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1.  Dietary and genetic manipulations of folate metabolism differentially affect neocortical functions in mice 
Converging evidence suggests that folate-mediated one-carbon metabolism may modulate cognitive functioning throughout the lifespan, but few studies have directly tested this hypothesis. This study examined the separate and combined effects of dietary and genetic manipulations of folate metabolism on neocortical functions in mice, modeling a common genetic variant in the MTHFD1 gene in humans. Mutant (Mthfd1gt/+) and wildtype (WT) male mice were assigned to a folate sufficient or deficient diet at weaning and continued on these diets throughout testing on a series of visual attention tasks adapted from the 5-choice serial reaction time task. WT mice on a deficient diet exhibited impulsive responding immediately following a change in task parameters that increased demands on attention and impulse control, and on trials following an error. This pattern of findings indicates a heightened affective response to stress and/or an inability to regulate negative emotions. In contrast, Mthfd1gt/+ mice (regardless of diet) exhibited attentional dysfunction and a blunted affective response to committing an error. The Mthfd1gt/+ mice also showed significantly decreased expression levels for genes encoding choline dehydrogenase and the alpha 7 nicotinic cholinergic receptor. The effects of the MTHFD1 mutation were less pronounced when combined with a deficient diet, suggesting a compensatory mechanism to the combined genetic and dietary perturbation of folate metabolism. These data demonstrate that common alterations in folate metabolism can produce functionally distinct cognitive and affective changes, and highlight the importance of considering genotype when making dietary folate recommendations.
PMCID: PMC5096640  PMID: 23684804
Folate; MTHFD1; Attention; Impulsivity; Affect
2.  Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model 
Diabetologia  2012;56(3):644-653.
We sought to determine the impact of longstanding type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells.
Wild-type, gp130−/− and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp+ chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood.
BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b+/CD45hi/CCR2+/Ly6Chi inflammatory monocytes. Diabetic gp130−/− mice were protected from development of diabetes-induced changes in their HSCs.
The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.
PMCID: PMC3773610  PMID: 23192694
Bone marrow microenvironment; Endothelial progenitors; GP130 deletion; Haematopoietic stem cells; IL-1β and IL-10; MMPs; Monocytes; Side population cells; Type 1 diabetes
3.  Analysis of PRA1 and Its Relationship to Candida albicans- Macrophage Interactions▿ †  
Infection and Immunity  2008;76(9):4345-4358.
Phagocytosis of Candida albicans by either primary bone marrow-derived mouse macrophages or RAW 264.7 cells upregulated transcription of PRA1, which encodes a cell wall/membrane-associated antigen previously described as a fibrinogen binding protein. However, a pra1 null mutant was still able to bind fibrinogen, showing that Pra1p is not uniquely required for fibrinogen binding. As well, Pra1 tagged with green fluorescent protein did not colocalize with AlexaFluor 546-labeled human fibrinogen, and while PRA1 expression was inhibited when Candida was grown in fetal bovine serum-containing medium, Candida binding to fibrinogen was activated by these conditions. Therefore, it appears that Pra1p can play at most a minor role in fibrinogen binding to C. albicans. PRA1 gene expression is induced in vitro by alkaline pH, and therefore its activation in phagosomes suggested that phagosome maturation was suppressed by the presence of Candida cells. LysoTracker red-labeled organelles failed to fuse with phagosomes containing live Candida, while phagosomes containing dead Candida underwent a normal phagosome-to-phagolysosome maturation. Immunofluorescence staining with the early/recycling endosomal marker transferrin receptor (CD71) suggested that live Candida may escape macrophage destruction through the inhibition of phagolysosomal maturation.
PMCID: PMC2519410  PMID: 18625733
4.  A diffusion of innovations model of physician order entry. 
OBJECTIVE: To interpret the results of a cross-site study of physician order entry (POE) in hospitals using a diffusion of innovations theory framework. METHODS: Qualitative study using observation, focus groups, and interviews. Data were analyzed by an interdisciplinary team of researchers using a grounded approach to identify themes. Themes were then interpreted using classical Diffusion of Innovations (DOI) theory as described by Rogers [1]. RESULTS: Four high level themes were identified: organizational issues; clinical and professional issues; technology implementation issues; and issues related to the organization of information and knowledge. Further analysis using the DOI framework indicated that POE is an especially complex information technology innovation when one considers communication, time, and social system issues in addition to attributes of the innovation itself. CONCLUSION: Implementation strategies for POE should be designed to account for its complex nature. The ideal would be a system that is both customizable and integrated with other parts of the information system, is implemented with maximum involvement of users and high levels of support, and is surrounded by an atmosphere of trust and collaboration.
PMCID: PMC2243456  PMID: 11825150
5.  Multiple perspectives on physician order entry. 
OBJECTIVE: Describe the complex interplay of perspectives of physicians, administrators, and information technology staff regarding computerized physician order entry (POE) in hospitals. METHODS: Linstone's Multiple Perspectives Model provided a framework for organizing the results of a qualitative study done at four sites. Data from observation, focus groups, and formal and informal interviews were analyzed by four researchers using a grounded approach. RESULTS: It is not a simple matter of physicians hating POE and others loving it. The issues involved are both complex and emotional. All groups see both positive and negative aspects of POE. CONCLUSION: The Multiple Perspectives Model was useful for organizing a description to aid in understanding all points of view. It is imperative that those implementing POE understand all views and plan implementation strategies accordingly.
PMCID: PMC2243815  PMID: 11079838
7.  Factors affecting the diffusion of online end user literature searching. 
OBJECTIVES: The aim of this study was to identify factors that affect diffusion of usage of online end user literature searching. Fifteen factors clustered into three attribute sets (innovation attributes, organizational attributes, and marketing attributes) were measured to study their effect on the diffusion of online searching within institutions. METHODS: A random sample of sixty-seven academic health sciences centers was selected and then 1,335 library and informatics staff members at those institutions were surveyed by mail with electronic mail follow-up. Multiple regression analysis was performed. RESULTS: The survey yielded a 41% response rate with electronic mail follow-up being particularly effective. Two dependent variables, internal diffusion (spread of diffusion) and infusion (depth of diffusion), were measured. There was little correlation between them, indicating they measured different things. Fifteen independent variables clustered into three attribute sets were measured. The innovation attributes set was significant for both internal diffusion and infusion. Significant individual variables were visibility for internal diffusion and image enhancement effects (negative relation) as well as visibility for infusion (depth of diffusion). Organizational attributes were also significant predictors for both dependent variables. No individual variables were significant for internal diffusion. Communication, management support (negative relation), rewards, and existence of champions were significant for infusion. Marketing attributes were not significant predictors. CONCLUSIONS: Successful diffusion of online end user literature searching is dependent on the visibility of the systems, communication among, rewards to, and peers of possible users who promote use (champions). Personal image enhancement effects have a negative relation to infusion, possibly because the use of intermediaries is still seen as the more luxurious way to have searches done. Management support also has a negative relation to infusion, perhaps indicating that depth of diffusion can increase despite top-level management actions.
PMCID: PMC226524  PMID: 9934530
8.  Perceptions of house officers who use physician order entry. 
OBJECTIVE: Describe the perceptions of housestaff physicians about their experience using computerized physician order entry (POE) in hospitals. METHODS: Qualitative study using data from participant observation, focus groups, and both formal and informal interviews. Data were analyzed by three researchers using a grounded approach to identify patterns and themes in the texts. RESULTS: Six themes were identified, including housestaff education, benefits of POE, problems with POE, feelings about POE, implementation strategies, and the future of POE. CONCLUSION: House officers felt that POE assists patient care but may undermine education. They found that POE works best when tailored to fit local and individual workflow. Implementation strategies should include mechanisms for engaging housestaff in the decision process.
PMCID: PMC2232743  PMID: 10566403
9.  The dissemination of clinical practice guidelines over an intranet: an evaluation. 
This study compares two clinical practice guideline dissemination systems. It was hypothesized that placing guidelines on an intranet would make this information easier to retrieve. Retrieval time, retrieval accuracy, and ease of use were empirically evaluated. Sixteen clinicians from Kaiser Permanente volunteered to complete tasks that measured these variables. Time values were significantly longer for tasks completed with intranet guidelines (Intranet = 6.7 minutes, Paper = 5.7 minutes). Tasks completed with paper guidelines had a significantly higher percentage of perfect scores than those completed with the intranet (Paper = 85%, Intranet = 59%). There was no significant difference in reported ease of use. Simply placing clinical information on an electronic system does not guarantee that the information will be easier to retrieve. Such information needs to be fully integrated into the clinical decision making process. Computerizing guidelines may provide a necessary initial step toward this goal, but it does not represent the final solution.
PMCID: PMC2232537  PMID: 10566503
10.  Physician order entry in U.S. hospitals. 
OBJECTIVE: Determine the percent of U.S. hospitals where computerized physician order entry (POE) is available and the extent of its use. METHODS: A survey was sent to a systematic sample of 1,000 U.S. hospitals asking about availability of POE, whether usage is required, percent of physicians using it, and percent of orders entered by computer. RESULTS: About 66% do not have POE available. Of the 32.1% that have it completely or partially available, 4.9% require its usage, over half report usage by under 10% of physicians, and over half report that fewer than 10% of orders are entered this way. Analysis of comments showed that many hospitals have POE available for use by non-physicians only, but that they hope to offer it to physicians after careful planning. CONCLUSION: Most U.S. hospitals have not yet implemented POE. Complete availability throughout the hospital is rare, very few require its use, low percentages of physicians are actual users, and low percentages of orders are entered this way. On a national basis, computerized order entry by physicians is not yet widespread.
PMCID: PMC2232213  PMID: 9929217
11.  Factors affecting the diffusion of the Computer-Based Patient Record. 
A survey of the perceptions of 629 informatics experts representing 67 institutions with accredited schools of medicine was used to identify factors most important in implementing the Computer-Based Patient Record. A model outlined three theoretical factors: Innovation Attributes (attributes inherent in the CPR itself); Organizational Attributes; and Boundary-Spanning Attributes (related to marketing efforts). The model was explored using multiple regression techniques to delineate the relative importance of 15 variables within the three sets of factors and their effect on two measures of diffusion. The two dependent variables were internal diffusion, or spread of usage of the CPR, and infusion, or depth of usage. Data from the 144 respondents indicate that for diffusion, the organizational variables of "decision making" and "planning" had a significant impact, although the relation between "planning" and diffusion was negative. For infusion, the Innovation Attributes variable "visibility" was significant. The major implication is that successfully encouraging usage of the CPR entails attention and resources devoted to managing the organizational aspects of implementation.
PMCID: PMC2233519  PMID: 9357712
12.  Splicing signals are required for S-phase regulation of the mouse thymidylate synthase gene. 
Molecular and Cellular Biology  1996;16(1):376-383.
The thymidylate synthase (TS) gene is expressed at a much higher level in cells undergoing DNA replication than in nondividing cells. In growth-stimulated mammalian cells, TS mRNA content increases 10 to 20-fold as cells progress from G1 through S phase. However, the rate of transcription of the TS gene does not increase during this interval, indicating that the gene is regulated at the posttranscriptional level. We have shown that both the promoter of the mouse TS gene and TS introns are necessary (although neither is sufficient) for S-phase-specific regulation of TS mRNA content. In the present study, we examined in more detail the role of introns in regulating TS mRNA levels in growth-stimulated cells. TS minigenes that contain normal or modified introns were stably transfected into mouse 3T6 fibroblasts, and the regulation of the minigenes was compared with that of the endogenous TS gene. TS minigenes that contain TS intron 1 or 2 maintain S-phase regulation. Deletion of most of the interior of the introns had only minor effects on regulation. However, when splicing of the intron was inhibited by alteration of the splice donor and acceptor sites, the minigene was expressed at a constant level following growth stimulation. Minigenes consisting of the TS promoter linked to either a luciferase or a human beta-globin indicator gene were growth regulated when spliceable introns were included in the minigenes. However, when the introns were eliminated, the minigenes were expressed at a constant level. These observations indicate that the splicing reaction itself, rather than a control sequence within the intron, is important for growth-regulated expression of the TS gene. Possible mechanisms to account for the dual requirement for the TS promoter and intron splicing for proper regulation of the TS gene are discussed.
PMCID: PMC231012  PMID: 8524318
13.  The impact of IAIMS on the work of information experts. Integrated Advanced Information Management Systems. 
Integrated Advanced Information Management Systems (IAIMS) programs differ but have certain characteristics in common. Technological and organizational integration are universal goals. As integration takes place, what happens to those implementing the vision? A survey of 125 staff members, or information experts, involved in information or informatics at an IAIMS-funded institution was conducted during the last year of the implementation phase. The purpose was to measure the impact of IAIMS on the jobs of those in the library and related service units, and the computing, telecommunications, and health informatics divisions. The researchers used newly developed scales measuring levels of integration (knowledge of and involvement with other departments), customer orientation (focus on the user), and informatedness (changes in the nature of work beyond automation of former routines). Ninety-four percent of respondents indicated that their jobs had changed a great deal; the changes were similar regardless of division. To further investigate the impact of IAIMS on librarians in particular, a separate skills survey was conducted. The IAIMS librarians indicated that technology and training skills are especially needed in the new, integrated environment.
PMCID: PMC226064  PMID: 8547905
14.  Cross-site study of the implementation of information technology innovations in health sciences centers. 
An interpretive oral history technique was used to identify factors most important in the implementation stage of information technology innovation diffusion. Electronic mail, end user literature searching, and aspects of the computer-based patient record were the innovations selected for study at academic health sciences centers. Transcripts of thirty-four interviews with key individuals were analyzed to determine six categories of factors. Word counts were then used to determine underlying emphases. Analysis of variance tested whether there were significant differences in uses of words by categories of individuals, by those at different institutions, and when different innovations were described. Results indicate that the innovations themselves correlate significantly with different word categories, where category of individual and institution do not. Words related to the computer based patient record characterize further critical factors in implementing that particular innovation.
PMCID: PMC2579203  PMID: 8563400
15.  Can primary care physicians' questions be answered using the medical journal literature? 
Medical librarians and informatics professionals believe the medical journal literature can be useful in clinical practice, but evidence suggests that practicing physicians do not share this belief. The authors designed a study to determine whether a random sample of "native" questions asked by primary care practitioners could be answered using the journal literature. Participants included forty-nine active, nonacademic primary care physicians providing ambulatory care in rural and nonrural Oregon, and seven medical librarians. The study was conducted in three stages: (1) office interviews with physicians to record clinical questions; (2) online searches to locate answers to selected questions; and (3) clinician feedback regarding the relevance and usefulness of the information retrieved. Of 295 questions recorded during forty-nine interviews, 60 questions were selected at random for searches. The average total time spent searching for and selecting articles for each question was forty-three minutes. The average cost per question searched was $27.37. Clinician feedback was received for 48 of 56 questions (four physicians could not be located, so their questions were not used in tabulating the results). For 28 questions (56%), clinicians judged the material relevant; for 22 questions (46%) the information provided a "clear answer" to their question. They expected the information would have had an impact on their patient in nineteen (40%) cases, and an impact on themselves or their practice in twenty-four (51%) cases. If the results can be generalized, and if the time and cost of performing searches can be reduced, increased use of the journal literature could significantly improve the extent to which primary care physicians' information needs are met.
PMCID: PMC225885  PMID: 7772099
16.  Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene. 
Molecular and Cellular Biology  1993;13(3):1565-1571.
The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.
PMCID: PMC359468  PMID: 8095091
17.  A systems approach to planning biomedical information services. 
A systems approach to planning was applied within the Biomedical Information Communication Center at Oregon Health Sciences University when its User Services division launched a strategic planning effort. By looking at the choice subsystem, the organizational structure, and the behavioral subsystem, those engaged in planning attempted to assure that desired change would permeate the entire system. The challenge of applying a theoretically ideal planning model within an environment averse to planning is delineated.
PMCID: PMC2247578  PMID: 1666968
18.  Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells. 
Molecular and Cellular Biology  1989;9(1):116-123.
Measurements of internal ion concentrations, amino acid pools, and membrane potential were made across a series of HeLa subclones which are amplified for the genes for the sodium- and potassium-activated ATPase (Na,K-ATPase). These subclones expressed heterogeneous levels of ouabain-binding sites, allowing us to construct a graded amplification series. While [K+]i levels did not vary systematically across the series studied, [Na+]i ranged from 9 to 20 mM as a function of Na,K-ATPase expression. Steady-state accumulation of tetraphenylphosphonium in low versus high potassium was used to measure membrane potential. Values for [Na+]i and the membrane potential were used to calculate the sodium electrochemical potential, which was also found to be a function of Na,K-ATPase expression. Measurements of acid-soluble amino acid pools in cell lysates demonstrated that amino acids which are substrates for sodium-dependent transport systems, or which can potentially exchange through system L for a substrate of a sodium-dependent system, varied as a function of the sodium electrochemical potential. This confirmed our prediction of increased amino acid pool sizes in Na,K-ATPase-amplified lines based on observations of elevated flux through the sodium-independent system L. Finally, we measured lactate production and glycolytic potential in a subset of clones and found that both were reduced in subclones with elevated Na,K-ATPase.
PMCID: PMC362152  PMID: 2538714
19.  Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells. 
Molecular and Cellular Biology  1986;6(7):2476-2481.
We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.
PMCID: PMC367801  PMID: 3023935
20.  Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells. 
Molecular and Cellular Biology  1986;6(4):1164-1171.
Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.
PMCID: PMC367628  PMID: 3023874
22.  Regulation of mouse thymidylate synthase gene expression in growth-stimulated cells: upstream S phase control elements are indistinguishable from the essential promoter elements. 
Nucleic Acids Research  1995;23(22):4649-4656.
Expression of the mammalian thymidylate synthase (TS) gene in growth-stimulated cells is closely coordinated with entry into S phase. Previous studies with transfected TS minigenes have shown that sequences upstream of the coding region as well as an intron in the transcribed region are both necessary for proper regulation of TS mRNA content in growth-stimulated cells. The goal of the present study was to identify the upstream regulatory elements. Minigenes consisting of TS 5' flanking sequences linked to the TS coding region (interrupted by introns 1 and 2) were stably transfected into mouse 3T6 cells. Deletion and site-directed mutagenesis of the 5' flanking region revealed that there is a close correspondence between the upstream sequences that are necessary for S phase regulation and the 30 nucleotide region that is essential for promoter activity. These observations raised the possibility that regulation of the TS gene occurs at the transcriptional level. However, nuclear run-on assays showed that the rate of transcription of the TS gene changed very little during the G1-S phase transition. Furthermore, when the TS promoter was linked to an intron-less luciferase indicator gene, there was no change in expression following growth-stimulation. Therefore it appears that the TS gene is controlled primarily at the posttranscriptional level, and that the TS essential promoter region is necessary (although not sufficient) for proper S phase regulation.
PMCID: PMC307439  PMID: 8524656
23.  Bidirectional promoter of the mouse thymidylate synthase gene. 
Nucleic Acids Research  1994;22(20):4044-4049.
The promoter of the mouse thymidylate synthase (TS) gene lacks both a TATAA box and an initiator element and directs transcriptional initiation at multiple sites over a 90 nucleotide initiation window. Earlier studies defined an essential region near the 5' end of the initiation window that is required for promoter activity. The essential region contains possible binding sites for Sp1 and Ets transcription factors. In the present study we show that this essential region stimulates transcription with approximately equal strength in both directions. Transcription is initiated over a broad initiation window in the reverse direction. The same elements are important for the reverse promoter and for the normal TS promoter. Sequences upstream of the essential region partially suppress expression in the reverse direction. The TS 5' flanking region, in either the normal or inverted orientation, directs S phase-specific expression of a TS minigene. This raises the possibility that an upstream gene and the TS gene may be coordinately induced at the G1/S phase boundary by a common set of control elements.
PMCID: PMC331888  PMID: 7937129
24.  Concerning “A Central Medical Registry” 
PMCID: PMC1473595  PMID: 18747133

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