Learning the exact demographic characteristics of a neighborhood in which a public library serves, assists the collection development librarian in building an appropriate collection. Gathering that demographic information can be a lengthy process, and then formatting the information for the neighborhood in question becomes arduous.As society ages and the methods for health care evolve, people may take charge of their own health. With this prospectus, public libraries should consider creating a consumer health collection to assist the public in their health care needs. Using neighborhood demographic information can inform the collection development librarians as to the dominant age groups, sex, and races within the neighborhood. With this information, appropriate consumer health materials may be assembled in the public library. the data was manipulated with ArcView GIS and queried to produce maps displaying the requested neighborhood demographics to view in respect to libraries.In order to visualize the demographics of a neighborhood, the computer program ArcView GIS (geographic information systems) was used to create maps for specified areas. The neighborhood data was taken from the U.S. Census Department's annual census and library addresses were accumulated through a free database. After downloading the census block information from ArcView GIS produced maps displaying public libraries and requested demographics. After viewing the maps the collection development librarian can see exactly what populations are served by the library and adjust the library's collection accordingly.ArcView GIS can be used to produce maps displaying the communities that libraries serve, spot boundaries, be it "man-made or natural," that exist prohibiting customer service, and assist collection development librarians in justifying their purchases for a dedicated consumer health collection or resources in general. Libraries have the objective to build collections that support the communities they serve. To build a viable collection the collection development librarian, outreach/marketing librarian, and others, must determine exactly what populations reside in the neighborhood, in respect to race, spoken language, educational level, and age groups. One method collection development librarians use to gather demographic information is to physically visit the communities and integrate into the neighborhoods. They may attend events within the community in order to analyze the attendees or walk around the neighborhood to get a feel for the community. Another method for collection development is to perform an informal survey with people that visit the library and learn about their preferences and/or what they like to read or look for on the Internet. Using census information can be a third way to gather community/neighborhood information. The US Census Bureau takes a census of the United States every 10 years and publishes the results on the Internet. Taking the Census information and transforming it into a graphical format provides an objective view of the communities surrounding a library. One way to visualize the census data is through the use of GIS, Geographic Information Systems. GIS are databases arranged by spatial coordinates that, when programmed, can produce maps ."Many disciplines have used GIS and many large academic libraries even support GIS by establishing a GIS department and employing a librarian to specialize in GIS information. In the field of library science articles have been published describing the establishment of GIS departments in libraries and what GIS librarians do -7, but lGovernments use GIS to visualize land use planning, tax appraisal, utility and infrastructure planning and more . BusinesBuilding a collection for a library has become a sophisticated art. Johnson states that "collection building consists of four steps: identifying the relevant items, assessing the item to decide if it is appropriate for the collection and evaluating its quality, deciding to purchase, and preparing an order ." Each sThis paper reports the steps taken to use GIS in assisting a large metropolitan public library system in visualizing neighborhoods that surround branch libraries in order to make an educated decision on whether a dedicated consumer health collection should be established to support the community. The objective of the project was to determine if GIS could be used to improve collection development.After comparing large library systems in major metropolitan areas, (Chicago, Los Angeles, and New York City) Chicago was selected as a convenient sample to be mapped with GIS because of its size, number of public libraries, their geographic distribution within the urban area, and ease of access to data. Compared to other large cities, the New York Public Library system has 86 libraries and Los Angeles has 67 public libraries . The citWith the selection of the city of Chicago, the next step was to create a map of the metropolitan area. ESRI ArcGIS v9.0 geographic information software (GIS)  was seleIn order to create the city map of Chicago, data files were obtained from the U.S. Census Bureau website. The Census Bureau produces TIGER  Files available for free to the public . These fThe next step required mapping distances between neighborhoods and neighborhood demographics. In order to achieve this, files were obtained from the U.S. Census Bureau website  which di. The output of this search was imported into Excel, cleaned, and converted into a dBase file. The dBase file was imported into ArcView to coordinate the library addresses to the street addresses of the base map. This enabled the software to place an image for each public library in the city map of Chicago.The final piece of information needed to build a map showing neighborhoods in respect to public library locations, was to map the public libraries within Chicago. Because the base map of Cook County was a street map, addresses of public libraries were entered into ArvView to mark all the public libraries within the city of Chicago. The state of Illinois provides a database available on the Internet that permits searching types of libraries and locations of libraries, With the city of Chicago and its public libraries now in a GIS application, the location of libraries was displayed in respect to neighborhoods and distances from library to library . The age ranges presented in the maps varied by query and census information. The groups analyzed were: women, men, white women (group age 40–49), African American women (group age 40–49), Asian (men/women, Latinos (men/women). The color schemes used represent the number of people that live within the census block as to the specified GIS query. In Figure In this project ArcGIS was used to build maps of neighborhoods in the city of Chicago, display public libraries and spatially represent distances from libraries to populations. The US Census demographic information was incorporated into ArcGIS and queried to produce 15 different maps. Queries were built to display neighborhood demographics through colors showing the percentages of the population(s). Each query produced a map for a specific age or race. Once the map was produced the public libraries were added to the map and analysis was made regarding collection needs according to the neighborhood population surrounding the library.To decide if a public library should establish a consumer health collection, a map showing the breakdown of women in their 40s was built. As in Figure By creating maps showing the demographic breakdown of the city, a public library can build, adjust, or verify its collection in respect to the neighborhoods it supports . It is eMany studies have been conducted analyzing what people search for on the Internet and who is searching. Studies have found that women in their 40s search for and utilize health information from the Internet more than men -24. UsinUsing ArcGIS can assist in defining the exact make-up of the population that a library serves. By turning the census information into a graphic the library is provided a chance to see how far  their services may reach. Library systems that have branch libraries may have one collection development librarian who buys for each library. By using ArcGIS the central library can analyze the communities surrounding each library to purchase materials appropriately without having to spend time in each library learning the environment. Purchase decisions may now be justified with hard data from ArcGIS. For example, if ArcGIS shows that the largest population surrounding a library speaks a minority language, materials should be purchased in the dominant language to best meet the needs of the community.There is a fairly high learning curve to use ESRI Arcview GIS. There are five programs in ESRI Arcview and each program has separate features that must be used within the specific program to be imported into the primary map. Learning what each program does and then how to incorporate the data into the map requires hours of reading. Acquiring the necessary data to build a map requires background study of the geographic area in order to assure that the map is configured correctly. Upon attaining all necessary data for the map, one must build queries to manipulate the data according to the information need, and then configure the map accordingly.While GIS presents a new technique to use in performing collection development, there was not a way to test the differences from a collection built using GIS information and the current methods of collection development.In addition, research has discovered that knowing the service area radius of the library doesn't mean that people within the service area will actually use or belong to the neighborhood library ,25. WhenWhile GIS can take the census information and demonstrate the population surrounding a library, the census does not describe the specific users of the library . To builBudget constraints will add a need for justification of purchases for a library's collection. Using GIS may provide one method to justify additions to collections. As health care changes and individuals take more control of their health, libraries need to have materials that provide valid information in an age and language appropriate format and be culturally sensitive. Branch libraries respond to the demands of the neighborhood  and knowGIS = geographical information systems


Chronic ethanol exposure has been shown to result in changes in neuronal cyto-architecture such as aberrant sprouting and alteration of neurite outgrowth. In PC12 cells, chronic ethanol treatment produces an increase in Nerve Growth Factor (NGF)-induced neurite outgrowth that appears to require the epsilon, but not delta, isoform of Protein Kinase C (PKC). Neurites contain a core of microtubules that are formed from polymerization of free-tubulin. Therefore, it would be expected that an increase in neurite outgrowth would correlate with an increase in microtubule content. We examined the effect of chronic ethanol exposure on microtubule content in PC12 cells and the role of PKC epsilon and delta in ethanol's effect on microtubule levels.Chronic ethanol exposure of wild-type and vector control PC12 cells resulted in a significant increase in microtubule content and a corresponding decrease in free tubulin. There was also a significant increase in microtubule content in PC12 cells expressing a dominate-negative inhibitor of epsilon PKC; cells which have previously been shown to have no ethanol-induced increase in neurite outgrowth. In contrast, ethanol had no effect on microtubule content in PC12 cells expressing a dominate-negative inhibitor of delta PKC.These results suggest that chronic ethanol exposure alters the relative ratio of free tubulin to microtubule-associated tubulin, an important component of the cytoskeleton. Further, the data from the PKC dominant-negative cell lines suggest that the effects of ethanol on microtubule content do not correlate with the effects of ethanol on neurite outgrowth. The delta isoform of PKC appears to be necessary for the ethanol-induced increase in microtubule content. These studies demonstrate an effect of chronic ethanol exposure which may contribute to previously documented alterations of neuronal cyto-architecture. For eing rats , increasing rats , the sizing rats , and theing rats . Further neurons . Contrarin vitro ,9. HowevPC12 cells have been used as a cell culture model system to study the underlying mechanisms of ethanol's alteration of neurite outgrowth ,11. PC12Nerve growth factor-induced neurite outgrowth in PC12 cells involves an induction of microtubule assembly ,14. MicrSpecific isoforms of PKC have also been implicated in mediating NGF-induced neurite outgrowth. Using both antisense oligonucleotides and specific inhibitors of PKC delta, Corbit et al.  have demIt is possible that the enhancement of NGF-induced neurite outgrowth produced by chronic ethanol may be due to ethanol's known alterations of PKC signaling. Chronic ethanol has multiple effects on PKC, including altered PKC subcellular localization following chronic exposure ,29. MessIn this study, we examined the effect of chronic ethanol exposure on the neuronal microtubule cytoskeleton using PC12 cells as a model system. Here we show that chronic ethanol exposure increases microtubule content, while decreasing free-tubulin content. Thus, it appears that ethanol enhances microtubule polymerization in PC12 cells. We also investigated the role of microtubule polymerization in mediating ethanol's effects on neurite outgrowth using PC12 cells which over-express an inhibitory fragment of either delta or epsilon PKC. Importantly, it is the PKC epsilon isoform which is required for ethanol's enhancement of neurite outgrowth. Here, we found that the PKC delta isoform, but not the PKC epsilon isoform, is required for the enhancement of microtubule polymerization following treatment with chronic ethanol. Thus, it appears that neurite outgrowth does not correlate with enhanced microtubule polymerization in PC12 cells.9 = 5.2; p < 0.001; n = 10). Similarly, there was a significant decrease (about 15%) in free-tubulin concentration  following 96 hours of ethanol exposure. There was no effect on total tubulin expression following four days of chronic ethanol exposure .For the following studies, we used 100 mM ethanol for four days as a chronic exposure; a dose and duration used by previous researchers to demonstrate ethanol's enhancement of neurite outgrowth [5 = 7.15; p < 0.001; n = 6) and decreased tubulin content  (similar to control PC12 cells). In the cells which express the inhibitor of PKC delta (DND cells), there was no significant effect on microtubule  or tubulin  content. Ethanol had no effect on total tubulin content in either the DNE (t5 = 0.2) or DND cells (t5 = 0.013). There was no difference between the control vector transfected PC12 cells and wild-type PC12 cells, thus these groups were combined and expressed in Figure We next used PC12 cells which over-express the first variable domain of PKC epsilon or delta, which acts as an isozyme specific inhibitor of PKC epsilon or delta , respectCells maintain a balance between free tubulin in the cytoplasm and tubulin which is polymerized into microtubules of the cytoskeleton. In this study, we find that chronic ethanol exposure increases microtubule content while decreasing free tubulin content in PC12 cells. Ethanol appears to be enhancing polymerization of tubulin into microtubules. While there was no increase in total tubulin within the cells, there was a change in the proportion of tubulin in the polymerized (microtubule) versus non-polymerized state.We initially hypothesized that the increase in microtubule content was due to ethanol's enhancement of neurite outgrowth. Presumably, neurite outgrowth is not due to simple stability of microtubules, but to an increase in dynamic microtubule growth. Microtubules are particularly abundant along the axons of nerve cells and multiple labs have show that chronic ethanol exposure increases NGF-induced neurite outgrowth in PC12 cells ,11. TherOur data from experiments utilizing PC12 cells which express dominant negative inhibitors of PKC do not seem to support the idea that the increased microtubule content reflects increased neurite outgrowth. Hundle et al.  have demHundle et al  has showBased on our data, we speculate that ethanol's enhancement of microtubule polymerization may involve phosphorylation of tubulin by PKC. However, it should be noted that we have, thus far, not directly measured phosphorylation of tubulin. Studies have shown that chronic ethanol increases expression of delta and epsilon PKC in PC12 cells  and PKC Our studies demonstrate that chronic ethanol alters the relative ratio of free versus microtubule-associated tubulin content in PC12 cells, resulting in an increase in microtubule content and a corresponding decrease in free tubulin. This alteration was found to occur in wild type cells, as well as those expressing a dominant-negative inhibitor of epsilon PKC but not in cells expressing a dominant-negative inhibitor of delta PKC. These ethanol-induced changes could be important during activity-dependent remodeling of synapses or developmental growth of axons and dendrites which may lead to cognitive dysfunction.Wild type PC12 cells were a gift from Nicholas Pantazis, Ph.D (University of Iowa). PC12 cell lines expressing dominant negative inhibitors of delta or epsilon PKC were a gift from Robert Messing, M.D . RPMI, Dulbecco's Modified Eagle Medium, and PenStrep were purchased from Gibco, NGF from R&D Systems, horse and fetal bovine serum was purchased from Hyclone, and laminin was purchased from Invitrogen. The microtubule assay kit was purchased from Cytoskeleton, Inc (#BK038).PC12 cells were cultured in RPMI medium supplemented with 10% horse serum, 5% fetal bovine serum, and 1% PenStrep. Medium for the dominant-negative PC12 cells also included G418 (250 μg/ml) for selection purposes. The dominant-negative lines are cells that have been transfected to express isozyme specific dominant-negative inhibitors of delta or epsilon PKC and have been used to show specific PKC isozyme involvement in multiple ethanol effects ,33. Thes2. For all experiments, PC12 cells were plated into six-well, laminin-coated (5 μg/ml) plates at a density of 270,000 cells/well. The cells were differentiated into neuronal-like cells with Nerve Growth Factor (25 ng/ml) for four days prior to addition of any chronic drug exposure. For chronic exposures, ethanol (100 mM) was added to the culture media  for 96 hours and plates (control and ethanol) were wrapped in parafilm to prevent ethanol evaporation. Media were changed every other day.The cells were maintained in an incubator at 37°C in 5% CO2 (200 μM) and incubated for one hour. CaCl2 acts to enhance microtubule depolymerization. Thus, the microtubules remaining in the pellet were depolymerized to free-tubulin. The samples were then centrifuged at 14,000 × g (4°C) for 10 minutes. The supernatant  was collected and frozen. Tubulin concentrations in both fractions were measured using Western blotting as described below.Measurement of microtubule and free-tubulin contents in cells are well-established assays. The materials are available as a commercially kit (Cytoskeleton, Inc). Microtubules are very sensitive to changes in temperature; therefore, all equipment and buffers were warmed to 37°C before use (unless otherwise indicated). To prevent free tubulin from polymerizing onto existing microtubules during the assay, lysis buffer was added at a ratio of 10 volumes of buffer to 1 volume of cell pellet. Cells were homogenized via syringe trituration and incubated for 10 minutes in lysis buffer (contents listed below). 10 μL of cell homogenates were saved for protein measurement using the Bradford Assay. Homogenized cells were then centrifuged at 100,000 × g for 30 minutes to separate microtubules from free-tubulin. The polymerized microtubules settle in the pellet, while the free-tubulin remains in the supernatant. Following centrifugation, the supernatant (free-tubulin) was removed and frozen until Western blot analysis. The pellet was resuspended in ice-cold water containing CaClLysis buffer: LMS1 solution containing GTP (100 mM) + ATP (100 mM) + protease inhibitor cocktail (10 μM) + Okadaic Acid (100 nM).LMS1: PIPES (100 mM) containing MgCl2 (5 mM) + EGTA (1 mM) + glycerol (30%)+ Nonidet P40 (0.1%) + Triton X-100 (0.1%) + Tween 20 (0.1%) + beta-mercapto-ethanol (0.1%) + Antifoam (0.001%).Protease inhibitor cocktail: Pepstatin (1 μg/ml) + Leupeptin (1 μg/ml) + benzamidine (10 μg/ml) + tosyl arginine methyl ester (500 μg/ml).Western blot analysis was used to determine tubulin content. Samples for Western blot were boiled with electrophoresis sample buffer (containing 25% glycerol, 5% beta-mercaptoethanol, 2% SDS, and 0.01% bromophenol blue in 62.5 mM Tris-HCl, pH 6.8). Equal amounts of protein (25 μg) were separated by SDS-PAGE using a 12% polyacrylamide gel. Molecular weight standards were also loaded. The separated proteins were transferred to nitrocellulose membranes using an electroblotting apparatus. The membrane was blocked [Tris-Buffered Saline (TBS) containing 4% nonfat dry milk and 0.05% Tween-20] for 20 minutes at room temperature. Membranes were then incubated in primary antibody  overnight at 4°C. Following incubation in the primary antibody, membranes were washed with TBS/0.05% Tween-20 and incubated in secondary antibody . Membranes were again washed and tubulin bands were visualized by Enhanced Chemiluminescence (ECL) using standard luminol reagents captured using a BioRad Gel Doc 1000. The computer analysis software Molecular Analyst  was used to quantify tubulin concentrations . All blots were very clean and no filtering was needed.Tubulin concentrations in both the free-tubulin and microtubule fractions were expressed as a percent control relative density and presented as mean (+/-SEM). The data represent multiple replicates of the same experiment . Student's t-tests were used to make comparisons between control and ethanol-treated PC12 cells.Each individual band analyzed was an independent extract from the same experiment, with the experiment being replicated 5 times. Each replication of the experiment was composed of 2 separate wells of cells for each treatment condition. For each of the 5 replications, the 2 control bands were averaged and each sample, including the controls, from that replication normalized to the average. The data was combined after all 5 replications.CKR-F conceived and performed the experiments and participated in writing the manuscript. DPD participated in the design of the study and writing of the manuscript. Both authors read and approved the final manuscript.


High prevalence rates for rheumatoid arthritis, spondyloarthopathies, and systemic lupus erythematosus have been described in American Indian and Alaskan Native adults. The impact of these diseases on American Indian children has not been investigated.We used International Classification of Diseases-9 (ICD-9) codes to search two Indian Health Service (IHS) patient registration databases over the years 1998–2000, searching for individuals 19 years of age or younger with specific ICD-9-specified diagnoses. Crude estimates for disease prevalence were made based on the number of individuals identified with these diagnoses within the database.Rheumatoid arthritis (RA) / juvenile rheumatoid arthritis (JRA) was the most frequent diagnosis given. The prevalence rate for JRA in the Oklahoma City Area was estimated as 53 per 100,000 individuals at risk, while in the Billings Area, the estimated prevalence was nearly twice that, at 115 per 100,000. These rates are considerably higher than those reported in the most recent European studies.Chronic arthritis in childhood represents an important, though unrecognized, chronic health challenge within the American Indian population living in the United States. As a group, the rheumatic diseases of childhood represent one of the most common chronic disease conditions in children . These iPrevalence rates of rheumatic disease in North American Indian/First Nations populations have been reported in small studies from single tribes. From these studies, significantly higher prevalence rates for rheumatoid arthritis (RA) have been reported in adults from tribes living in the Great Lakes region , the PacTo our knowledge, a comprehensive survey of rheumatic diseases affecting children, adolescents, and young adults has not been reported in any non-Caucasian population. Because both our clinical experience here in Oklahoma suggests that rheumatic diseases in children may also be more prevalent in the American Indian population compared with Caucasians, we undertook a search of the Oklahoma City Indian Health Service (IHS) user population databases in order to develop prevalence estimates of rheumatic diseases in American Indian children and adolescents. We performed the same queries from the database in the Billings Area IHS office as a basis of comparison.The Oklahoma City Area IHS serves a population of 291,288 individuals, most of whom reside in Oklahoma, with small numbers living in the neighboring states of Kansas and Texas . IHS serThe Billings Area IHS serves a population of 72,591 individuals. The tribes in this service area, a significant proportion of whom live on 8 reservations located in Montana and Wyoming, consist largely of northern plains tribes (e.g., Crow, Sioux, Blackfeet).In both Areas, the population is younger than the population of the United States as a whole, with 40 percent of the population 19 years of age or younger .International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9) codes were used to search the Oklahoma City and Billings Area IHS National Patient Information Reporting Systems and Patient Registration user databases  over a tOutpatient data over this three-year period was gathered for the user population ≤ 19 years of age and included: patient chart number (with the chart number scrambled to protect patient identity), date of birth, sex, date of visit and diagnosis . The codes used and the diagnoses denoted by those codes are listed in Table A case was defined as any person who was 19 years of age or younger on January 1,1998, January 1, 1999 and January 1, 2000 and whose diagnoses included at least one of the entities listed in Table service population, which includes all individuals who are eligible to receive IHS services. Estimation of disease prevalence was based on three assumptions: (1) that the diagnoses recorded were, in fact, accurate; (2) that the population at risk did not change significantly over the three-year period; (3) that the three-year mortality rate for the diseases of interest was no greater in the IHS user population than for all races in the United States.The population at risk was defined as the number of individuals ≤19 years of age within the IHS user population  . The useFor the Oklahoma City Area, accuracy of the IHS database information was assessed by matching the IHS identification number of known JRA cases followed at the Children's Hospital of Oklahoma with the same number in the IHS patient databank to be sure that known patients were identified and coded accurately.et al, manuscript in preparation). The absence of a prevalence peak in the preschool years may reflect the almost complete absence of children with monoarticular or pauciarticular JRA in the IHS user population, consistent with previous studies in non-European populations [Rheumatoid arthritis and juvenile rheumatoid arthritis  were the most frequent rheumatic disease diagnoses recorded in individuals ≤ 19 years of age in both IHS areas. In the Oklahoma City Area, we identified 62 individuals  with these diagnoses. Assuming a population at risk of 117,409 , this gives a crude prevalence rate of 53 cases per 100,000 at risk. The prevalence rate for Billings was considerably higher. The 714.0 and 714.3 codes identified 33 individuals in the Billings Area. Based on a population at risk of 28,724, the prevalence estimate for Billings was 115 per 100,000 at risk  by IHS identification number with patients in the database. All 15 of the children followed at CHO were identified within the database and correctly identified by subtype.We did not have access to patient records in the Billings Area, but do have access to databases at other IHS facilities. At a large facility in the Aberdeen Area (comprising the states of North Dakota, South Dakota, Nebraska, and Iowa and serving a patient population very similar to that served in the Billings Area) we identified 20 children with JRA using the same search strategy as that used in for the Billings and Oklahoma City Areas. Subsequent chart review demonstrated that 17 of these children had strong clinical evidence to confirm the diagnosis of JRA, while diagnosis could not be supported or excluded in the other three. The estimated prevalence for JRA in the population served by this facility calculates to 236/100,000, within the same order of magnitude by considerably higher than the Billings Area estimate.The second most common diagnoses identified in each Area database were the group of illnesses collectively denoted spondyloarthropathies (#ICD-9 # 720.0, 720.1, 720.2, 720.8, 720.89). Although these illnesses are sometimes viewed as distinct entities, they share sufficient common features that allow them to be grouped for purposes of this analysis. These common features include: (1) male sex preponderance; (2) arthritic involvement of the axial skeleton (e.g., sacroiliac joints); (3) extra-articular musculoskeletal involvement (e.g., bursitis, enthesitis); and (4) extra-articular (e.g., ocular, genito-urinary) inflammation. In both white -38 and AWe identified 20 patients  with spondyloarthropathy in the Oklahoma City Area IHS database, giving an overall crude prevalence rate of 17 per 100,000 at risk. Included in this group are three patients  with psoriatic arthritis (ICD-9 Code # 696.0). In the Billings Area, we identified 12 individuals  with ICD-9 codes used to identify patients with spondyloarthopathy, yielding a prevalence rate of 42 per 100,000 at risk. These included one patient  with ankylosing spondylitis (#ICD-9 # 720.0), one  with psoriatic arthritis (#ICD-9 #696.0), and 10 patients  with nonspecific sacroiliitis (#ICD-9 # 720.2). The Oklahoma and Billings Area estimates were within the range previously reported for childhood spondyloarthropathies (e.g. ankylosing spondylitis) in the United States and United Kingdom (12 to 33 per 100,000) and Mexico (13 to 65 per 100,000) , but sliAlthough rare individually, the rheumatic diseases, taken together, are among the most common chronic health conditions affecting children . Exact pThe reasons for the discrepancy in prevalence estimates between the Oklahoma City and Billings Areas are not clear. One possibility is that the northern plains tribes are particularly susceptible to rheumatoid disease in ways that other groups (e.g., Eastern Woodlands or Southwestern tribes) are not. It is also possible that the difference in ethnic composition of the two populations accounts for this difference. While many Oklahoma tribes require at least a 25% blood quantum of tribal ancestry (e.g., the Kiowa tribe ), other Our study once again points out the rarity of the pauciarticular form of JRA in non-European populations. In studies of European and European-descended populations, pauciarticular JRA is the most common form of chronic childhood arthritis ,8.The Oklahoma City Area database listed a single child with ICD-9 codes #714.32 (pauciarticular JRA) or 714.33 (monoarticular arthritis), the codes used to identify such children. These findings are consistent with reports from the Indian subcontinent,, Turkey The slight female-to-male preponderance for spondyloarthopathy is also worth noting. High prevalence rates for spondyloarthopathies have been noted in both Northwestern and Southwestern tribes ,41-44. HAn important limitation to this study is the fact that we did not have the means to verify every individual case listed in Oklahoma City database and were unable to confirm any diagnosis in the Billings Area database. However, our limited test of the accuracy of the Oklahoma City data provided surprising confirmation of the accuracy of coding for known cases. While we could not confirm any of the Billings cases, our search of the database of a single IHS facility in the neighboring Aberdeen Area corroborated the prevalence statistics we derived from the Oklahoma City and Aberdeen databases. Indeed, our experience suggests that a search strategy like the one we used is likely to under-estimate rather than over-estimate the prevalence of rheumatic disease in the IHS user population.We are aware that there are many factors that might overestimate disease prevalence using this type of database search. The first is the possibility that a given ICD-9 code might have been used to designate a "working" diagnosis that was never established by the patient's clinical course. The second opportunity for overestimation of prevalence would occur if children were systematically misdiagnosed. This could occur easily if physicians use serologic data as the sole criterion for diagnosis. For example, many physicians routinely screen children with musculoskeletal complaints using antinuclear antibody (ANA) tests. However, the prevalence of low-titer positive ANA tests is extraordinarily high in the pediatric population . Thus, iSimilarly, there are factors that might have led to underestimation of JRA prevalence by relying solely on a three-year database search. Children or adolescents with well-controlled JRA may not have seen an IHS physician during the relevant time period, and thus would have been excluded. Similarly, physicians who rely on rheumatoid factor tests as a diagnostic criterion for JRA might fail to diagnose the disease in a child, since only a small number of children with JRA have detectable IgM rheumatoid factor .The ideal method for obtaining true disease prevalence rates would include rigorous, pro-active case finding in a known population at risk. This approach was taken by Manners and Diepeveen in a study of school children in Australia [61]. Using such an approach, these authors reported a prevalence rate of 4 per 1,000 for JRA, significantly higher than any previous estimates. We are now preparing a similar project involving American Indian communities on the northern plains.We conclude that the rheumatic diseases of childhood may represent a significant burden of morbidity in these two IHS user populations. More detailed studies with rigorous case ascertainment are required to follow up these preliminary data.None declared.Dan Cameron provided data from the IHS database in Oklahoma City, and Diane Jeannotte provided the Billings Area data. Joyce Mauldin and Glenn Solomon performed the database searches. Dr. Jarvis directed this study and assisted in data analysis and interpretation.The pre-publication history for this paper can be accessed here:


Anesthesia is produced by a depression of central nervous system function, however, the sites and mechanisms of action underlying this depression remain poorly defined. The present study compared and contrasted effects produced by five general anesthetics on synaptic circuitry in the CA1 region of hippocampal slices.At clinically relevant and equi-effective concentrations, presynaptic and postsynaptic anesthetic actions were evident at glutamate-mediated excitatory synapses and at GABA-mediated inhibitory synapses. In addition, depressant effects on membrane excitability were observed for CA1 neuron discharge in response to direct current depolarization. Combined actions at several of these sites contributed to CA1 circuit depression, but the relative degree of effect at each site was different for each anesthetic studied. For example, most of propofol's depressant effect (> 70 %) was reversed with a GABA antagonist, but only a minor portion of isoflurane's depression was reversed (< 20 %). Differences were also apparent on glutamate synapses-pentobarbital depressed transmission by > 50 %, but thiopental by only < 25 %.These results, in as much as they may be relevant to anesthesia, indicate that general anesthetics act at several discrete sites, supporting a multi-site, agent specific theory for anesthetic actions. No single effect site (e.g. GABA synapses) or mechanism of action (e.g. depressed membrane excitability) could account for all of the effects produced for any anesthetic studied. A-mediated inhibitory synapses and postsynaptic chloride channels [General anesthetics have been shown to depress neuronal responses in virtually all brain areas studied and this depression has been proposed to result from actions at GABAchannels -4, potaschannels -7, or cachannels -11, and/channels -17. The channels ,19. It ichannels -26 at cochannels -29. The All five anesthetics depressed synaptically evoked discharge, measured as a block of population spike (PS) responses recorded from CA1 neurons  reversed anesthetic-induced PS depression to varying degrees for each agent: isoflurane – 16.2 ± 7.4 %, halothane – 22.3 ± 18.4 %, pentobarbital – 56.2 ± 12.4 %, thiopental – 64.9 ± 12.9 % and propofol – 69.5 ± 14.3 %. Similar degrees of reversal were observed using the GABA-chloride channel blocker, picrotoxin . A GABAB receptor antagonist, CGP 55845A (10 μM) did not reverse PS depression for any of the anesthetics studied , less so for pentobarbital (~ 50 %), and contributed only partially to the depressant effects of the volatile anesthetics  were recorded from dendritic regions in stratum radiatum. All five anesthetics depressed EPSP responses  were observed in all CA1 neurons studied (n = 15) and were completely blocked by bicuculline  facilitation of Schaffer-collateral evoked EPSPs were studied. In the presence of either halothane or isoflurane no apparent change in EPSP rise time or decay kinetics were observed Fig. , contrasine Fig. . This inin vivo findings [Agent-specific effects were observed for paired pulse inhibitory responses (120 ms separation) recorded from CA1 neurons Fig. . Halothafindings  and is tfindings .per se.To determine whether the anesthetics could alter postsynaptic membrane excitability, effects on action potentials evoked by direct current injection into CA1 neurons were studied. Differences in effect were apparent across anesthetic agents – hardly any effect was evident for halothane and isoflurane, but the barbiturates and propofol produced a significant depression of action potential discharge Fig. . When meTwo conclusions can be drawn from these results: 1) for a given anesthetic, like halothane, multiple sites of action contributed in an additive manner to produce an overall depression of transmission through the CA1 neuronal circuitry Fig. ; 2) for Differing degrees of action (efficacy) were evident at both glutamate and GABA synapses for each anesthetic. For example, our results demonstrate that the two barbiturates studied appear to have differing degrees of effect at GABA synapses since thiopental's depressant effects were reversed ~ 65 % by a GABA antagonist, but pentobarbital's effects were only reversed by ~ 55 %. Similarly, these two barbiturates exhibited differing degrees of depression for glutamate-mediated excitatory inputs to the CA1 neurons, pentobarbital produced a 45 % depression in contrast to thiopental with only a 25 % depression. It was interesting that opposite actions were seen at presynaptic sites (GABA release was increased by anesthetics, while glutamate release was depressed) and at postsynaptic sites (GABA-mediated synaptic currents were prolonged, glutamate-mediated currents were not). The MAS theory can readily account for the unique agent-specific profiles of effects observed in various experimental models, and also seen clinically – a long standing weakness of Unitary theories . Finallyin vivo support comes from studies utilizing a GABA beta 3 receptor mutant mouse model – proprofol-induced anesthesia was blocked in these mice, while volatile anesthetic effects were not [in vivo findings, our results indicate that effects on GABA synapses play a role in anesthetic actions, especially for propofol, thiopental and pentobarbital; but the results also indicate that effects on glutamate synapses and postsynaptic membrane excitability contribute to the CNS depression produced by all anesthetics. Given the multiple effects observed for anesthetic actions on the two types of synapses studied here, it is likely that effects on other neurotransmitter systems also contribute to anesthetic-induced depression of the CNS.Glutamate and GABA synapses in the hippocampus are among the best characterized synapses in the brain and appear to utilize receptor subtypes which are similar to those in neocortex, thalamus and other higher brain regions. Thus, the effects described in the present study would be expected to occur in these other brain regions as well, but it should be noted that different GABA and glutamate receptor subtype distributions are known to occur in cerebellum, spinal and some brain stem nuclei, and it remains to be determined whether anesthetics alter these synapses in a similar manner to their hippocampal counterparts. Ted Eger's group at UCSF has recently found that enhanced GABA-mediated synaptic transmission at the spinal level plays an important role for propofol-induced immobility in response to a noxious stimulus , but thiwere not . Taken t2/CO2, carbogen) artificial cerebral spinal fluid (ACSF). The ACSF had the following composition (in mM): Na 151.25; K 2.5; Ca 2.0; Mg 2.0; Cl 131.5; HCO3 26.0; SO4 2.0; H2PO4 1.25; and glucose 10. Whole brain coronal slices (450 μm) were cut using a vibratome (Campden Instruments), following careful removal of the dura and pia membranes. Hemisected brain slices were equilibrated for at least one hour at room temperature in an incubation chamber filled with ACSF and continually bubbled with carbogen. Individual slices were transferred to a recording chamber and equilibrated for an additional 10 minutes prior to electrophysiological recording. Oxygenated ACSF solution was continuously perfused through the chamber at a flow rate of 3.0 ml/min and maintained at 22 ± 1°C. The present studies were carried out at room temperature because synaptic responses recorded from cooler brain slices exhibit considerably better baseline stability and the tissue remains viable for many more hours in vitro compared to slices maintained at physiological temperatures. Room temperature also facilitates the use of submerged preparations (oxygen solubility and delivery to slices is increased), which allows the use of 60× optics to visualize single neurons for the patch clamp recordings used in some experiments. Previous studies comparing both volatile and intravenous anesthetic effects at physiological and cooler temperatures in brain slices found that there were no apparent differences in effects [vs. 35°C and our methods for measuring and compensating for changed aqueous solubility, as well as the remarkably similar physiological responses recorded from brain slices at these two temperatures [Male Sprague-Dawley rats were anesthetized with ether (22 vol % in air) and the brain was rapidly removed and placed in ice cold (5°C) and pregassed  were placed in stratum pyramidale to record stimulus-evoked population spike field potentials, or in stratum radiatum to record field EPSPs. Single stimulus pulses  were delivered via constant current isolation units (Grass Instruments, SIU 6D) from a Grass S8800 two channel stimulator; at stimulus rates of 0.05 Hz. Field potential signals were amplified (× 1000), filtered (1 Hz to 10 KHz, bandpass), conditioned (DC offset), and digitally stored for later analysis .Whole cell patch-clamp recordings were made using thin-walled borosilicate capillaries (1.5 mm O.D.) pulled in two stages on a Narishige PP83 pipette puller. Patch electrodes were filled with the following intracellular solution (in mM): potassium gluconate or CsCl2 – 100, EGTA – 10, MgCl2 – 5, HEPES free acid – 40, ATP disodium salt – 0.3, and GTP sodium salt -0.3. The electrode solution also contained the local anesthetic QX 314 (1.0 mM) in some experiments, to prevent action potential discharge that would contaminate recordings of IPSCs. Electrode solutions were filtered and pH adjusted to 7.2 using KOH or CsOH and had a final osmolarity of 260 to 270 mOSM. Patch electrodes with a DC resistance of 4 to 5 MOhm were used. Recordings were made using an Axoclamp 2A preamplifier (Axon Instruments) in single electrode voltage clamp mode with > 80 % series resistance compensation and > 5 GOhm seals. Patch-clamp current signals were filtered (0.1 Hz to 10 KHz, bandpass), amplified (× 100) and digitized (10 KHz) for storage and analysis. Frequency and amplitudes of IPSCs were analyzed using Data Wave Technologies and Strathclyde Electrophysiological software in a continuous data recording configuration.The intravenous anesthetics  were made fresh for each experiment, solubilized using 0.5% dimethyl sulfoxide (DMSO) and sonicated immediately prior to test administration in stock solutions and serially diluted into ACSF to achieve the final concentrations for testing. Volatile anesthetics  were applied in the perfusate at equilibrated concentrations, delivered from calibrated vaporizers and bubbled into the perfusate for at least 10 min prior to switching from control ACSF, to ensure steady-state concentrations were achieved. The concentration of volatile anesthetics in the gas phase were continually measured using a Puritan-Bennett anesthetic monitor. Only a single concentration of a given anesthetic was tested on each brain slice.Data are expressed as the mean ± standard deviation and statistical analysis (ANOVA with post Tukey test) was performed using Instat from GraphPad Software. For drug effects on paired pulse inhibition, the percent change was first calculated as: Drug 1st/Control 1st = (0.5 × 100) - 100 % = 50 % depression; and Drug 2nd/Control 2nd = (X × 100) - 100 % = X % depression. Then the percent increase in paired pulse inhibition was = (X / 50) × 100 %. This approach has the advantage of normalizing paired responses with respect to varying degrees of population spike facilitation observed during control recordings, i.e. differing degrees of EPSP facilitation on a background of differing degrees of inhibition from preparation to preparation.SP and AMH contributed equally to this study by conducting most of the experimental work and they also contributed to data analysis and helped with figure preparation. MBM conceived of the study and contributed to experimental design, data analysis and interpretation of results; as well as wrote and prepared the manuscript.


Reinforcement - a process that helps prevent interbreeding between hybridising populations - is an important and little understood mechanism driving the completion of speciation Reinforcement, like sympatric speciation see , has chaSympatry—Area(s) of overlap in the ranges of populations, enabling potential interbreeding.Allopatry—Area(s) of population ranges that do not overlap with one another, preventing interbreeding.Sympatric speciation—Speciation that occurs within a range of sympatry.Reinforcement—The evolution of mechanisms that prevent interbreeding between newly interacting incipient species, as a result of selection against hybrids (narrow definition) or interspecific matings (broad definition) (See ion) See .Conspecific sperm precedence—Disproportional fertilization of a female by sperm of a conspecific male, when that female has mated with both conspecific and heterospecific males.Much of the appeal of both reinforcement and sympatric speciation lies in the way they unite micro- and macroevolution. Reinforcement, a concept popularized by Reinforcement provides a pathway toward the completion of the speciation process. Imagine that two divergent populations  come into contact after a period of allopatry . If the If the isolating mechanisms between these populations are only partially complete, extensive hybridization may occur. This can result in fusion back into a single population, or in the swamping of one population's gene pool by the genes of the other (extinction). But there is another possibility, one that can cause the speciation between the two populations to proceed. Remember that if the populations have been separated for long enough, it is likely that hybrids between them will have relatively low fitness. Individuals who mate with members of the opposing population will therefore produce offspring of poor quality, and hence have lower fitness than individuals that mate within their own population. This favors the evolution (or further divergence) of characteristics that cause mating within, rather than between, populations . SpeciatThis process, the evolution of premating isolation after secondary contact due to selection against hybrids, is reinforcement sensu Despite the substantial progress in our understanding of reinforcement that has been achieved over the last few decades, many questions remain about the process. These questions lend themselves to exploration by a broad variety of disciplines (evolution, ecology, behavior, phylogenetics, phylogeography, genetics), approaches  and taxonomic systems.Doubtless, the most important unanswered question about reinforcement is how often it occurs. It is very difficult to prove that reinforcement is occurring, or has occurred, between two species. Reinforcement occasionally leaves a signature, called reproductive character displacement, in which mating characteristics have diverged between populations in areas of sympatry but not areas of allopatry  the rel. In sympDrosophila pseudoobscura and D. persimilis (Several isolated examples of reinforcement between specific pairs of species have been demonstrated, fairly conclusively, in a variety of taxa including rsimilis , flycatcrsimilis , sticklersimilis , spadeforsimilis , and walrsimilis  (see alsrsimilis . These sDrosophila (While examples such as these provide essential information about reinforcement, their slow rate of compilation and biased reporting do not provide efficient ways to assess how often reinforcement occurs in general. Comparative approaches, which examine patterns across a broader taxonomic group, can also provide support for reinforcement without these detailed mechanistic analyses (review in osophila . By compAnother area where further research is essential is the determination of which biological factors promote reinforcement, as opposed to population fusion. Theoretical studies, using mathematical models and computer simulations, are proving useful in pinpointing the effects of many factors such as migration rates and patterns, the type of selection against interspecific mating, and the genetic basis of premating isolation (reviews in In recent years, exciting developments have started to take place in the analysis of the genetics of reinforcement (reviewed in PLoS Biology illustrates the extent of the insights that can be made with this approach. Using high-resolution genetic mapping the authors have identified the locations of genes that cause increased discrimination against Drosophila persimilis males by D. pseudoobscura females, due to reinforcement in sympatry. Surprisingly, these genes map to very different areas of the chromosomes than do genes that cause a basal level of mating discrimination between the species in allopatry. Among other insights, the position of these genes suggests that the reinforced discrimination is based on odor, not on the mechanism used in allopatry, male song. This leads to the novel conclusion that reinforcement is not just increasing the strength of an already existing mechanism of species discrimination, but is occurring through the development of a new discrimination system. These kinds of developments can also motivate more realistic theoretical models of the reinforcement process.Genetic analysis is also allowing a new understanding of the mechanisms by which reinforcement might be taking place in specific cases. Work by What if, when our assessment of the frequency of reinforcement is improved, it turns out to have been a rare occurrence in the generation of current biological diversity? The study of reinforcement is broad and varied enough that many of our findings about the process would still have wide-reaching implications.First, recall the claim, at the start of this article, that studying reinforcement reveals much about the role of microevolution in the macroevolutionary process of speciation. Knowledge gained about this relationship is not only directly applicable to the very similar process of sympatric speciation, but can also tell us a great deal about speciation caused by ecological adaptation and sexual selection, which are critical components of reinforcement in many systems e.g., . StudiesSecond, analysis of reinforcement clarifies the interactions between levels of reproductive isolation that occur at different stages in the life cycle. Reinforcement, broadly defined, can be driven by isolation at the postzygotic level or by incompatibilities that occur between mating and zygote production (postmating-prezygotic incompatibilities; Finally, regardless of whether reinforcement has been a common pathway in speciation, its relevance may be increasing. Reinforcement is a possible outcome anytime species that are capable of hybridization come into contact. Human activity is increasing the incidence of secondary contact by altering habitat and introducing invasive species. This contact often results in hybridization (reviews in


Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake. Using mutated dopamine transporters, the authors find that they can separate the actions of amphetamine from the normal function of the dopamine transporter The dopamine transporter (DAT) plays a critical role in the synaptic clearance of dopamine (DA) by mediating the reuptake of DA released into the presynaptic terminal . It ther32P incorporation into DAT in response to PKC activation  , 2003. Tn = 5) in FLAG-HA-DAT relative to FLAG-DAT. Surprisingly, the DAT-mediated whole-cell currents gated by AMPH, which have been shown to be uncoupled from the transport process (In a cell suspension (or in a population of adherent cells), it is difficult to assess the potential effects on efflux of a change in ionic gradients or membrane potential because the membrane potential and ionic gradients change freely depending on the stimuli. Indeed, AMPH has been shown to induce depolarization through a DAT-mediated uncoupled chloride conductance that can be gated by substrates such as AMPH . Therefo process , were aln = 5).This reduction in current and efflux resulted from the N-terminal deletion and not from the presence of the HA epitope, since a FLAG-tagged construct lacking the first 22 amino acids of DAT (FLAG-del22-DAT) but without any other added sequence showed a reduction in current and efflux similar to that of FLAG-HA-DAT. max for uptake of the substrate tyramine by FLAG-del22-DAT was 146% that by FLAG-DAT  or cocaine  was significantly different in FLAG-DAT and FLAG-del22-DAT. Cell-surface biotinylation studies revealed that the increased Vmax in FLAG-del22-DAT was accounted for by an increased number of DAT molecules at the cell surface  that is (are) phosphorylated should lead to an effect similar to that of the truncation. Since it is not known which of the serines are phosphorylated, we simultaneously mutated all five serines to alanine in the full-length FLAG construct (FLAG-S/A-DAT). To obtain further evidence that phosphorylation of one or more of the N-terminal serines is essential for AMPH-induced DA efflux, we also created a construct in which all five of these serines were simultaneously mutated to aspartate (FLAG-S/D-DAT), in an attempt to simulate phosphorylation of the serines.m nor the Vmax for tyramine uptake was significantly different in FLAG-S/A-DAT and FLAG-S/D-DAT  or by cocaine  were not significantly different.Neither the K-DAT see . The sma-DAT see , suggestn = 6)   . Thus, the ability of AMPH to induce DAT-mediated currents and DA efflux was impaired dramatically, either by N-terminal truncation, or by substitution of the five N-terminal serines to alanine. Remarkably, substituting these five serines to aspartate to mimic phosphorylation restored the ability of AMPH to induce voltage-dependent DA efflux and to produce currents, indicating that negative charges in the DAT N-terminal region are essential for these actions of AMPH.Similarly, in FLAG-S/D-DAT cells the AMPH-induced currents and DA efflux were much greater than those generated in FLAG-S/A-DAT cells . In contrast, amperometric currents in FLAG-S/A-7D-DAT and FLAG-S/A-12D-DAT were 29.8% ± 12.6% and 45.1% ± 9.6%, respectively, of that seen in FLAG-S/D-DAT . Thus, negative charge at either position 7 or position 12 restores a substantial fraction of the efflux seen with aspartate at all five positions, and the size of the resulting efflux relative to FLAG-S/D-DAT and FLAG-DAT suggests that both of these serines may be phosphorylated in vivo (see below).To explore which serine or serines are critical to the effect on efflux, we created five additional mutants in the FLAG-S/A-DAT background in which we mutated each of the five positions, one at time, to aspartate, and we created stable pools of EM4 cells expressing each of these mutants. At +100 mV the amperometric currents in FLAG-S/A-DAT, FLAG-S/A-2D-DAT, FLAG-S/A-4D-DAT, and FLAG-S/A-13D-DAT were 7.4% ± 2.6%, 8.4% ± 5.7%, 11.2% ± 3.1%, and 12.3% ± 7.0%, respectively, of that seen in FLAG-S/D-DAT  That Dmax for uptake is unaltered in the mutants, it is possible that phosphorylation might alter the ionic coupling of DAT. The ratio of whole-cell to amperometric current . (Similar results were obtained at +60 and +80 mV [data not shown].) This ratio is a microscopic property of an individual transporter that is inversely proportional to the fraction of charge carried by dopamine  gene in pCIHyg was described previously . In the max and Km values, increasing concentrations of tyramine from 0.02 to 50 μM were used. Km and Vmax values for [3H]tyramine and [3H]dopamine uptake were determined by nonlinear regression analysis using GraphPad Prism 4. IC50 values were determined using increasing concentrations of AMPH between 0.002 and 2 μM and of cocaine between 0.001 and 10 μM in competition with approximately 60 nM [3H]tyramine. Ki values were calculated from the IC50 values as described by Uptake assays with adherent EM4 cells stably expressing the appropriate DAT construct were performed as described previously . TyraminEM4 cells stably expressing the DAT constructs were incubated with cleavable sulfo-NHS-S-S-biotin  to label surface-localized transporter, and the biotinylated material was prepared and immunoblotted as described previously .2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, and 5.6 mM glucose) and incubated at 37oC with 15 μM DA for 30 min. Following incubation, cells were washed with KRH, harvested, resuspended in 0.20 ml of KRH and superfused in a Brandel superfusion apparatus (Brandel SF-12, Gaithersburg, Maryland, United States) as described by Confluent 100-mm plates of HEK-293 cells stably expressing FLAG-DAT or FLAG-HA-DAT were washed twice with KRH (25 mM HEPES [pH 7.4], 125 mM NaCl, 4.8 mM KCl, 1.2 mM KHWhole-cell and amperometric currents were recorded as described previously . The AMPAccession Numbershttp://ca.expasy.org/cgi-bin/niceprot.pl?Q01959) entry name for the gene discussed in this paper is S6A3_HUMAN, accession number Q01959.The Swiss-Prot (


Background to the debate: In the United States, the passage of the Bayh–Dole Act in 1980 encouraged universities to license inventions for commercial development. Although this financial incentive can stimulate academic researchers to discover new drugs and devices, there is concern that the possibility of monetary reward could distort investigators' objectivity. In the US, universities are encouraged to license inventions for commercial development. Although this financial incentive can stimulate academics to discover new treatments, it might also distort the investigators' objectivity One of the principal missions of an academic health center is to advance the understanding and treatment of disease through clinical research. In this pursuit, there is a need for checks and balances. When Jesse Gelsinger, a relatively healthy young adult, died in Philadelphia during a clinical trial of a novel adenovirus-based genetic therapy for ornithine transcarbamylase deficiency, it was a tragedy Attorney Alan Milstein made the case that the investigator and institution were both blinded to these problems by their heavy financial investment in the technology, an investment worth millions of dollars At its core, the issue revolves around conflicts of interest. In clinical research, the investigator should be primarily an advocate for the patient or volunteer. The core reason to perform clinical research is to create generalizable knowledge about a therapy, patient population, or a disease process with the long-term intent of improving human health. The interests of the patient and investigator should be fully aligned. However, most physicians in clinical research have other, more personal motivations, intermixed with the desire for progress. Successful research projects can lead to publications, promotions, grant renewals, and per case clinical trial enrollment fees. Some investigators have intellectual property rights that may have very substantial financial value if the drug or device reaches the level of approval by the United States Food and Drug Administration. These investigators stand to gain personally if the clinical trial is successful, a situation that has the potential to distort the investigator's objectivity, and may lead to a less honest relationship with study volunteers.In order to ensure that investigators are honest with potential research volunteers, the system of institutional review boards (IRBs) evolved. The IRB approves the informed consent document, which should describe the clinical experiment in a clear and dispassionate way to patients and their families. IRBs are largely made up of faculty and staff from the institution, although there are also public members and nonscientists on most IRB panels. The IRB must remain autonomous and be able to hold up or stop an investigation. There is an obligation that the IRB first and foremost think about patient rights and safety.The passage of the Bayh–Dole Act in 1980 enabled universities to license inventions for commercial development In terms of performing clinical trials using new technologies in which it has a financial interest, how is a university different from a corporate sponsor? In regard to patient safety, one primary distinction rests with the IRB. The corporate sponsor will present the research protocol to an independent commercial IRB, the university to its own IRB. Yet in both cases, there are potential conflicts of interest. The university IRB members will have a conflict of interest between the investments of their employer and the rights of the research volunteers. Independent commercial IRBs depend on pleasing corporate customers for their continued existence, and there is an unstated expectation that they will both be fast and produce rulings consistent with corporate expectations .At the university level a logical and conservative solution to the problem of institutional conflicts is to require that an IRB from outside the institution become the IRB of record when such a conflict arises. This external IRB could be either an independent commercial IRB or one of another university. The key is to grant the IRB independence and the authority to provide real oversight.There are other elements of conflicts of interest that need to be considered when the institution has a commercial interest, but most have more to do with the management of personal conflicts than institutional conflicts. The institution needs to assure the presence of an independent data safety monitoring board, thorough audits of good clinical practice, and a publications committee that will ensure submission of all meaningful study results, whether positive, negative, or neutral. Anyone subjectively evaluating patient data should be as free of conflicts as possible. These steps can be formulaically required, which should allow for performance of clinical research despite the presence of institutional commercial interests.United States research universities, and especially their academic medical centers, have greatly benefited from their uniquely privileged status in our society. That status is rooted in public confidence and trust that these institutions and their faculties will be independent and impartial in fulfilling both their academic mission to create, transmit, and preserve knowledge, and their duty to the general society to serve as credible, trustworthy arbiters of knowledge. One important mark of this status has been the remarkably consistent generosity of public support for biomedical research. Another has been the noteworthy deference of the federal government to university autonomy, and the light hand with which the sponsoring agencies historically have overseen the conduct of university research.When federal interposition occurred, it typically responded to widely publicized episodes of research misconduct, sometimes intertwined with egregious financial self interests of investigators; these episodes legitimately questioned the effectiveness of institutional oversight. Nevertheless, regulations consistently focused more on defining the metes and bounds of the permissible than on prescriptive mandates, and their implementation was effected largely through the mechanism of “assurances”—commitments that institutions would faithfully safeguard the specified perimeters of acceptable conduct.Awardee institutions thus bear primary responsibility for assuring the credibility and integrity of federally sponsored research. Public confidence in the trustworthiness of these institutions is critical, and yet nowhere is it more fragile than in biomedical research involving human participants. That confidence eroded in the 1980s and 1990s because of reports of scientific misconduct and of individual and institutional financial self interests in clinical trials. Scathing reports from federal oversight agencies and angry congressional hearings questioned whether financially self-interested institutions could any longer be trusted to guard the welfare of research participants or the integrity of clinical research.In 2001, the Association of American Medical Colleges convened a task force to examine and make recommendations on individual and institutional financial self interests in clinical research. The task force began by recognizing four important trends over the past three decades. First, the nature and culture of academic biomedical research have changed, bringing the potential of commercial relevance even to the most fundamental of scientific discoveries. Second, there has been enormous growth in the extent and depth of interactions between research universities and industry, especially in biomedicine. Third, the public has become increasingly impatient that its extraordinary investments in research yield more effective disease preventions and therapies. Fourth, the involvement of academic researchers in the translation of their discoveries has been essential in bringing those discoveries to market and to the benefit of public health.But the task force, in its two reports, asserted that both individual and institutional financial conflicts of interest in clinical research could be problematic ,5. It reBoth reports rest on a common set of core principles. The most important is that institutions should regard all significant financial interests in research involving human participants as potentially problematic. Where such interests exist, there should be a rebuttable presumption that the concerned individual or institution should not conduct the research, absent compelling circumstances. Importantly, the task force, after intense debate, rejected categorical prohibitions lest they unintentionally impede the translation of research discoveries into tangible public benefits.The task force acknowledged that the issue of institutional financial self interests is extraordinarily complex and sensitive, since it touches the very core of institutional autonomy. But the fact that an institution has a financial interest per se should raise a strong presumption against its participation in the clinical testing of that product. Public accountability and scientific integrity require that all research results emanating from academic medicine be as free as possible from the taint of financial compromise. Adding human participants to the research mix should raise the barrier to the highest level and require compelling justification for any participation by a financially self-interested institution.The task force did not define “compelling,” believing that each institution should make that determination based on disinterested scrutiny of the facts and circumstances of each case. For example, there may exist in a given institution a unique capability, without which the proposed research involving human participants could not be conducted as effectively or safely, or at all. In these instances, the public and science deserve access to that capability, provided the necessary safeguards are put in place to mediate the conflicting interests. In all such instances, protection of scientific integrity and the welfare of research participants must remain the foremost priority of both investigator and institution.This narrow window avoids absolute prohibition while striving to prevent institutional participation where credible alternatives exist. Only by such stringent self-policing can we sustain the trustworthiness and credibility of biomedical research, researchers, and their institutions, while continuing vigorously to promote the translation of biomedical discovery for the public's benefit.The public has every right to expect that academic institutions are working first for the public's interest. This value is even codified in the laws granting these institutions tax-exempt status. The public also expects that new, more effective therapies will be developed swiftly as a consequence of its support for academic research.The inventor of a new technology is always more motivated to see it through to widespread use than anyone else. This motivation, which may be as simple and benign as curiosity or as easy to understand as a financial incentive, is a powerful force driving human research. This force can be disciplined and controlled by the IRB and policies on conflicts of interest. Personal investment in research is, nevertheless, an important driver of scientific progress.When society makes an unnecessarily broad assumption that nearly all research with financial implications for investigators or their institution is potentially corrupted, a brake is placed on progress. Society will be better served by establishing clear guidelines and formalizing oversight of the research process than by rigidly limiting clinical research affected by conflict of interest. As examples, clinical trials should have independent data safety monitoring boards charged to review the study design, execution, data analysis, and publication of results. The IRB system should be strengthened in its independence through the use of community members. And, to be certain that institutional conflict of interest is avoided, an IRB from outside the institution in most cases will be preferableSociety wants better treatments. The fact that an inventor has mixed motives for developing a new treatment has always been acknowledged. The need to carefully manage the experimental process in human studies has always been understood. However, when rules minimize the role of inventors at academic centers, by forcing trials of their new ideas to go to outside institutions, society loses more than it gains. The incremental gain in safety is likely to be small (particularly if oversight is well established), while the decrease in speed of development will be significant.There are many similarities between the position espoused by Ross McKinney and my position. Most saliently, we both recognize the critically important role played by academic biomedical scientists in making discoveries and in facilitating their efficient translation into beneficial products. Neither of us proposes that academic investigators, or their institutions, should be flatly prohibited from trying to foster that translation in the presence of financial self interests.But there is an important difference. McKinney's approach for dealing with institutional conflicts of interest depends critically on the engagement of external agents to monitor closely both scientific integrity and the welfare of human participants. That, in my view, would require such deep interposition of those agents into the conduct of academic research as to be not only unprecedented but unfeasible. Beyond that, the approach falls short with respect to the maintenance of institutional trustworthiness and protection of public trust.Routine clinical assessment of technologies by financially interested institutions fosters public cynicism and distrust of the motives of academic biomedical researchers. The protective mechanisms recommended by McKinney are opaque to the public and reflect a “business as usual” image that fails fully to account for the markedly changed circumstances and perceptions of academic biomedical research. Most important, the mechanisms appear to be aimed primarily at protecting institutions' financial interests.By contrast, the American Association of Medical Colleges formulation urges that any institutional involvement in clinical research involving human participants in the presence of financial conflicts must be predicated on the presence or absence within the institution of demonstrably unique capability. This approach offers a much higher and more credible standard that aims to protect not only participant well-being and scientific integrity, but also institutional trustworthiness and public trust.


In this study, we investigated the effect of genetic background on expression profiles. We analysed the transcriptome of mouse hindlimb muscle of five frequently used mouse inbred strains using spotted oligonucleotide microarrays.Through ANOVA analysis with a false discovery rate of 10%, we show that 1.4% of the analysed genes is significantly differentially expressed between these mouse strains. Differential expression of several of these genes has been confirmed by quantitative RT-PCR. The number of genes affected by genetic background is approximately ten-fold lower than the number of differentially expressed genes caused by a dystrophic genetic defect.We conclude that evaluation of the effect of background on gene expression profiles in the tissue under study is an effective and sensible approach when comparing expression patterns in animal models with heterogeneous genetic backgrounds. Genes affected by the genetic background can be excluded in subsequent analyses of the disease-related changes in expression profiles. This is often a more effective strategy than backcrossing and inbreeding to obtain isogenic backgrounds. Due to their isogenicity, inbred mouse strains demonstrate low biological variability within each strain,2. Genetet al., manuscript in preparation). We, therefore, determined gene expression levels in hindlimb muscles from five frequently used wildtype mouse inbred strains, and compared these to the differential gene expression levels in affected muscle tissue from a mouse model (mdx) for Duchenne muscular dystrophy with healthy muscle tissue. Both the number of differentially expressed genes between strains as well as the fold-change levels are lower when compared to the differences found in affected versus healthy muscle tissue.From literature -9, it apet al., 2002), which requires an input of only 1 μg total RNA. Labelled cDNA was hybridised to murine microarrays containing 7,776 65-mer oligonucleotides spotted in duplicate.Gene expression levels in hindlimb muscle tissue from five different inbred strains (CBA, BALB, BL6, DBA, and BL10) were determined. Total RNA from two individuals per strain was isolated, reversed transcribed, and subsequently labelled according to a recently developed protocol  replicate numbers[Significance levels  between the five mouse inbred strains were calculated using analysis of variance. Signifie numbers. The hige numbers.Using an FDR of 10% we selected 88 out of 6144 (1.4%) expressed genes that are differentially expressed between strains  was determined to allow a direct comparison with identical statistical methods. Selection with an FDR of 10% showed an approximately ten-fold decrease in the number of differentially expressed genes (126). Absolute fold changes were calculated and subsequently a comparison of the distribution was made  on five genes to verify our microarray data. Two genes myomesin 1 and tropomodulin 1, which were 2.2-fold and 1.8-fold lower expressed in BL6 compared to the other strains on our microarrays, were also found to be lower expressed (2.0-fold and 2.2-fold respectively) in our qPCR assay Fig. . Three omdx vs. wild-type, 1298 genes with 221 genes having a fold-change >3). Therefore, the use of mice with deviating genetic background may be justified in disease-related studies. Alternatively, strain-dependent gene expression differences may be evaluated in the initial study phase of gene targeting experiments, although the effect of hybrid backgrounds is difficult to assess.This study shows that variation in overall gene expression levels between mouse inbred strains is relatively low in hindlimb muscle tissue. This is particularly evident when the number of differentially expressed genes between two mouse inbred strains (C57 vs. Bl6, 126 genes with 7 genes having a fold-change > 3) is compared to that between diseased and healthy muscle tissue  occurring in specific blocks between mouse inbred strains. The presence of cis-acting (single nucleotide) polymorphisms may be associated with regulatory variation affecting gene expression levels. It was estimated that probably a consistent amount (up to 6%) of the roughly estimated 35,000 mouse genes contain such functional regulatory variants. We inveThis study suggests an additional method for phenotyping mouse inbred strains and provides a list of genes with significant differential expression based upon false discovery rate selection. Although overall gene expression profiles are highly similar, most significant differences are determined by low gene expression levels of BL6 compared to the other strains. A large proportion of these BL6-specific genes function as structural muscle proteins . To date, however, no major differences in muscle physiology in BL6-mice have been described which can be attributed to these reduced gene expression levels.mdx) we study. As such, the genetic background will only marginally interfere with data analysis. Determination of gene expression profiles between mouse strains enables flagging a modest number of differentially expressed genes, and is an efficient and sensible approach to circumvent tedious backcrossings, necessary to obtain isogenic animals.Perfectly isogenic backgrounds are sometimes difficult to obtain. This explorative study demonstrates that the effect of genetic background on muscle expression profiles is significant but rather limited compared to other effects, e.g. the dystrophic genetic defect (mdx/J (mdx) mice from Jackson Laboratory at the age of 6 weeks. Mice were kept under standard conditions and were sacrificed by cervical dislocation when 8 weeks old. Hindlimb muscles (m. quadriceps femoris) were dissected and promptly snap-frozen in isopentane at -80°C. Total RNA was prepared by disrupting tissue using mortar and pestle and subsequent homogenisation by a rotor-stator homogenizor (Ultra-Turrax T25, Janke & Kunkel IKA-Labortechnik) in RNA-Bee (Campro Scientific) until uniformly homogenous (15–45 sec). Total RNA was isolated according to manufacturer's instructions followed by purification using RN-easy columns (Qiagen). Quality and yield was determined using Lab-on-a-chip .We obtained CBA/CaOlaHsd (CBA), BALB/cOlaHsd (BALB), C57Bl/6JOlaHsd (BL6), DBA/2OlaHsd (DBA), and C57Bl/10ScSnOlaHsd (BL10) mice from Harland Laboratories, and C57Bl/10ScSn-Dmd6 primer (5'-NH2-(CH2)6-TN6, Eurogentec) were adjusted to a volume of 21 μl with DEPC-treated H2O (diethyl pyrocarbonate, Sigma), heated for 10 minutes at 70°C and chilled on ice for 10 minutes. Reverse transcription mastermix (1.8 μl RevertAid RNaseH-M-MuLV reverse transcriptase (200 U/μl, MBI Fermentas), 6 μl 5x first-strand buffer (MBI Fermentas), and 1.2 μl 25x aa-dUTP / dNTP solution ) was added per reaction and incubated at room temperature for 10 minutes followed by 2 hours at 42°C. RNA was hydrolysed by addition of 10 μl 0.5 M EDTA and 10 μl 1 M NaOH and incubation at 65°C for 30 minutes followed by neutralization by addition of 10 μl 1 M HCl. Aminoallyl labelled cDNA was then purified by combining 300 μl of PB-buffer (Qiagen) to 60 μl of the neutralized sample and centrifuged through a Qiaquick column (Qiagen) at 13000 rpm for 1 minute. Two washing steps were performed by spinning 500 μl of 75% EtOH at 13000 rpm for 1 minute while discarding the flow-through. To remove ethanol-traces the columns were centrifuged for an additional minute. cDNA was recovered by eluting three times using 30 μl basic H2O (3.3 mM NaHCO3 buffer, pH 9.0) and concentrated to a volume of 6.66 μl using a speedvac. Aliquots of Cy3 and Cy5 reactive dyes (PA23001, PA25001, Amersham) were prepared by dissolving each vial of monoreactive dye in 40 μl fresh anhydrous DMSO (Sigma) and dividing into aliquots of 2 μl followed by vaccuumdrying until dry and subsequent storage at 4°C in the presence of silica. Fluorescent dyes were coupled by adding 3.33 μl of bicarbonate buffer (1 M NaHCO3 buffer, pH 9.0) to the aa-cDNA sample and dissolving the dried aliquot of reactive dye, followed by incubation at room temperature for 1 hour in the dark. To the samples 4.5 μl 4 M hydroxylamine (Sigma) was added and incubated at room temperature in the dark for 15 minutes, followed by addition of 186 μl TE-3-buffer. Hybridisation mixtures were prepared by combining a Cy3-labeled cDNA sample with a Cy5-labeled cDNA sample and 10 μl Mouse-Hybloc (1 μg/μl, Applied Genetics Laboratories) followed by removing uncoupled dyes by spinning through a pre-wetted Microcon column (YM30, Amicon) for 8 minutes at 13000 rpm. Hybridisation mixture was washed by spinning 500 μl TE-3-buffer through the column and discarding the flow-through. This step was repeated two times as 2 μl yeast-tRNA (10 μg/μl, Sigma) and 2 μl polyA-RNA (10 μg/μl, Sigma) were added during the last step. Mixture was collected by inverting the column and spinning for 1 minute at 13000 rpm. Hybridisation mixture was finalized by adding TE-3-buffer to 84 μl together with 17 μl 20x SSC and 3 μl 10% SDS followed by denaturing at 100°C for 2 minutes, renaturing at room temperature for 15 minutes and spinning at 13000 rpm for 10 minutes. Labelled target was hybridised overnight on murine oligonucleotide microarrays (65-mer with 5'-hexylaminolinker, Sigma-Genosys mouse 7.5 K oligonucleotide library, spotted in duplicate). Hybridisation occurred in a automatic hybridisation station (GeneTac, Perkin Elmer) and was followed by washing with 5x 2xSSC + 0.1% SDS at 30°C, 5x 1xSSC at 30°C, 3x 0.2xSSC at 30°C, 1x 0.2xSSC at 65°C, 2x 0.2xSSC at 30°C, and subsequently scanned as described previously[Aminoallyl labelled cDNA (aa-cDNA) was prepared based on a previously described protocol. AliquotMAANOVA2.0 The Jackson Laboratory ), hierarchical t-test [Gene expression profiles from hindlimb muscle derived from 2 male animals of each strain were generated using dye-swap experiments. Subsequent duplicate spots on each array resulted in 8 replicate measurements per gene. Targets were assigned at random to the arrays, while avoiding co-hybridisation of samples from the same strain. GenePix Pro 3.0 (Axon) was used for feature extraction and quantification. Genes were considered as being expressed when the corresponding feature was not flagged by the algorithm provided by GenePix. Local background corrected spot intensities were normalized using Variance Stabilization and Normalization (VSN) in R . Array dl t-test  and the l t-test  selectio- MuLV reverse transcriptase and accompanying buffer (MBI-Fermentas) using 1 mM dNTPs. The mixture was incubated at room temperature for 10 minutes before a 2 hour incubation step at 42°C, followed by 10 minutes at 70°C. Quantitative PCR was performed using the Lightcycler (Roche). PCR mixture was prepared by combining cDNA dilution, 10 pmol forward and reverse primer, MgCl2 (4 mM) with 4x home-made LC mastermix (0.9 mM dNTPs, BSA (1 μl/μl, Pharmacia Biotech), Taq polymerase (0.8 U/μl), 4x SYBR Green I (Molecular Probes), 4x AmpliTaq Reaction Buffer (Perkin Elmer)) to a total volume of 20 μl. Amplicons were generated during 45 cycles with annealing temperature set at 55°C. Optimal cDNA dilutions and relative concentrations were determined using a dilution series per gene. Replicate experiments (n = 4) were normalized to 1 and relative expression values were determined by calculating the ratio per gene over the average relative expression of genes, which show no differential expression on both microarray and qPCR (dysferlin, cystatin B, and thrombospondin 4). Significance levels were calculated with a one-sample t-test. PCR primer pairs were designed using the Primer3 search engine, available at: Primer3 Software Distribution . The screened genes and the oligonucleotide primer pairs used for each of the genes in this study correspond to the following nucleotides: myomesin1, 4761–4780 and 4865–4884 (NM_010867); tropomodulin1, 670–689 and 878–897 (NM_021883); dysferlin, 4218–4237 and 4353–4372 (AF188290); cystatinB, 3–22 and 151–170 (NM_007793); thrombospondin4, 2167–2186 and 2289–2308 (NM_011582).qPCR was performed in duplicate for each individual resulting in four measurements per strain per gene. cDNA was prepared by reverse transcription using 1 μg total RNA as template. Random hexamers (40 ng) were used to prime the transcription after heating 10 minutes at 70°C followed by chilling on ice for 10 minutes. cDNA was synthesized by RevertAid RNaseHRT carried out the tissue preparation, total RNA isolation, target preparation, hybridisations, experimental design, data extraction, data analysis, rt-PCR, and the drafting of the manuscript. PH participated in the experimental design, analysis, rt-PCR, and study coordination. ES participated in the experimental design and analysis. RM provided statistical support. EM was responsible for mouse breeding and tissue preparation. JM participated in experimental design. GO and JD coordinated the study. All authors read the final manuscript.Differentially expressed genes between mouse inbred strains are selected with a false discovery rate of 10, 15, and 20%. Selected genes are indicated with 1, genes not selected by the specified criteria are indicated with 0. The mean of the relative gene expression levels of each of the five mouse strains is shown. For each gene the GenBank accession number is shown as well as the UniGene ID, gene description and the gene ontology description. The additional file is formatted as Comma Separated Values (CSV) file, and is named Turketal2004_Additional_File.csv.Click here for file