(CN)6(3-)/Fe(CN)6(4) system, indicating that the ssDNA-SWNTs film should be a good analytical electrode for electrochemical detection or sensing. This was demonstrated by highly selective and sensitive detection of a low concentration of dopamine in the presence of excess ascorbic acid.

Carbon↗
PubMed2005

Adult chronic sleepwalking and its treatment based on polysomnography.

Adult sleepwalking affects 2.5% of the general population and may lead to serious injuries. Fifty young adults with chronic sleepwalking were studied prospectively. Clinical evaluation, questionnaires from patients and bed partners, and polysomnography were obtained on all subjects in comparison with 50 age-matched controls. Subjects were examined for the presence of psychiatric anxiety, depression and any other associated sleep disorder. Isolated sleepwalking or sleepwalking with psychiatric disorders was treated with medication. All other patients with other sleep disorders were treated only for their associated problem. Prospective follow-up lasted 12 months after establishment of the most appropriate treatment. Patients with only sleepwalking, treated with benzodiazepines, dropped out of follow-up testing and reported persistence of sleepwalking, as did patients with psychiatric-related treatment. Chronic sleepwalkers frequently presented with sleep-disordered breathing (SDB). All these patients were treated only for their SDB, using nasal continuous positive airway pressure (CPAP). All nasal CPAP-compliant patients had control of sleepwalking at all stages of follow-up. Non-compliant nasal CPAP patients had persistence of sleepwalking. They were offered surgical treatment for SDB. Those successfully treated with surgery also had complete resolution of sleepwalking. Successful treatment of SDB, which is frequently associated with chronic sleepwalking, controlled the syndrome in young adults.

Adolescent↗
PubMed2005

Adaptive finite-element method for diffraction gratings.

A second-order finite-element adaptive strategy with error control for one-dimensional grating problems is developed. The unbounded computational domain is truncated to a bounded one by a perfectly-matched-layer (PML) technique. The PML parameters, such as the thickness of the layer and the medium properties, are determined through sharp a posteriori error estimates. The adaptive finite-element method is expected to increase significantly the accuracy and efficiency of the discretization as well as reduce the computation cost. Numerical experiments are included to illustrate the competitiveness of the proposed adaptive method.

Journal Article↗
PubMed2005

Computational design for guided-mode grating resonances.

We are concerned with optimal design of guided-mode grating resonant structures (GMGRs). A typical structure is the integration of a zeroth-order grating and a planar waveguide. Our approach has two main steps. The first is to find the resonant wavelength. For any fixed grating structure the resonant wavelength is found by solving a nonlinear eigenvalue problem. The second step is to develop a Newton-type local optimization method. A crucial step is to determine an appropriate initial guess of the design parameters. Numerical design examples for both TE and TM polarization are presented. The design algorithm is expected to provide systematic guidance in engineering design of GMGRs.

Journal Article↗
PubMed2004

Dual FRET molecular beacons for mRNA detection in living cells.

The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT-PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.

Binding Sites↗
PubMed2004

Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells.

Real-time visualization of specific endogenous mRNA expression in vivo has the potential to revolutionize medical diagnosis, drug discovery, developmental and molecular biology. However, conventional liposome- or dendrimer-based cellular delivery of molecular probes is inefficient, slow, and often detrimental to the probes. Here we demonstrate the rapid and sensitive detection of RNA in living cells using peptide-linked molecular beacons that possess self-delivery, targeting and reporting functions. We conjugated the TAT peptide to molecular beacons using three different linkages and demonstrated that, at relatively low concentrations, these molecular beacon constructs were internalized into living cells within 30 min with nearly 100% efficiency. Further, peptide-based delivery did not interfere with either specific targeting by or hybridization-induced fluorescence of the probes. We could therefore detect human GAPDH and survivin mRNAs in living cells fluorescently, revealing intriguing intracellular localization patterns of mRNA. We clearly demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.

Amino Acid Sequence↗
PubMed2004

Upper airway resistance syndrome--one decade later.

PURPOSE OF REVIEW: The term upper airway resistance syndrome (UARS) was coined to describe a group of patients who did not meet the criteria for diagnosis of obstructive apnea-hypopnea syndrome and thus were left untreated. Today, most of the patients with UARS remain undiagnosed and are left untreated. RECENT FINDINGS: Today, the clinical picture of UARS is better defined. We have learned that patients usually seek treatment with a somatic functional syndrome rather than sleep-disordered breathing or even a disorder of excessive daytime sleepiness. Therefore, most of these patients are seen by psychiatrists. In addition, recent technologic advances have allowed a better recognition of the problem. We have learned that obstructive apnea-hypopnea syndrome is associated with a local neurologic impairment that is responsible for the occurrence of the hypopnea and apneas. In contrast, patients with UARS have an intact local neurologic system and have the ability to respond to minor changes in upper airway dimension and resistance to airflow. New treatment options including internal jaw distraction osteogenesis are used and are promising for treatment of patients with UARS. SUMMARY: The clinical presentation of patients with UARS is similar to the presentation of subjects with functional somatic syndrome. To diagnose UARS, nocturnal polysomnography should include additional measurement channels.

Airway Resistance↗
PubMed2004

[The study of the invasive biologic behavior of ameloblastoma].

OBJECTIVE: To investigate the invasive biologic behavior of ameloblastoma (AB) and to analyze its correlative factors. METHODS: The specimens of 43 cases of AB (primary AB 16 cases, recurrent AB 21 cases, malignant AB 6 cases) were examined immunohistochemically using the streptavidin-biotin method to determine the expression of E-cadherin (E-cad), matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF). RESULTS: The cells in malignant AB scattered more, grew invasively, and the basal membrane ruptured or lost. The expression of E-cad in AB descended, MMP-2 and MMP-9 were strongly expressed in the epithelia cells of 28/41, 30/43 cases of AB, respectively. The positive rate and intensity of VEGF increased as AB recurred and transformed malignantly. The expression of E-cad, MMP-9 and VEGF were related to recurrence or malignant transformation of AB (r(s) = 0.309, 0.519, 0.381, P < 0.05). CONCLUSION: AB is a high invasive tumor. The biological behavior of AB is related to lost or abnormal expression of E-cad, the high expression of MMP-2, MMP-9, and VEGF.

Adolescent↗
PubMed2004

The effects of BclXL and Bax over-expression on stretch-injury induced neural cell death.

The Bcl-2 family of proteins has recently been implicated as a possible player in the complex cascade of neural cell death due to traumatic brain injuries. However, it is unclear if the Bcl-2 pathways are activated in mechanically injured neurons. Here we report the effects of BclX(L) and Bax over-expression on stretch-induced neural cell death using an in vitro uniaxial stretch model of traumatic axonal injury. Specifically, YFP, YFP-tagged Bax and YFP-tagged BclX(L) proteins were expressed in differentiated NG108-15 cells and stretch-injury assays were carried out at different strain and strain rate combinations. As a control, insults known to act within the Bcl-2 pathways were used to study cell viability and to compare with the results of cell death due to mechanical stretching. Surprisingly, under the stretch-injury conditions in this study, BclXL did not provide protection against cell death. Further, translocation of Bax could not be identified after stretch-injury. The implications of these findings to cell death pathways in traumatic brain injury are discussed.

Animals↗
PubMed2004

Engineering nanostructured probes for sensitive intracellular gene detection.

The ability to detect, localize, quantify and monitor the expression of specific genes in living cells in real-time will offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery, and medical diagnostics. However, current methods for quantifying gene expression employ either selective amplification (as in PCR) or saturation binding followed by removal of the excess probes (as in microarrays and in situ hybridization) to achieve specificity. Neither approach is applicable when detecting gene transcripts within living cells. Here we review the recent development in engineering nanostructured molecular probes for gene detection in vivo, describe probe design approaches and its structure-function relations, and discuss the critical issues and challenges in performing living cell gene detection with high specificity, sensitivity and signal-to-background ratio. The underlying biological and biochemical aspects are illustrated.

Genes↗
PubMed2003

A filtration-based protein microarray technique.

Protein microarrays are an emerging technology for studying protein expression profiling and protein functions. However, with the current design approaches, the overall performance of protein microarrays can be compromised by diffusion-limited kinetics. We developed a new protein microarray platform that utilizes a filtration assay with protein microarrays printed on protein-permeable nitrocellulose filter membranes. Compared with protein microarrays assayed with the conventional incubation-shaking method, this new approach overcomes the diffusion limit. We demonstrated that this novel technique can improve the overall reaction kinetic rate by 10-fold, yield a dynamic range of 4 decades, and enhance the assay sensitivity and specificity. Further, using multistacking protein chips, at least 14 chips can be probed simultaneously, with 22400 different reactions in a single assay. The advantages of large fluorescent dyes, such as phycobilisome and quantum dots, can be better exploited using the filtration assay. The potential clinical applications of the filtration-based protein microarrays were demonstrated by detecting carcinoembryonic antigen in human plasma samples.

Antibodies↗
PubMed2003

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons.

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.

Base Sequence↗
PubMed2003

Local spectral time-domain method for electromagnetic wave propagation.

We explore the feasibility of using a local spectral time-domain (LSTD) method to solve Maxwell's equations that arise in optical and electromagnetic applications. The discrete singular convolution (DSC) algorithm is implemented in the LSTD method for spatial derivatives. Fourier analysis of the dispersive error of the DSC algorithm indicates that its grid density requirement for accurate simulations can be as low as approximately two grid points per wavelength. The analysis is further confirmed by numerical experiments. Our study reveals that the LSTD method has the potential to yield high resolution for solving large-scale electromagnetic problems.

Journal Article↗
PubMed2003

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets.

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.

DNA↗