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Biomedical subjects

David Brown

Publications and source records attributed to David Brown.

At least 37 records · Page 2Linked to original sources
PubMed2005

An optimized isolation and labeling platform for accurate microRNA expression profiling.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than ~40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from <1 mug of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.

Adult↗
PubMed2005

Induction of prosurvival molecules by apoptotic stimuli: involvement of FOXO3a and ROS.

Most cancer therapeutics fails to eradicate cancer because cancer cells rapidly develop resistance to its proapoptotic effects. The underlying mechanisms remain incompletely understood. Here we show that three representative apoptotic stimuli, that is, serum starvation, a mitochondrial toxin, and a DNA-damaging agent (etoposide), rapidly induce several distinct classes of prosurvival molecules, in particular, Bcl-2/Bcl-X(L) and superoxide dismutase (SOD; including both MnSOD and Cu/ZnSOD). At the population level, the induction of these prosurvival molecules occurs prior to or concomitant with the induction of proapoptotic molecules such as Bim and Bak. Blocking the induction using siRNAs of the prosurvival or proapoptotic molecules facilitates or inhibits apoptosis, respectively. One master transcription factor, FOXO3a, is involved in the transcriptional activation of some of these prosurvival (e.g., MnSOD) and proapoptotic (e.g., Bim) molecules. Interestingly, in all three apoptotic systems, FOXO3a itself is also upregulated at the transcriptional level. Mechanistic studies indicate that reactive oxygen species (ROS) are rapidly induced upon apoptotic stimulation and that ROS inhibitors/scavengers block the induction of FOXO3a, MnSOD, and Bim. Finally, we show that apoptotic stimuli also upregulate prosurvival molecules in normal diploid human fibroblasts and at subapoptotic concentrations. Taken together, these results suggest that various apoptotic inducers may rapidly mobilize prosurvival mechanisms through ROS-activated master transcription factors such as FOXO3a. The results imply that effective anticancer therapeutics may need to combine both apoptosis-inducing and survival-suppressing strategies.

Apoptosis↗
PubMed2005

RAS is regulated by the let-7 microRNA family.

MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.

3' Untranslated Regions↗
PubMed2005

SiRNA strategy against overexpression of extracellular matrix in diabetic retinopathy.

Increased synthesis of extracellular matrix (ECM) contributes to the development of vascular BM thickening, a prominent abnormality in diabetic retinopathy. RNA interference (RNAi) approach was used in this study to examine the effect of small interfering RNAs (siRNAs) for their ability to inhibit ECM-specific gene overexpression under high glucose condition in rat microvascular endothelial cells (RMECs). Four fibronectin (FN)-siRNAs, three collagen IV (Coll IV)-siRNAs, and four laminin (LM)-siRNAs, a total of 11 siRNAs were screened. RMECs were transfected with 10, 30, or 100 nm of each siRNAs in the presence of 8 microm lipofectin and subjected to analysis 72 hr after transfection. In long-term studies siRNA-transfected cells were examined after 12 days. Two FN siRNAs, two Coll IV siRNAs, and two LM siRNAs significantly reduced the respective target expressions. Findings from this study indicate that high glucose-induced abnormal expression of BM components may contribute to increased vascular permeability. SiRNA may be a useful tool in preventing excess vascular permeability, a characteristic feature of early diabetic retinopathy.

Animals↗
PubMed2005

Development and evaluation of Varicella zoster virus ELISA for oral fluid suitable for epidemiological studies.

An enzyme-linked immunosorbent assay (ELISA) for detection of Varicella zoster virus (VZV) antibodies in oral fluid is described. The assay was optimised and evaluated using paired serum and oral fluid from healthy adult volunteers (n = 205) and preschool children (n = 98), oral fluid samples collected for routine measles, mumps and rubella testing (n = 537) and samples from a study of atopic dermatitis (n = 252). As chickenpox is predominantly a childhood disease and most adults are immune, it was crucial to have samples from children aged 1-5 years to evaluate the assay. Mixture modelling of the oral fluid results was used to determine the optimum cut-off, sensitivity and specificity of the assay. Compared to paired sera tested by the same ELISA the sensitivity of the oral fluid assay was 93% and specificity 95.7% overall, varying slightly with age group. The assay was shown to have good potential for use in large-scale epidemiological studies.

Antibodies, Viral↗
PubMed2005

High-throughput RNAi screening in vitro: from cell lines to primary cells.

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.

Animals↗
PubMed2005

Personalized exposure assessment: promising approaches for human environmental health research.

New technologies and methods for assessing human exposure to chemicals, dietary and lifestyle factors, infectious agents, and other stressors provide an opportunity to extend the range of human health investigations and advance our understanding of the relationship between environmental exposure and disease. An ad hoc Committee on Environmental Exposure Technology Development was convened to identify new technologies and methods for deriving personalized exposure measurements for application to environmental health studies. The committee identified a "toolbox" of methods for measuring external (environmental) and internal (biologic) exposure and assessing human behaviors that influence the likelihood of exposure to environmental agents. The methods use environmental sensors, geographic information systems, biologic sensors, toxicogenomics, and body burden (biologic) measurements. We discuss each of the methods in relation to current use in human health research; specific gaps in the development, validation, and application of the methods are highlighted. We also present a conceptual framework for moving these technologies into use and acceptance by the scientific community. The framework focuses on understanding complex human diseases using an integrated approach to exposure assessment to define particular exposure-disease relationships and the interaction of genetic and environmental factors in disease occurrence. Improved methods for exposure assessment will result in better means of monitoring and targeting intervention and prevention programs.

Biomarkers↗
PubMed2005

Alloys for metal-ceramic restorations.

UNLABELLED: The requirements of a metal-ceramic dental restoration consisting of a rigid substrate, which supports an aesthetic veneer, are assessed. The contemporary, cast, bonding alloys that have good track records as substrates are categorized and their properties are compared. Several alternatives to cast metals are considered. CLINICAL RELEVANCE: Despite developments in all-ceramic restoration technology, the metal-ceramic dental restoration still has a significant place in the dentist's armamentarium.

Dental Alloys↗
PubMed2004

Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant.

BACKGROUND: Highly publicised outbreaks of norovirus gastroenteritis in hospitals in the UK and Ireland and cruise ships in the USA sparked speculation about whether this reported activity was unusual. METHODS: We analysed data collected through a collaborative research and surveillance network of viral gastroenteritis in ten European countries (England and Wales were analysed as one region). We compiled data on total number of outbreaks by month, and compared genetic sequences from the isolated viruses. Data were compared with historic data from a systematic retrospective review of surveillance systems and with a central database of viral sequences. FINDINGS: Three regions (England and Wales, Germany, and the Netherlands) had sustained epidemiological and viral characterisation data from 1995 to 2002. In all three, we noted a striking increase in norovirus outbreaks in 2002 that coincided with the detection and emergence of a new predominant norovirus variant of genogroup II4, which had a consistent mutation in the polymerase gene. Eight of nine regions had an annual peak in 2002 and the new genogroup II4 variant was detected in nine countries. Also, the detection of the new variant preceded an atypical spring and summer peak of outbreaks in three countries. INTERPRETATION: Our data from ten European countries show a striking increase and unusual seasonal pattern of norovirus gastroenteritis in 2002 that occurred concurrently with the emergence of a novel genetic variant. In addition to showing the added value of an international network for viral gastroenteritis outbreaks, these observations raise questions about the biological properties of the variant and the mechanisms for its rapid dissemination.

Caliciviridae Infections↗
PubMed2004

Synthesis of flexible sulfur-containing heteroarotinoids that induce apoptosis and reactive oxygen species with discrimination between malignant and benign cells.

Regulation of growth, differentiation, and apoptosis by synthetic retinoids can occur through mechanisms that are dependent and independent of their ability to bind and activate nuclear retinoic acid receptors. The objective of this study was to determine if increasing flexibility of the heteroarotinoid structure would affect the specificity of the synthetic retinoids for the receptors and for their regulation of cancerous and nonmalignant cells. Methods were developed to produce the first examples of heteroarotinoids 15a-15h, which contain urea and/or thiourea linking groups between two aryl rings. Substituents at the para position of the single phenyl ring were either an ester, a nitro group, or a sulfonamide group. Ovarian cancer cell lines Caov-3, OVCAR-3, SK-OV-3, UCI-101, and 222 were utilized, and the inhibitory prowess of the heteroarotinoids was referenced to that of 4-HPR (25). Similar to 4-HPR (25), the heteroarotinoids inhibited growth of all cell lines at micromolar concentrations. Although the heteroarotinoids did not activate retinoic acid receptors, the agents induced potent growth inhibition against the cancer cells with weak activity against normal and benign cells. The growth inhibition was associated with cell loss and induction of reactive oxygen species.

Antineoplastic Agents↗
PubMed2004

Effect of rocker soles on plantar pressures.

OBJECTIVE: To examine the effect of different types of rocker soles on plantar pressures. DESIGN: In-shoe plantar pressures were measured in subjects without deformity with baseline shoes and 3 types of rockers: toe-only, negative heel, and double. SETTING: Medical college. PARTICIPANTS: Forty healthy patients (20 men, 20 women) without foot deformity. INTERVENTIONS: Plantar pressures were recorded over a 2 1/2-hour test period with over 400 steps analyzed for each type of rocker sole. Peak pressures, pressure-time integral (PTI), and sensor contact duration were computed for each step. MAIN OUTCOME MEASURES: Peak plantar pressure PTI, and contact duration were compared for each rocker with a baseline shoe. RESULTS: Significant reduction (P<.01) in peak pressure and PTI were recorded across the forefoot for all 3 rockers. The reduction of pressure at the forefoot was balanced by shifting pressure to the midfoot with the negative heel and toe-only rockers. CONCLUSION: This study lends scientific credence to the prescription of rocker soles for patients who need forefoot pressure reduction, such as in diabetic neuropathy and possible ulceration.

Diabetic Neuropathies↗
PubMed2004

Seroepidemiology of group C rotavirus infection in England and Wales.

A total of 3199 serum samples collected in 1993--1994 from two population cohorts from England and Wales were tested for the prevalence of IgG antibodies specifically directed against group C rotavirus VP6. Seroprevalence was 39% (95% confidence intervals: 37.0-40.4%). Seroprevalence was highest (46.0%) in the oldest age group (61-70 years of age). Infection with group C rotaviruses occurred at an earlier age and with higher incidence in rural than in urban populations. These results may suggest transmission from animals to humans, however further work is required to identify the reservoir of group C rotavirus for human infection.

Adolescent↗
PubMed2004

Evolution of hepatitis B virus during primary infection in humans: transient generation of cytotoxic T-cell mutants.

BACKGROUND & AIMS: Acute hepatitis B is a highly dynamic human viral infection during which the hepatitis B virus can generate many genetic variants. METHODS: We analyzed the evolution of the hepatitis B virus genome in sequential serum samples from a unique cohort of patients with acute infection acquired from a single source. RESULTS: We showed that most mutations were nonsynonymous, that genetic diversity was greatest at the peak of viremia, and that patients who resolved their infection ("resolvers") showed a significantly higher level of diversity in the core, surface, and polymerase genes compared with those who progressed to chronic infection. Overall, the core gene showed the greatest genetic diversity. In resolvers who possessed an HLA-A*0201 haplotype, the emergence of mutants in the immunodominant HLA-A*0201-restricted core 18-27 epitope was observed. Functional studies showed that these mutants were less able to stimulate interferon-gamma release from core 18-27 specific CD8 + T-cell lines. However, they appeared only as a transient low-abundance species and were rapidly displaced by wild-type sequences before resolution of infection, and their overall significance is uncertain. CONCLUSIONS: Overall, genetic evolution of the hepatitis B virus differs at early time points between patients who experience acute resolving hepatitis B and those who progress to chronicity. These observations suggest that the rapid development of broadly reactive host immune responses leads to clearance of hepatitis B virus, even in the presence of possible CD8+ T-cell immune escape variants.

Biological Evolution↗
PubMed2004

Musculoskeletal injection skills competency in physical medicine and rehabilitation residents: a method for development and assessment.

An educational module was developed in attempt to satisfy two main objectives: to develop and implement a methodology for instruction of generally encountered injection techniques in the practice of physical medicine and rehabilitation and to implement an objective assessment format to measure attainment of these skills. Guidelines for this module were developed in consideration of Accreditation Council for Graduate Medical Education (ACGME) physical medicine and rehabilitation residency program requirements and ACGME Outcome Project. Eleven physical medicine and rehabilitation residents participated in a 1-day didactic/laboratory workshop covering injection education and techniques, followed by a supervised 2-wk practice session. Knowledge acquisition and skill attainment were measured via before and after multiple-choice written examination and simulated patient encounters utilizing anatomic models. An increase in knowledge was observed on the multiple-choice examination from pretest to posttest (pretest, 59.3%; posttest, 90.6%). Before instruction, 0 of 11 residents were able to perform any of the injection techniques (14 injection techniques and one sterile technique). At completion of the module, 7 of 11 residents (64%) competently performed 15 of 15 techniques (100%), 3 of 11 (26%) competently performed 14 of 15 techniques (93%), and 1 of 11 (9%) competently performed 11 of 15 techniques (73%). Participation in this module resulted in substantial acquisition of knowledge and skills regarding musculoskeletal injections for physical medicine and rehabilitation residents. This workshop provided a framework for the definition of baseline competency in this clinical skill area.

Attitude of Health Personnel↗
PubMed2004

Validity of the alcohol use disorders identification test in college students.

BACKGROUND: High-risk alcohol use among college students is associated with accidents, partner violence, unwanted sexual encounters, tobacco use, and performance issues. The identification and treatment of high-risk drinking students is a priority for many college campuses and college health centers. The goal of this study was to test the psychometric properties of the Alcohol Use Disorders Identification Test (AUDIT) in college students. METHODS: A convenience sample of students coming into a college health clinic was asked to complete the 10-question AUDIT and then participate in a research interview. The interview focused on assessing students for alcohol abuse and dependence by using the Composite International Diagnostic Interview Substance Abuse Module and timeline follow-back procedures to assess a 28-day drinking history. RESULTS: A total of 302 students met the eligibility criteria and agreed to participate in the study. The sample consisted of 185 females (61%) and 117 males (39%), with a mean age of 20.3 years. Forty students were abstinent, 88 were high-risk drinkers, and 103 met criteria for a 12-month history of dependence. Receiver operator curves demonstrated that the AUDIT had the highest area under the cure for detecting high-risk alcohol use (0.872) and the lowest for identifying persons with a lifetime history of alcohol abuse or dependence (0.775). An AUDIT cutoff score of 6 or greater demonstrated a sensitivity of 91.0% and a specificity of 60.0% in the detection of high-risk drinkers. CONCLUSIONS: The AUDIT has reasonable psychometric properties in sample of college students using student health services. This study supports the use of the AUDIT in this population.

Adolescent↗