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Biomedical subjects

Samuel Wang

Publications and source records attributed to Samuel Wang.

12 recordsLinked to original sources
PubMed2006

Bioluminescence imaging after HSV amplicon vector delivery into brain.

BACKGROUND: Firefly luciferase (Fluc) has routinely been used to quantitate and analyze gene expression in vitro by measuring the photons emitted after the addition of ATP and luciferin to a test sample. It is now possible to replace luminometer-based analysis of luciferase activity and measure luciferase activity delivered by viral vectors directly in live animals over time using digital imaging techniques. METHODS: An HSV amplicon vector expressing Fluc cDNA from an inducible promoter was delivered to cells in culture and into the mouse brain. In culture, expression of Fluc was measured after induction in a dose-dependent manner by a biochemical assay, and then confirmed by Western blot analysis and digital imaging. The vectors were then stereotactically injected into the mouse brain and Fluc expression measured non-invasively using bioluminescence imaging. RESULTS: Rapamycin-mediated induction of Fluc from an HSV amplicon vector in culture resulted in dose-dependent expression of Fluc when measured using a luminometer and by digital analysis. In mouse cortex, a single injection of an HSV amplicon vector (2 microl, 1x10(8) transducing units (t.u.)/ml) expressing Fluc from a viral promoter (CMV) was sufficient to detect robust luciferase activity for at least 1 week. Similarly, an HSV amplicon vector expressing Fluc under an inducible promoter was also detectable in the mouse cortex after a single dose (2 microl, 1x10(8) t.u./ml) for up to 5 days, with no detectable signal in the uninduced state. CONCLUSIONS: This HSV amplicon vector-based system allows for fast, non-invasive, semi-quantitative analysis of gene expression in the brain.

Animals↗
PubMed2006

Telestroke: extending stroke expertise into underserved areas.

Telestroke systems offer the opportunity to extend stroke-care expertise into rural and underserved areas. These systems are being used to give alteplase to patients with stroke in previously underserved areas safely, effectively, and rapidly. Telestroke will probably play a large part in improving the quality of stroke care and in enrolling patients into clinical trials in rural and community hospitals. One such telestroke system, REACH (remote evaluation of acute ischaemic stroke), is a low-cost, web-based system that allows the consultant to access the system from work, home, or on the road. REACH is presently being used to give alteplase and guide acute stroke care in eight rural community hospitals in Georgia.

Female↗
PubMed2005

REACH: clinical feasibility of a rural telestroke network.

BACKGROUND AND PURPOSE: Development of stroke networks is critical to bringing guideline-driven stroke care to rural, underserved areas. METHODS: A Web-based telestroke tool, REACH, was developed to provide a foundation for a rural stroke network that delivered acute stroke consults 24 hours per day 7 days per week to 8 rural community hospitals in Georgia. RESULTS: There were 194 acute stroke consults delivered. Thirty patients were treated with tissue plasminogen activator (tPA). The mean National Institutes of Health Stroke Score (NIHSS) was 15.4, and the median NIHSS was 12.5. The mean onset to treatment time (OTT) was 122 minutes. The OTT dropped from 143 minutes in the first 10 patients treated to 111 minutes in last 20 patients. Of the 30 patients treated with tPA, 23% (7) were treated in < or =90 minutes and 60% (18) were treated within 2 hours. There were no symptomatic intracerebral hemorrhages. CONCLUSIONS: The REACH telestroke system permits the rapid and safe use of tPA in rural community hospitals. Over time, the system became more efficient and OTT decreased.

Aged↗
PubMed2004

Primary care physician time utilization before and after implementation of an electronic health record: a time-motion study.

Despite benefits associated with the use of electronic health records (EHRs), one major barrier to adoption is the concern that EHRs may take longer for physicians to use than paper-based systems. To address this issue, we performed a time-motion study in five primary care clinics. Twenty physicians were observed and specific activities were timed during a clinic session before and after EHR implementation. Surveys evaluated physicians' perceptions regarding the EHR. Post-implementation, the adjusted mean overall time spent per patient during clinic sessions decreased by 0.5 min (p=0.86; 95% confidence interval [-5.05, 6.04]) from a pre-intervention adjusted average of 27.55 min (SE=2.1) to a post-intervention adjusted average of 27.05 min (SE=1.6). A majority of survey respondents believed EHR use results in quality improvement, yet only 29% reported that EHR documentation takes the same amount of time or less compared to the paper-based system. While the EHR did not require more time for physicians during a clinic session, further studies should assess the EHR's potential impact on non-clinic time.

Attitude of Health Personnel↗
PubMed2004

Targeting HSV amplicon vectors.

Several techniques have been developed to deliver DNA directly into mammalian cells, spanning in difficulty from simple mixing procedures to complex systems requiring expensive equipment. Viral vectors have proven able to deliver genes into mammalian cells with high efficiency and low toxicity. In particular, herpes simplex virus type-1 (HSV-1) amplicon vectors are well suited for gene transfer studies as they can infect many cell types, both non-dividing and dividing, have a large transgene capacity and are easy to manipulate. For some applications, it may be desirable to target gene delivery to specific cell populations or to transduce normally non-susceptible cells. This can be achieved by modifying one or more of the glycoproteins found in the viral envelope. Glycoprotein C (gC) has a well-characterized heparan sulfate binding domain (HSBD) necessary for HSV binding to cells. Replacing this region with unique ligands can result in less efficient binding to natural target cells and increase binding to cells which express receptors for these ligands. A method to retarget amplicon vectors by replacing gC HSBD with a model ligand, the hexameric histidine-tag, is described, as well as means to evaluate the binding of modified vector as compared to wild-type virus to cells with or without the appropriate receptor, in this case, a his-tag pseudo-receptor. This protocol demonstrates increased binding of modified virus to receptor-positive cells (at levels greater than wild-type) with no loss of infectivity. Retargeted vectors can provide an additional tool for increasing the efficiency of gene delivery to specific cell types.

Animals↗
PubMed2004

HSV-1 virions engineered for specific binding to cell surface receptors.

Expression of specific peptide epitopes on the surface of virions has significant potential for studying viral biology and designing vectors for targeted gene therapy. In this study, an HSV-1 amplicon plasmid expressing a modified glycoprotein C (gC), in which the heparan sulfate binding domain was replaced with a His-tag, was used in generating HSV-1 virions. Western blot analysis demonstrated the presence of modified gC in the purified virions. The amplicon vectors were packaged using a gC-, lacZ+ helper virus to generate a mixture of high-titer helper virus (lacZ+) and amplicon vectors (GFP+), which expressed modified gC in the virion envelope. His-tagged virions bound to 293 6H cells expressing a cell surface pseudo-His-tag receptor four-fold more efficiently than to parental 293 cells and also proved more effective than wild-type virus in binding to both cell types. Binding resulted in productive infection by the modified virions with expression of reporter genes and cytopathic effect comparable to those of wild-type virions. Thus, not only can HSV-1 tropism be manipulated to recognize a non-herpes simplex binding receptor, but it is also possible to increase the infective capacity of the vectors beyond that of the wild-type virus via specific ligand receptor combinations.

Animals↗
PubMed2003

Ten commandments for effective clinical decision support: making the practice of evidence-based medicine a reality.

While evidence-based medicine has increasingly broad-based support in health care, it remains difficult to get physicians to actually practice it. Across most domains in medicine, practice has lagged behind knowledge by at least several years. The authors believe that the key tools for closing this gap will be information systems that provide decision support to users at the time they make decisions, which should result in improved quality of care. Furthermore, providers make many errors, and clinical decision support can be useful for finding and preventing such errors. Over the last eight years the authors have implemented and studied the impact of decision support across a broad array of domains and have found a number of common elements important to success. The goal of this report is to discuss these lessons learned in the interest of informing the efforts of others working to make the practice of evidence-based medicine a reality.

Decision Making, Computer-Assisted↗
PubMed2003

Single HSV-amplicon vector mediates drug-induced gene expression via dimerizer system.

A variety of viral vectors have been used to deliver genes into various tissues. Most have typically relied on either viral or cell-specific mammalian promoters to express transgenes. More recently, regulated promoter systems have been developed to fine-tune gene expression. Due to limited transgene capacity in most viral vectors, regulatory elements are typically subcloned into two separate vectors, which must be delivered simultaneously to a target cell. Here, we have cloned all the components of the rapamycin-based "dimerizer" system into the pantropic HSV-amplicon vector and used it to deliver and regulate red fluorescent protein (RFP) expression in cultured cells in a drug-dose-dependent manner. 293T/17 cells infected at an m.o.i. of 1 transducing unit/cell and induced with 20 nM rapamycin resulted in a 25-fold increase in RFP mRNA levels after 24 h as assessed by quantitative RT-PCR. However, due to a reduced ability to detect RFP optically, only a 5-fold induction in the number of RFP-expressing cells was noted by FACS analysis 48 h after infection. Further, there was at least 100-fold variation in the levels of RFP in individual, infected cells in the induced state. Gene induction in several neuronal models, including primary cell culture and organotypic cultures, as well as in rodent brain, was observed.

Animals↗
PubMed2002

Modified herpes simplex virus delivery of enhanced GFP into the central nervous system.

Controlled expression of proteins is a key experimental approach to a deeper understanding of the molecular basis of neuronal function. Here we evaluate the HSV-1 (herpes simplex virus) amplicon vector for gene delivery into the brains of living rats. We demonstrate that HSV-1 amplicon vectors expressing enhanced green fluorescent protein (EGFP) can reliably infect neurons after it is injected into cortex, striatum and thalamus in rats, producing sufficient numbers of infected neurons for electrophysiological experiments in acute brain slices. Expression of EGFP delivered by the HSV-1 amplicon was detected for up to 5 weeks post-infection. We detected no changes in the morphology or the electrophysiological properties of thalamic, striatal or cortical neurons within a period of at least 2 weeks after HSV-1 amplicon injection. We conclude that the HSV-1 amplicon is a valuable tool for gene delivery in the rat central nervous system.

Animals↗
PubMed2002

Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain.

Previous studies demonstrated that the rat neuron-specific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken beta-actin hybrid (CBA) promoter-based systems. NSE promoter-driven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10(7) particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted detection of cells distal to the injection site that had retrogradely transported the vector from their terminal areas. Incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into a CBA promoter vector induced greater expression levels in the hippocampus, as measured by stereological estimates of cell numbers and by Western blots, which demonstrated an 11-fold increase. Incorporation of the WPRE also improved transgene expression in primary neuronal cultures. The increased efficiency obtained with vector elements such as the CBA promoter and the WPRE may enhance the ability to genetically modify larger portions of the brain while requiring smaller doses and volumes.

Actins↗