In contrast to gene-mapping studies of simple Mendelian disorders, genetic analyses of complex traits are far more challenging, and high quality data management systems are often critical to the success of these projects. To minimize the difficulties inherent in complex trait studies, we have developed GeneLink, a Web-accessible, password-protected Sybase database.GeneLink is a powerful tool for complex trait mapping, enabling genotypic data to be easily merged with pedigree and extensive phenotypic data. Specifically designed to facilitate large-scale (multi-center) genetic linkage or association studies, GeneLink securely and efficiently handles large amounts of data and provides additional features to facilitate data analysis by existing software packages and quality control. These include the ability to download chromosome-specific data files containing marker data in map order in various formats appropriate for downstream analyses . Furthermore, an unlimited number of phenotypes  can be stored and analyzed. Finally, GeneLink generates several quality assurance reports, including genotyping success rates of specified DNA samples or success and heterozygosity rates for specified markers..GeneLink has already proven an invaluable tool for complex trait mapping studies and is discussed primarily in the context of our large, multi-center study of hereditary prostate cancer (HPC). GeneLink is freely available at  In the past decade, hundreds of genes involved in the etiology of simple Mendelian disorders such as cystic fibrosis and Huntington's disease have been identified -3. The gEfforts to identify susceptibility genes involved in complex traits such as cancer, diabetes, hypertension, or Alzheimer's disease are complicated by genetic heterogeneity, incompplete penetrance, phenocopies, and the later age of onset of disease . Each of these factors results in a significant reduction in power for any given study. Therefore, gene-mapping studies of complex traits require high-throughput genotyping performed on large collections of DNA samples using hundreds to thousands of polymorphic markers. The significant amounts of data generated during these genome surveys pose numerous data management challenges. In order to address these challenges, which are inherent in any large, collaborative genotyping study, we have developed a robust, easy-to-use database system named GeneLink.GeneLink was initially developed to facilitate our studies of genetic susceptibility to prostate cancer, whose aims are to identify novel high- and moderate- penetrance genes involved in hereditary prostate cancer risk. These studies are multi-center collaborative efforts involving researchers from the United States, Finland, and Sweden -7. The padmin (administrator) privilege provides a user the ability to view data as well as manage access to the data. Users without admin privileges can be assigned the following privileges by the administrator: export, import, modify and view, with obvious permissions. Project- or user-specific reports summarizing assigned privileges can be generated. Second, as GeneLink provides the ability to associate a "group" (of users) with a collection site-specific set of families, collaborators can be provided the ability to view and/or manipulate all or only a subset of data.GeneLink is a platform-independent, Web-accessible Sybase database that can manage complete genotypic, phenotypic and pedigree data for genetic linkage or association studies. Figure Currently, GeneLink's database uses Sybase SQL server ASE 12.5.1, which runs on a Sun V880 computer running Unix. GeneLink's Web scripts to access the database require Perl version 5.6.1 or greater. The necessary CPAN Perl modules required by GeneLink are DBI, DBD::Sybase, CGI, and Carp. These modules are usually included in standard Perl 5 releases. A Web server such as Apache 1.3.29 is also required to run the GeneLink Web scripts. GeneLink can operate on a Sun Enterprise 6500 or similar machine configured to operate as a Web server.Families, Pedigrees, Trait Score, Traits Translations, Markers, Primers, Maps, Genotypes, Allele Translations, Liability Classes and DNA. Each table can be populated either by importing multiple records from a delimited text file or by inserting a single record at a time through a Web interface. The Families table stores pertinent clinical information regarding each family as a whole; each record reflects a single family included in the linkage or association study. For example, in our HPC study, we used the Families table to store information regarding evidence of male-to-male transmission of disease as well as the occurrence of other cancer types in the family. The Pedigrees table stores one record per individual within a family and contains the biological relationships  for each individual. Also included in the Pedigrees table is age, sex, whether the individual has been or will be genotyped , two ways to store qualitative phenotype information , and individual liability class information. In our HPC study, we defined prostate cancer affection status in two ways; specifically, we used the StatusBroad field to classify individuals as affected, unaffected or unknown, while the StatusNarrow definition was used for an affecteds-only analysis in which individuals were coded either as affected or unknown. More extensive trait or covariate information can also be stored in the Trait Score table. This table stores an unlimited number of qualitative or quantitative phenotypes as trait 1, trait 2, trait 3, and so forth. Definitions of the traits are stored in the Traits Translation table.Figure Markers table stores information regarding all markers typed in a given project, including the panel in which the marker was run. A panel is defined as a group of microsatellite or SNP markers which can be electrophoresed simultaneously by taking advantage of different fluorescent dye labels and varying amplicon sizes. Also stored in the Markers table is the allele size range (ASR) of the marker, the fluorescent dye used to label the forward primer, and the marker-specific genotype for a CEPH control individual . The Primers table provides additional information for each specific marker. This information includes UniSTS ID, GenBank accession number, forward primer sequence, reverse primer sequence, and primer purchasing and inventory information. The Primers table also contains comprehensive genetic map information  as well as physical location . The Maps table stores the genetic map location of a marker in the genome, as well as the relative order of markers along a chromosome and the distance between adjacent markers. The markers typed thus far in our HPC study are di-, tri-, or tetra- nucleotide repeats; however GeneLink is capable of handling any combination of microsatellite and single nucleotide polymorphisms (SNP) data.The Genotypes table is where final genotype data are stored. Each record represents an individual/marker combination and alleles are stored as size in base pairs. The Genotypes table also holds information regarding the laboratory run . When exporting data in LINKAGE or RelCheck format, GeneLink systematically recodes genotype labels  to provide properly-formatted data for analysis by these programs. The Allele Translations table provides a key or legend linking the newly "translated" genotype to the original size in base-pairs score. Future exports use identical "allele translation codes." New alleles identified after the first export will be added to the end of a marker's allele translation ensuring consistent recoding of alleles across exports . For example, in our HPC study we used five different liability classes  and that the genotype for the control individual  matches what is expected (Markers table).Each of GeneLink's 11 tables has measures built-in for quality control purposes. First, all changes  to GeneLink records are stamped with the date, time, and USER ID of the individual doing the editing. Changes to the families, pedigrees, or genotypes tables can be easily reviewed in a Pedigrees table or the Trait Score table). This facilitates exporting data in formats commonly used in downstream analyses . There are three ways data can be exported. First, any user with administrator (admin) privileges can export data by Panel in LINKAGE or GAS formats. We use this option when checking our data for Mendelian inconsistencies because most laboratory errors could be detected if multiple markers within a panel showed up as inconsistent. Next, other users with export privileges can export data by chromosome. All data are exported by chromosome in the map order specified in the maps table. Chromosomes are only available for exporting once data for all markers on that chromosome have been imported, though we did design GeneLink to accommodate the possibility that all markers on a chromosome may not have been typed for all sites. The export genotype data screen  to export, how to define liability classes if necessary, what file format is desired and which families to include in the export. Only the family collection sites to which a user's group has access will appear in this screen. The final way data can be exported by users with export privileges is directly from the Families table. Using the Export by chromosome option described above means that all families from the selected site(s) will be exported. Alternatively, exporting directly from the Families table makes it possible to export only a subset of families from a collection site or a subset of families from across sites. Regardless of the exporting mechanism employed, GeneLink generates an Export Report, which summarizes all the pertinent information regarding the export, including families, chromosome (exported markers and distance between them), phenotypes included, liability class definition, and file format  . We are To collect the necessary number of DNA samples needed to provide sufficient power to detect linkage or association, collaborative efforts are almost always required. The Web-based interface of GeneLink facilitates multi-center collaborations, as data can easily be accessed via the Internet. GeneLink's Web-based interface also makes it platform-independent, a feature that was essential given the number of researchers who would be accessing it using various hardware-browser combinations. Other publicly-available databases described in the literature do not have this advantage. In this paper, we have presented GeneLink in the context of a collaborative effort in which multiple sites will need access to data generated in a single laboratory. However, GeneLink would also be valuable in the context of a meta-analysis of data generated in more than one laboratory. Making data access easier for our collaborators translated into the need for a sophisticated security system. Specifically, in our study of hereditary prostate cancer, researchers are permitted access to only their own set of data. This is important because, in some cases, a site's internal review board (IRB) protocol may not allow for raw data to be shared with other analysts.GeneLink provides several other advantages for investigators performing linkage or linkage disequilibrium studies of complex traits. For example, the process by which genotypes can be imported into GeneLink was designed to be flexible enough to handle data from laboratories like our own which employ duplicated samples and double-scoring methods for quality control purposes. Using duplicated samples and double scoring aids us in keeping our genotyping error rate low (< 1%). In our HPC study, we included 92 duplicated samples  in order to evaluate our genotyping error rate. The entire import process is outlined in Figures Allele Translation table allows comparison of alleles across families or across analyses, as each allele for each polymorphic marker will only be recoded once. This is particularly important as linkage disequilibrium or association studies become more common. Fourth, GeneLink's ability to export only a subset of families is critical, as genetic heterogeneity is a significant factor contributing to the difficulty of mapping genes involved in many complex traits. Multiple genes  haveAdditional quality control measures were included in GeneLink's design. First, all changes to the database are recorded. As genetic studies of complex traits can be spread over many years, it is important to keep a detailed log of any changes made to the data. For example, an individual's affection status may change during the course of a study; therefore it is critical to track when this information was updated in the database. Second, in order to monitor data quality, GeneLink was also designed to perform several built-in checks, as described above.Given that genetic studies of complex traits will generate millions (or even billions) of genotypes, it is essential to have appropriate mechanisms in place to ensure data integrity. In our study of hereditary prostate cancer, these checks immediately discovered a typographical error, which, if left undiscovered, would have resulted in data from an affected individual never being exported or analyzed. Finally, GeneLink generates detailed reports storing pertinent information regarding all imports and exports Figures  and 8A, GeneLink was designed primarily in the context of family-based studies of complex traits. It is capable of handling both linkage and association data, and can be used for both whole genome scans and/or candidate-gene studies. Further development of GeneLink will focus on extending its capabilities in regard to the case-based design. We recognize that both the family-based and case-based study designs have unique advantages, so we see it as critical to make GeneLink flexible enough to accommodate a case-based design. Currently, there is no limitation in storing case-based data however changes to GeneLink's exporting mechanisms should be made. Finally, in the same way that GeneLink is capable of storing "exported" data input files, future work will center on the storage of analysis results. Again, this would be helpful for multi-center collaborative studies, which will continue to be critical to successful efforts to identify genes important in complex trait etiology.In summary, GeneLink was designed specifically to ease the data management burden of mapping complex traits. It provides many functions that make it a uniquely powerful tool for use in genetic linkage or association studies. GeneLink simplifies merging genotypic data with pedigree, phenotype, and genetic or physical map information. Specifically, GeneLink's design makes it ideal for large-scale, multi-center studies, which will become more and more common in efforts to dissect the genetic factors contributing to complex trait etiology.Project name: GeneLinkProject home page: Operating system(s): Platform independentProgramming language: PerlOther requirements: Sybase SQL server ASE 12.5.1, Perl version 5.6.1 or greater, CPAN Perl modules DBI, DBD::Sybase, CGI, and Carp, Web server such as Apache 1.3.29License: Sybase SQL server ASE 12.5.1Any restrictions to use by non-academics: noneEG, JT, JEBW and AB participated in database's design. AM, LU, KT and TW did all of the programming. EG, DG, PD, APK, MJ, DFL, and GI performed extensive testing of the database. EG, PD, TW, JEBW and AB drafted the manuscript. All authors read and approved the final manuscript.


The principle of "a calorie is a calorie," that weight change in hypocaloric diets is independent of macronutrient composition, is widely held in the popular and technical literature, and is frequently justified by appeal to the laws of thermodynamics. We review here some aspects of thermodynamics that bear on weight loss and the effect of macronutrient composition. The focus is the so-called metabolic advantage in low-carbohydrate diets – greater weight loss compared to isocaloric diets of different composition. Two laws of thermodynamics are relevant to the systems considered in nutrition and, whereas the first law is a conservation (of energy) law, the second is a dissipation law: something (negative entropy) is lost and therefore balance is not to be expected in diet interventions. Here, we propose that a misunderstanding of the second law accounts for the controversy about the role of macronutrient effect on weight loss and we review some aspects of elementary thermodynamics. We use data in the literature to show that thermogenesis is sufficient to predict metabolic advantage. Whereas homeostasis ensures balance under many conditions, as a general principle, "a calorie is a calorie" violates the second law of thermodynamics. The recent awareness of an epidemic of obesity coincides with, and may have contributed to a dramatic increase in the popularity of a variety of low carbohydrate diets. This rapid switch in dietary habits of a significant part of the population, and the virtual revolution in the food industry, is unusual in that it stands in direct opposition to long-standing recommendations of the majority of the nutritional and medical establishment e.g. ,2). Desp. Desp2])was found. In the absence of an identifiable methodological error, experimental data has to be accepted and numerous investigations, in fact, serve as precedents for Greene et al.'s findings .always be independent of macronutrient composition would violate the second law of thermodynamics.In our previous review of metabolic advantage  we showeBecause it is a colloquial phrase, it is important to understand exactly what it is meant by "a calorie is a calorie." The most common meaning is that is it impossible for two isocaloric diets to lead to different weight loss. Frequently, the concept is justified by reference to the "laws of thermodynamics", but an explicit connection has never been spelled out. More recently, Buchholz & Schoeller  appear ts of thermodynamics" it is important to be sure that one is including the second law. The first law is very different in character from the second law [dissipation law: it defines a quantity, the entropy, S, which we traditionally identify with disorder or high probability. The second law says that in any  irreversible process, the entropy must increase (ΔS > 0); balance is not expected. Entropy is, in fact, identifiable with irreversibility.The idea that "a calorie is a calorie" comes from a misunderstanding of the laws of thermodynamics. There are two laws of thermodynamics. (The zeroth law that establishes the concept of temperature and the third law that describes absolute zero are not relevant here). When speaking of "the lawcond law ,11,12. TIt is important to understand that it is the second law that drives chemical reactions. The first law is a bookkeeping law and tells us that the total energy attributed to work, heat and changes in chemical composition will be constant. It does not tell us whether such a reaction will occur, or if it does, what the relative distributions of the forms of energy will be. To predict the tendency of the reaction to occur, we must employ the second law that says the entropy must increase. In a chemical reaction, at constant temperature and pressure, the entropic and energetic effects are combined into the change in the Gibbs free energy, ΔG, whose sign predicts the direction of reaction, and whose magnitude indicates the maximum amount of work realizable from the reaction.in vivo values will differ from standard state values but they are continuous functions of the concentrations and there will be numerous conditions under which ΔG' is not zero. In other words, assuming that protein and carbohydrate are energetically equivalent leads to a contradiction.To understand the implications of "a calorie is a calorie," that energy yield could be path-independent and the same for all diets consider that it implies that carbohydrate and protein are equivalent fuels as shown in Figure is unrecoverable. The efficiency of a machine is dependent on how the machine works and, for a biochemical machine, the nature of the fuel and the processes enlisted by the organism. A simple example is the inefficiency of low-test gasoline in high compression gasoline engines. If a "calorie is a calorie" were true, nobody would pay extra for high test gasoline. . In weight loss diets, of course, inefficiency is desirable and is tied to hormonal levels and enzyme activitiesThe second law was developed in the context of the industrial revolution and the attempt to understand the efficiency of machines. The law describes the theoretical limits on the efficiency of engines and applies as well to living (irreversible) systems. The second law says that no machine is completely efficient. Some of the available energy is lost as heat and in the internal rearrangement of chemical compounds and other changes in entropy. In other words, although the first law holds even in irreversible processes – energy is still conserved – the second law says that something is lost, something possibility of metabolic advantage should not come as a surprise.In nutrition, one component of inefficiency is measured in thermogenesis (thermic effect of feeding), or the heat generated in processing food. There is a large literature on this subject and the general conclusion, as summarized in a recent review by Jéquier , is thatRecommendations for fighting obesity frequently call for small reductions in calories . In facttheoretically required in all cases is mistakenly based on equilibrium, reversible conditions that do not hold for living organisms and an insufficient appreciation of the second law. The second law of thermodynamics says that variation of efficiency for different metabolic pathways is to be expected. Thus, ironically the dictum that a "calorie is a calorie" violates the second law of thermodynamics, as a matter of principle.A review of simple thermodynamic principles shows that weight change on isocaloric diets is not expected to be independent of path (metabolism of macronutrients) and indeed such a general principle would be a violation of the second law. Homeostatic mechanisms are able to insure that, a good deal of the time, weight does not fluctuate much with changes in diet – this might be said to be the true "miraculous metabolic effect" – but it is subject to many exceptions. The idea that this is The analysis above might be said to be over-kill although it is important, intellectually, not to invoke the laws of thermodynamics inappropriately. There are also, however, practical consequences. The seriousness of the obesity epidemic suggests that we attack it with all the means at our disposal. Metabolic advantage with low carbohydrate diets is well established in the literature. It does not always occur but the important point is that it can occur. To ignore its possibilities and to not investigate the precise conditions under which it appears would be cutting ourselves off from potential benefit. The extent to which metabolic advantage will have significant impact in treating obesity is unknown and it is widely said in studies of low carbohydrate diets that "more work needs to be done." However, if the misconception is perpetuated that there is a violation of physical laws, that work will not be done, and if done, will go unpublished due to editorial resistance. Attacking the obesity epidemic will involve giving up many old ideas that have not been productive. "A calorie is a calorie" might be a good place to start.None declared.


Elective endotracheal intubations are still commonly performed without premedication in many institutions. The hypothesis tested in this study was that morphine given prior to elective intubations in neonates would decrease fluctuations in vital signs, shorten the duration of intubation and reduce the number of attempts.2<85%), duration of procedure, duration of hypoxemia (Sp02<85%), number of attempts and change in mean blood pressure were compared between groups.From December 1999 to September 2000, infants of all gestations admitted to a level III neonatal intensive care unit and requiring an elective endotracheal intubation were randomly assigned to receive morphine 0.2 mg/kg IV or placebo 5 minutes before intubation. Duration of severe hypoxemia (HR< 90/min and Sp034 infants (median 989 g and 28 weeks gestation) were included. The duration of severe hypoxemia was similar between groups. Duration of procedure, duration of hypoxemia, number of attempts and increases in mean blood pressure were also similar between groups. 94% of infants experienced bradycardia during the procedure.We failed to demonstrate the effectiveness of morphine in reducing the physiological instability or time needed to perform elective intubations. Alternatives, perhaps with more rapid onset of action, should be considered. Endotracheal intubation is a painful and stressful procedure, which is associated with acute increases in blood pressure and intracranial pressure, bradycardia and hypoxemia . These pMost premature infants and many term infants admitted to neonatal intensive care units (NICU) will require one or more endotracheal intubations during their stay. In 1994, 84% of Canadian NICUs, including ours, rarely or never used premedication for elective intubations . In 2000A literature review revealed six randomized controlled trials -14, compWe reviewed our policy, which did not include premedication for elective endotracheal intubations, in light of the current evidence. As morphine has been used for years in neonates with apparent safety and efficacy for pain and as staff in our unit were comfortable with this medication, we aimed to evaluate the efficacy of morphine, in achieving better intubation conditions and success while maintaining vital signs stability.Infants of all gestations, admitted to McMaster University Medical Center level III NICU and considered likely to need an elective oral or nasotracheal intubation during their hospital stay, were candidates for inclusion in this study. Families were approached for consent as soon as possible after birth when an elective intubation during their hospital stay seemed likely: if their infant(s) was less than 30 weeks gestation, already ventilated , was on NCPAP for respiratory distress or was needing an elective surgery. Others were approached when an elective intubation was needed. At the time of this study, our unit was a 33-bed level 3 NICU, caring for both inborn and outborn patients, and the referral center for 25000 annual deliveries, with 900–1000 admissions per year.Infants were excluded if they met any of the following conditions: 1) absence of an intravenous access, 2) upper airway anomaly potentially leading to a difficult intubation, 3) cyanotic heart disease, 4) upper gastrointestinal obstruction (which would require a rapid sequence intubation) or 5) concurrent opioid administration.Infants requiring an elective intubation were randomly assigned to receive either morphine 0.2 mg/kg IV or placebo (0.9% NaCl), given over 1 minute, followed 5 minutes later by the intubation. This larger dose of morphine was chosen for the perceived acuity of pain produced by an intubation; a larger dose may be more effective to decrease the struggling by infants during the procedure, which is caused by pain. Infants were randomized according to a computer-generated random number table with random block sizes. Morphine and placebo were supplied in identical unidose vials, labeled PIN Rx, which were prepared by one pharmacist according to the randomization sequence and placed in sealed, consecutively numbered envelopes, which were opened just before intubation. Thus, randomization occurred just prior to intubation.2 and hand-ventilated with a self-inflatable bag for 1–2 minutes prior to intubation. Infants having their endotracheal tube replaced were ventilated through their existing tube until it was removed. Vital signs  were captured to a laptop computer from the infant's monitor  every 5 seconds (except blood pressure which was obtained with a self inflating cuff every minute) using Procom Plus Communication Software, from the time the study medication was given (which was considered the baseline) to 5 minutes after the infant's vital signs returned to pre-procedure values. One of three investigators, not involved in the procedure collected the following data manually: duration of the procedure  and the number of intubation attempts (defined as number of times the laryngoscope was inserted in the mouth). If there was more than one attempt, the clock continued between attempts and was stopped only when tube placement was confirmed by auscultation. In our NICU, the preferred method of intubation is via the nasotracheal route if mechanical ventilation is expected for more than a few hours.Three to four minutes after receiving the study medication, infants were preoxygenated with 100% 0a, neonatal fellows b, pediatric residents c, clinical nurse specialists d, clinical nurse specialist students e and transport nurses f. After 2 unsuccessful attempts by a junior team member , a more experienced intubator  was called.All team members performed the intubations: staff neonatologists Institutional ethics committee approval and informed consent from the parents were obtained for this study.2 < 85% with a HR< 90/min. This was felt to be the most undesirable side effect of endotracheal intubation as cerebral blood flow in neonates is highly dependent upon heart rate. Secondary outcomes included: (1) duration of the procedure, (2) duration of hypoxemia (Sp02 < 85%), (3) number of attempts, (4) maximum change in blood pressure from baseline, (5) occurrence of bradycardia (HR<90/min).The study aimed to test the hypothesis that morphine 0.2 mg/kg would decrease fluctuations in vital signs, shorten the duration of the procedure and reduce the number of attempts. The primary outcome was the duration of severe hypoxemia, defined as Sp0The study group's impression was that a majority of infants experience some degree of severe hypoxemia during an elective intubation, which was clinically undesirable. It was estimated to be 30 seconds, based on experience. In order to detect a one standard deviation difference in duration of severe hypoxemia between the 2 groups , 17 patients per group were required.Because the distribution of the main outcome was skewed and groups were small, continuous variables were compared using the Mann-Whitney U test. Dichotomous variables were compared using Fisher's exact test or Chi-square test. A p value < 0.05 (2-sided) was considered significant for the primary outcome; p < 0.01 was considered significant for secondary outcomes to account for multiple analyses in a small sample. Level of experience of the intubator, birth weight and gestational age were separately explored as potential confounders of the primary outcome using ANOVA or linear regression.Patients were recruited from December 1999 to September 2000. Patient flow in the study is depicted in figure Baseline characteristics are presented in Table 2 < 85%) during intubation. The median duration of hypoxemia was 235 sec in the treatment group and 90 sec in the control group (p = 0.04). Because of our small sample and the likelihood of finding a significant result by chance alone when assessing multiple outcomes, it was decided a priori that a p value of 0.01 would be considered significant for secondary outcomes. Nevertheless, this represents an interesting but somewhat worrisome trend. No difference was found in the maximal increase in blood pressure. Ninety-four percent of patients experienced bradycardia (HR<90/min) during the procedure with no difference between groups.All patients in the treatment group and 14/17 in the control group experienced hypoxemia , was similar to controls (median 1); total number of attempts was 38 in the premedicated infants versus 31 in the controls. Success rate at first attempt or need to call a more senior intubator after 2 failed attempts did not differ between groups. Because of the higher than usual dose of morphine that was used, we monitored the need to increase ventilator support over the next 24 h, in infants having their tubes changed, but found no difference between groups.Newborn infants, especially premature ones have adverse physiological responses to routine care procedures ,4. EndotOur hypothesis was that a moderate dose of morphine would facilitate intubation and stabilize vital signs better than placebo. Our data does not support this hypothesis. No significant difference was identified between the treatment and the control group in any prespecified outcome. The choice of severe hypoxemia as the primary outcome, although clinically very important, significantly limited the number of observations and increased the possibility of a type 2 error, as few infants met the criteria defining this outcome. The onset of action of morphine is about 5 minutes in infants, but the peak action occurs only at 15 to 30 minutes , perhapsThe only trend we identified was the duration of hypoxemia, which appeared longer in the treatment group. Most desaturations were in the mid 70's to low 80's range, but this is still a worrisome finding. We were unable to identify if birth weight, gestation or experience level of the intubator were significant contributors. Our sample size likely did not permit to identify such a contributor. Although morphine may not be potent enough to significantly relax infants to permit quicker and easier intubations, it may lead to decreased functional residual capacity in partially sedated infants, which could account for prolonged desaturations. The hypoxemia could have been compounded by the use of self-inflating bag and masks, which cannot provide a positive end-expiratory pressure (PEEP). Also, the larger dose of morphine used in this study could have contributed to this potential problem, by further decreasing the FRC in partially sedated infants. This trend is in keeping with the finding that, although not statistically significant, median duration of the procedure was 3 times as long in the treatment group as in the controls.Ninety four percent of infants experienced bradycardia, mostly vagal, during their intubation. As cerebral blood flow in infants is greatly dependent on heart rate, our data adds to the current knowledge that including atropine in the premedication appears warranted. Although there may be concern that atropine could mask hypoxia-induced bradycardia, the now universal use of oxygen saturation monitors should ensure that hypoxemia is identified.Previous trials have used various combinations of drugs for premedication and overall, they suggest that premedication is effective and safe. Kelly  and BarrOur study has several limitations. First, our sample size is relatively small, which precludes us from eliminating a type 2 error. We began this project with the assumption that severe hypoxemia would occur for about 30 seconds, which was not the case. An observational study would have been useful before making this assumption. Although limited in size, results of this trial should be useful for future investigators and clinicians in their choice of premedication. Second, due to limited resources , as this study was planned as a pilot, to assess feasibility and adequacy of equipment to obtain data, we decided not to stratify for gestational age. This could have been very useful in refining the findings, as more immature infants may respond differently to premedication in general and have less strength to struggle during an unpremedicated or not sufficiently premedicated painful procedure. Third, as we wanted to mimic our actual NICU practices, in view of modifying such practices, we did not restrict the study intubations to very experienced operators. As a result, there was substantial variability in the level of experience between individuals. Given our small number of patients, this might have impacted on the outcomes. Fourth, several eligible infants were not enrolled. This was due to unavailability of either trial investigators or parents, as many intubations occurred at night. The infants enrolled and those not enrolled had similar birth weights, gestation and reason for intubation, which is reassuring, but does not eliminate the potential for enrolment bias.Infants are entitled to effective pain management strategies . It seem2 oxygen saturation, ETT endotracheal tube, IVH intraventricular hemorrhage, PVL periventricular leukomalacia, NICU Neonatal Intensive Care Unit, PEEP positive end-expiratory pressureHR heart rate, BP blood pressure, Sp0The authors declare that they have no competing interests.BL led the study design and manuscript preparation and contributed to data collection. MM contributed to the study design and manuscript preparation. JD contributed to the study design, manuscript preparation and data collection. AK contributed to data gathering and manuscript editing. SG prepared the study medication and contributed her pharmaceutical expertise to the choice of medication dose for the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Cystic fibrosis (CF) lung disease manifest by impaired chloride secretion leads to eventual respiratory failure. Candidate genes that may modify CF lung disease severity include alternative chloride channels. The objectives of this study are to identify single nucleotide polymorphisms (SNPs) in the airway epithelial chloride channel, CLC-2, and correlate these polymorphisms with CF lung disease.The CLC-2 promoter, intron 1 and exon 20 were examined for SNPs in adult CF dF508/dF508 homozygotes with mild and severe lung disease (forced expiratory volume at one second (FEV1) > 70% and < 40%).PCR amplification of genomic CLC-2 and sequence analysis revealed 1 polymorphism in the hClC -2 promoter, 4 in intron 1, and none in exon 20. Fisher's analysis within this data set, did not demonstrate a significant relationship between the severity of lung disease and SNPs in the CLC-2 gene.CLC-2 is not a key modifier gene of CF lung phenotype. Further studies evaluating other phenotypes associated with CF may be useful in the future to assess the ability of CLC-2 to modify CF disease severity. Although greater than 1000 mutations of the CF gene product, CFTR are known, none of these can be used to make predictions about the occurrence of common complications, the severity, or course of pulmonary disease. The identification of a gene, which modifies the phenotypic expression of CF would be very important for understanding this complex disease. Because CF is a disease of chloride transport in respiratory epithelia, alternative chloride channels present in the airway may be able to partially compensate for the CF defect.CLC-2 is one candidate alternative chloride channel in respiratory epithelia. Localization to the luminal surface of the airway and perinatal downregulation of CLC-2 in mammalian lung suggests a role in lung morphogenesis ,2. PersiNasal polyps from CF patients were obtained at the time of elective surgery from 1989 to 1996. Genotypes of CF mutations for each patient was available, but not clinical status, according to approval by the Johns Hopkins Medical Institution Review Board. At harvest, the tissue was washed 3 times in HBSS, and incubated at 4°C overnight in Protease XIV (Sigma). Epithelial cells were isolated by gentle agitation and filtered through a 70-μm nylon cell strainer . Cells were grown on 1% collagen coated 35 mm dishes for 1 week. Cell lysates were prepared using 2% sodium dodecyl sulfate (SDS) at 65°C and a cell scraper. Equivalent amounts of total protein from primary CF nasal polyp cultured cell lysates were loaded onto an SDS-polyacrylamide gel electrophoresis (PAGE) system, electrophoresed and transferred to a nitrocellulose membrane. CLC-2 protein levels were detected using the polyclonal chicken anti-CLC-2 antibody and the enhanced chemiluminescent reaction as previously described  and human CLC-2 sequence [accession S7770] to amplify the promoter, intron 1 and exon 20 from adult CF subjects homozygous for delF508. Sequencing of the human CLC-2 promoter initially from one human genomic sample was performed by polymerase chain reaction using the 5'-flanking region of rat hpolE1 (dTCC GGG TCA ATA TCC TTC ACA TCG), which is approximately 2000 base pairs upstream from the rat CLC-2 coding sequence [Sequencing and identification of human CLC-2 promoter polymorphisms in 15 CF patients with severe obstructive lung disease (FEV1 ≤ 40% predicted) and 16 CF patients with mild disease (FEV1 ≥ 70% predicted) were performed by polymerase chain reaction using overlapping primers designed from the initial hCLC-2 clone. The only product that yielded a SNP was amplified using primers 15F dGTC CCA GGA GTA GAC TTC C and 16R dCAC TGC CCT CTG GCC TC providing a 760 base pair product, using cycling conditions of 94°C 6 mins, 35 cycles of 94°C 30 s, 59°C 30 s, 72°C 30 s and 72°C 6 min. A nested reaction with 20 uM primers 17F dTCC CCT CCG GCC TAC CCC TTC CGG T and 18R dGGA AGG ATT CGG AGA GGG TTG GGG C amplified both a 150 and 300 bp product using Epicentre MasterAmp™ buffer J  with cycling conditions 94°C 6 mins, 35 cycles of 94°C 30 s, 64°C 30 s, 74°C 30 s and 74°C 6 min.Because regulation of a gene may occur also through its first intron we amplified this region from all subjects using primers 1F' dCGC TGC AGC ACG AGC AGA C and 1R' dCCC AAG GTC CTG AGT GTA CC, which yielded a product 2273 bp product. Cycling conditions were 95°C 6 mins, 35 cycles of 95°C 30 s, 63°C 30 s, 72°C 3 minutes and 72°C 6 min. Finally, because exon 20 is alternatively spliced in rat lung [With PCR amplification of all 31 genomic CF samples using primers listed in Table CLC-2 protein is nearly undetectable in postnatal rat lung . High leIn order to minimize the confounding of genotype, race and age, all individuals were homozygous for delF508 mutation of CFTR, Caucasian, and over 17 years old. FEV1% determined 2 cohorts, one with mild CF lung disease with average FEV1% of 77.4 ± 3.18 SEM  as expected from the rat genomic structure , which mThere were five subjects with severe CF lung disease (FEV1 < 40%), who had the genotype A/G, whereas eleven had G/G at position -693 (Table The first intron of human CLC-2 was amplified and the 2273 bp product confirmed by gel electrophoresis. This sequence correlates with bp 319453 to 321725 of ref|NT_0292533|Hs_29412. Human CLC-2 intron 1 has regions with as much as 74% sequence identity with rat (gi 2873366) and 85% with mouse (gi28494743) Figure  and 3b. Primers used to examine the potential exon 20 splice variant region in hCLC-2 amplified a 481 bp fragment that correlates with bp 328123 to 328556 of human genomic sequence NT_0292533 and 2446 to 2617 of hCLC-2 cDNA (accession S7770). There were no SNPs identified using all 31 patient samples.With an autosomal recessive pattern of inheritance, CF was long considered a monogenic disease with 1 mutant allele inherited from each parent. While CF neonatal screening is offered in several states of the U.S., counseling of families has been difficult, because CF genotyping does not easily predict onset and severity of pulmonary complications . Strateg++-dependent chloride channel (CaCC) [There are several chloride conductances, which have been characterized in the mammalian lung: the cAMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR) , the Ca+l (CaCC) -18, the l (CaCC) , the purl (CaCC) ,21, and l (CaCC) ,22-24.++-dependent chloride channel [++-dependent chloride conductance is low in the intestine of the CF knock-out mouse. To take advantage of alternative chloride channels in the lung, UTP analogues have been used to stimulate chloride secretion in CF individuals via the purinergic receptor-mediated chloride channels [One or more of the chloride channels present in the respiratory epithelium may be able to partially compensate for defects in another. For example, there was no lung pathology in the first CF knock-out mouse models, where there is enhanced activity of a Ca channel -27, howe channel . The CF channels ,21.One member of the ClC family of chloride channels may also be an alternative chloride conductance in the airway epithelium. We have demonstrated that CLC-2 mRNA and protein are abundantly expressed in the fetal lung ,2 and thIn this study of 31 CF subjects, we identified 5 single nucleotide polymorphisms that have not previously been described for human CLC-2. One of these is -693 relative to the ATG start site of hCLC-2 (Genbank S7770). The -693 G/A polymorphism is a putative AP-2 binding site, predicted by TESS and MATINSPECTOR ,13 and mIn the rat, SP-1 sites are important for gene regulation ,29,30. CWe also identified 4 polymorphisms in hCLC-2 intron1. These did not appear more or less frequently in the mild CF subjects. Two of the polymorphisms were in complete linkage disequilibrium. The polymorphisms were not identified in areas that were highly conserved in rat or mouse. While splice variants of exons may be affected by intron/exon boundaries, we did not find any polymorphisms in the region of the rat exon 20 splice variant .These findings suggest that CLC-2 is not regulated differently at the genomic level in relatively healthy CF adults. Lack of an association in this study does not exclude the possibility that CLC-2 plays a role in modifying the CF phenotype as might be suggested by the variability of CLC-2 protein expression in primary respiratory epithelial cells from CF subjects in this study. Although we were limited by inadequate power with a small sample size and because phenotypic contrast was low, our data suggest that gene regulation of CLC-2 in relation to polymorphisms in regulatory domains does not play a major role in protection against CF lung disease. Studies which rely on recruitment of small numbers of patients have been shown to detect a difference when a strong relationship is present . AnotherThe identification of candidate genes, which may modify CF lung disease is important so that new therapies may be developed. Multi drug resistant genes have recently been identified that provide some "protection" to the CF lung phenotype . Ion traLastly, gene expression and function may be independent of genomic polymorphisms, as suggested by our data demonstrating variable expression of hClC-2 protein in CF nasal polyps and must also be considered as a mechanism whereby, CLC-2 could alter the course of CF disease. Haug et al. recently identified mutations in the CLC-2 coding region that are associated with idiopathic generalized seizures in humans . No lungThis study provides important information about the human CLCN2 genomic organization. Several polymorphisms of key regulatory domains of CLCN2 were identified in a cohort of subjects with cystic fibrosis, who carry the same CF genotype. While we have found no significant association of CLC-2 polymorphisms with FEV1 % predicted in adulthood, further study of potential polymorphisms in CF subjects at an earlier age and investigation of potential mutations in the coding region of CLC-2 that would lead to enhanced transepithelial chloride transport would be necessary to determine if CLC-2 can modify CF.The author(s) declare that they have no competing interests.CB provided overall study design, analysis, and drafted the manuscript. TH designed sequencing methods and analyzed alignment. AS and PB conducted experiments. EB contributed to the design of the study and OS designed sequencing methods and provided analysis including statistics.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A wealth of quality genomic and mRNA/EST sequences in recent years has provided the data required for large-scale genome-wide analysis of alternative splicing. We have capitalized on this by constructing a database that contains alternative splicing information organized as splicing graphs, where all transcripts arising from a single gene are collected, organized and classified. The splicing graph then serves as the basis for the classification of the various types of alternative splicing events. is a database of Drosophila melanogaster exons obtained from FlyBase arranged in a splicing graph form that permits the creation of simple rules allowing for the classification of alternative splicing events. Pfam domains were also mapped onto the protein sequences allowing users to access the impact of alternative splicing events on domain organization.DEDB DEDB's catalogue of splicing graphs facilitates genome-wide classification of alternative splicing events for genome analysis. The splicing graph viewer brings together genome, transcript, protein and domain information to facilitate biologists in understanding the implications of alternative splicing. Drosophila melanogaster genome in March 2000 [Drosophila melanogaster Exon Database) [The completion of the draft sequence of the rch 2000 ,2 and thrch 2000  presentsrch 2000 . We thenrch 2000  domains rch 2000 . All theatabase) . To aid Drosophila melanogaster genome annotations (release 3.2) were obtained from FlyBase [ FlyBase  as Game The transcripts contained in the database were retrieved and clustered on the basis that they occupy overlapping genomic positions. Splicing graphs are then constructed using these clusters of transcripts. In each cluster, exons and introns having identical start and end positions are merged into nodes and connections respectively. The nodes are then linked via connections to form the complete splicing graph that is loaded into the database. In cases where the transcripts are located on the negative strand, the entire splicing graph is reverse complemented so that all the splicing graphs contained in the database have sense strand nucleotide sequences. These steps are graphically depicted in Figure Rules are then derived to detect specific alternative splicing events as shown in Figure . Users can query the database using FlyBase gene names, FlyBase Gene Symbols, Pfam Accession Numbers or Pfam Identifiers via the query page. Users can also query the database using BLAST [Drosophila melanogaster homology together with alternative splicing information of a particular gene. Lists of splicing graphs for the various types of alternative splicing events are also provided on the website for users who are interested in a certain type of alternative splicing. For users who wish to use large subsets of the data, they can download the XML files available from the same site. To aid parsing and validation of the XML file, a XML schema is available. DEDB can also be accessed via links on Flybase gene records, under the external database links section. Correspondingly, the DEDB Splicing Graph Viewer provides links back to FlyBase Gene and Annotation records, where experimental evidence for the gene structure has also been provided. Basic statistical analysis of the database can be found at the DEDB website .The database together with the splicing graph viewer is freely available at ng BLAST  searchesThe splicing graph viewer consists of HTML pages created using a series of Python CGI (common gateway interface) scripts served by the Apache web server. The splicing graph viewer Figure  is a thrDrosophila moe gene, splicing graph 916, on the DEDB methodology link) resulting in the user having to correlate large amounts of data to comprehend all the alternative splicing events taking place. The DEDB schematic representation of the splicing graph is different from the one proposed by Heber et al. [Drosophila melanogaster homology of their gene of interest, which is made possible through a BLAST search on the DEDB query page. The splicing graph of the Drosophila melanogaster homology may provide insights into the possible splice variants in the gene of interest. It could also provide information on the level of conservation of alternative splicing between orthologs.By condensing all the various splicing variants into a single graph, where each splicing variant is a path through the graph, users can quickly establish the types and effects of various alternative splicing events present in the gene. Users can quickly pick up bifurcations which denote alternative splicing events far quicker than in the case of the traditional approach of presenting separate schematic representations of each splice variant, where the user has to correlate the splicing patterns from the transcript diagrams, to determine the impact and type of alternative splicing. The classical approach is particularly tedious in cases where the number of splice variants are numerous  are alternatively splicing. This is significantly less than the amount of alternative splicing found in higher eukaryotes like humans , but sufDrosophila melanogaster are also thought to result in greater intron definition. The data observed could be due to the splicing machinery adopting a definition model dependent on the length of the intron or exon in question [Failure of intron definition is more likely to result in intron retention as opposed to exon definition in which case, failure leads to cassette exons. Initial analysis of the DEDB data indicates a bias towards cassette exons  over intron retention (983) events, so that exon definition is less stringent than intron definition. The short introns in question . This isInformation content analysis indicates that alternative donor and acceptor sites  possess less information than constitutive sites . This observation is consistent with the general notion that alternatively spliced exons exhibit splicing motifs deviating more from the consensus motifs . CassettThe addition of Pfam domain information allows users to assess the impact of alternative splicing events on the proteins generated, enabling correlations not possible with the genome annotations alone.Future work would focus on integrating other relevant information onto the splicing graphs, such as three-dimensional structural information as well as DEDB analysis results. Expansion of the splicing graph representation available in DEDB to other organisms is also underway.The data housed in DEDB is organized as splicing graphs, which allows for ease of alternative splicing classifications. This has allowed DEDB to provide clean sets of data containing specific types of alternative splicing events. These specific sets of data could prove useful in understanding the biological basis of alternative splicing because different forms of alternative splicing have different biological basis. The splicing graph viewer provided allows biologists to quickly and intuitively understand the effects of alternative splicing on a gene of interest, thus aiding their research. suitable for most graphical web browser. XML files of the data contained in the database are also available together with an XML schema to aid parsing.The database is available at BTKL carried out the construction of the database as well as the splicing graph viewer. TTW and SR are responsible for the database concept and participated in its design and construction. All authors have read and approved the final manuscript.


Normalization is a critical step in analysis of gene expression profiles. For dual-labeled arrays, global normalization assumes that the majority of the genes on the array are non-differentially expressed between the two channels and that the number of over-expressed genes approximately equals the number of under-expressed genes. These assumptions can be inappropriate for custom arrays or arrays in which the reference RNA is very different from the experimental samples.We propose a mixture model based normalization method that adaptively identifies non-differentially expressed genes and thereby substantially improves normalization for dual-labeled arrays in settings where the assumptions of global normalization are problematic. The new method is evaluated using both simulated and real data.The new normalization method is effective for general microarray platforms when samples with very different expression profile are co-hybridized and for custom arrays where the majority of genes are likely to be differentially expressed. Microarray technology provides simultaneous measurements of expression levels for thousands of genes. Each step from sample preparation to data analysis, however, contains potential sources of bias and variability. Proper normalization adjusts for differences which interfere with the comparison of intensities of different labels at a given probe and with the comparison of intensities of corresponding probes on different arrays. Proper data normalization should allow for the comparison of expression levels across different arrays. Subsequent data analysis results are heavily dependent on effective normalization.Normalization issues differ for dual-labeled platforms compared to single labeled platforms such as the Affymetrix GeneChip arrays. In this paper we address normalization for dual-labeled arrays with either cDNA or oligonucleotide probes. The objective of normalization for dual-labeled arrays is to correct for differences in intensities for the two labels on the same array. These differences arise from factors such as differences in sample concentrations, differences in photomultiplier tube setting, and differences in the affinity of the two labels for DNA.Median or mean based global normalization methods use a single normalization factor applied to all genes on the array to adjust for labeling bias ,2. Such There are some methods which attempt to adaptively identify the subset of 'housekeeping' genes -8. Theseet al. proposed a Gamma-Gamma-Bernoulli model for identifying differentially expressed genes in dual labeled arrays [Newton d arrays . We haveAs described in the Methods section, we have applied our adaptive method to both the simulated data and real data. We have also compared our method with the global method and the intensity-based lowess method.2 scale) when the number of under-, null, and over-expressed genes were 200, 100, and 100, to more than a three fold difference  when the number of under-, null, and over-expressed genes were 200, 50, and 50. The RMSEs for the adaptive method ranged from 0.078 to 0.159 in log2 scale.Results of the simulation studies are shown as bar plots in Figure We compared the histogram of observed intensities to the fitted marginal density from the adaptive method as a simple check to see whether the proposed model and the estimation procedure are in line with available data. Figure 2 scale  Figure . Better Results comparing RMSEs for the adaptive method, the global method and the lowess method with real data are shown in Table In this paper, we propose a new method for normalization of dual-labeled arrays in cases where the number of differentially expressed genes is substantial and not necessarily symmetric in direction. The method performed effectively with both simulated and real data.c into Newton's Gamma-Gamma-Bernoulli model [We started our model building initially by introducing an unknown constant li model . The mixcRk) ~ Gammalog(Gk) ~ Gamma     (1A)logcRk) ~ Gammalog(Gk) ~ Gamma     (1B)log ~ GammaWe found that when the differential expression was symmetric between the two samples, the model worked well. However, the error increased significantly when the ratio of the numbers of under- to over-expressed genes shifted from 1.γR and γG for the red channel and green channel intensities. For this modified two-component mixture, the error still remained large. We then extended the model into a three-component mixture model listed as Equations 8A-8C in the additional material [see In order to make the model more flexible, we modified the model by assigning different scale factors rial see . The mod2(ratio) became larger . The mole 3 see  and Figule 3 see .We compared our adaptive method with the global method and the intensity-based lowess method. The lowess method assumes that in each intensity interval either the majority of genes are non-differentially expressed or the numbers of up- and down-regulated genes are equal. The global median normalization makes these assumptions only over the array as a whole. It is not surprising that our method performed much better that the above two methods, because the global median method only works well when the assumptions are valid while the intensity-based lowess method is only effective when there are intensity-dependent biases.et al. proposed using the lowess normalization separately within each grid on the array [Correlation structure is complicated for the thousands of genes on a microarray. In our model, the intensity of each channel is conditionally independent given the scale parameter, but not marginally independent.Therefore, we are not assuming the intensities in two channels are independent. Although we did not generate correlated genes in our simulated data sets, correlations of genes do exist in the real data sets we tested. Spatial correlations are also possible but our method is not designed for that purpose. Yang he array . Our algLimited simulations were performed in this study. We also tried to use real data to test our method. Since an appropriate data set with known normalization factor was not available, we synthesized such data sets by sub-setting large arrays in which the true normalization factor could be accurately estimated. In the process of synthesizing such small arrays we had to choose an empirical threshold to stratify the differentially expressed genes and non-differentially expressed genes. Although we do not believe that the superiority shown for our algorithm depends critically on the threshold chosen nor on details of the synthesis, it would be preferable to evaluate the algorithm on real data sets with know normalization factors.Although our method is designed for dual-labeled cDNA array, it can be extended to single channel Affymetrix chip data. The most popular normalization method for the Affymetrix chip compares each array to a single base line array for probe set summaries. The assumptions behind the normalization method are that the majority of the genes are non-differentially expressed and the numbers of over- and under-expressed genes are roughly equal; the same assumptions as those for dual-labeled cDNA arrays. We could treat the base line array as the 'reference channel' and the other array as the 'test' channel and apply our algorithm to probe set summaries. For Affymetrix chip data, there are multiple base pairs in a probe set and each probe has an intensity measurement. Several alternative normalization methods of Affymetrix arrays utilize the probe level information. For example, method based on an 'invariant set' proposed by Li and Wong assumes that a probe of a non-differentially expressed genes in two arrays to have similar ranks and uses an iterative procedure to identify the invariant set which presumably consists of points from non-differentially expressed genes .Our new normalization method does not require that the majority of genes be non-differentially expressed, and doesn't require multiple array replicates, dye swaps, spiked controls, or housekeeping genes. It appears much more effective than standard methods when the numbers of over- and under-expressed genes are unequal, and the majority of the genes are differentially expressed. It can be very useful for general microarray platforms when samples with very different expression profile are co-hybridized and for custom arrays where the majority of genes are likely to be differentially expressed. In both of these settings, standard normalization methods are problematic.k in two channels as Rk (red) and Gk (green). Let c be a positive constant which is related to the normalization constant. The observed intensities for gene k in two channels are cRk and Gk. We assume the logarithm of intensity in each channel has a Gamma distribution. The genes on the array belong to three different groups: 1) non-differentially expressed; 2) under-expressed; and 3) over-expressed. The overall data will be fitted into a mixture model listed below.We define true intensities for a specific gene k,For a non-differentially expressed gene cRk) ~ Gammalog(Gk) ~ Gamma     (2A)log.k,For an under-expressed gene cRk) ~ Gammalog(Gk) ~ Gamma     (2B)log ~ Gamma.k,For an over-expressed gene cRk) ~ Gammalog(Gk) ~ Gamma     (2C)log ~ Gamma.Gamma distributions, the parameters a and a0 are shape factors, and the parameters sk, γ, γ1,γ2, ,  are scale factors. The parameters a, a0, γ, γ1, γ2 will be estimated from the data.In the above pu, po and pn be the densities of  for under-expressed, over-expressed and non-differentially expressed genes, respectively. The joint distributions of  in three groups can be derived as follows [details see Let θ denote the unknown parameter vector , which can be estimated by maximizing the likelihood function of observed data. We used the EM algorithm [p1 be the proportion of under-expressed genes and p2 be the proportion of over-expressed genes. We define indicator binary variable zk1 to be 1 if the kth gene is under expressed, 0 otherwise; and zk2 to be 1 if the kth gene is over expressed, 0 otherwise. The complete-data loglikelihood for all spots can be derived as follows,Let lgorithm  for thisp1 and p2. This yieldsIn the M-step, we first take derivative on Equation (4) with respect to K is the total number of genes on the array.where θ.To maximize Equation (4), we only need to maximize Equation (6) because the left out terms do not depend on the parameter zk1 and zk2 given the other parameters from the M-step.In the E-step, we compute the conditional expectations of c was obtained, the normalization constant for log intensity ratio data can be calculated as log(1/c).Once the constant c = 1.5, a = 118, a0 = 410, γ = 31, γ1 = 23, and γ2 = 29. Six scenarios were included using different proportions of non-differentially expressed genes and different ratios of under- to over- expressed genes, as listed in Table Simulation studies were performed by generating two channel intensities from the mixture model with 2(1/) and the true log2(1/c) was calculated. The global method takes the median log2(ratio) of all genes in each data set as the normalization factor. The lowess method performs robust locally linear fits of M-A plot and corrects the biases that are dependent on spot intensity [2(ratio) using the lowess method and the normalized log2(ratio) using the global method with the true normalization factor (log2(1/c)) for all genes was also calculated for the same 100 data sets.One hundred data sets were generated for each scenario and the RMSE between the estimated logntensity . The RMS2 scale to reflect the experimental noise in inbred strains of mice or cell line data and 0.5 in log2 scale to reflect a larger experimental noise in human tissue data [Gaussian white noise was also added when generating the simulated data. We used standard deviation of 0.25 in logsue data .In-silico studies were performed on real data. We tested the method on ten arrays from publicly available breast cancer data [2(ratio) of all genes was considered as the true normalization factor c. The genes were then divided into three groups: over-expressed genes (log2(ratio)>1), non-differentially expressed genes(-1.5<log2(ratio)<1.5), and under-expressed genes (log2(ratio)<-1). We randomly sampled a specified number of genes from each group  and then combined them into an in-silico array. We constructed 100 datasets for each of the 10 arrays in this way and the RMSE between the estimated log2(1/ ) and the true log2(1/c) was calculated.cer data . Each arc = 1.5, a = 90, a0 = 120, γ = 8, γ1 = 6, and γ2 = 10 at three different noise levels (A) SD = 0; (B) SD = 0.25; and (C) SD = 0.50.Figure 4: Bar plots show comparison of RMSE by using the adaptive method (black bar) and global method (grey bar) with simulated data generated from a mixture model with Click here for filec = 1.5, a = 90, a0 = 120, γ = 8, γ1 = 6, and γ2 = 10. The superimposed curve on each plot is generated from the fitted model.Figure 5: Histograms and the estimated densities of log(ratio) and log(intensity) for a simulated data of a mixture model with Click here for fileEquations 8A-8C: a three-component mixture model.Click here for fileFigure 6: Histograms and the estimated densities of log(ratio) and log(intensity) for a set of real data generated from array svcc109. The superimposed curve on each plot is generated from the fitted model. The procedure to generate the data was described in the paper and the sampling rate was shown in Table 3 see .Click here for fileTable 3: Different number of genes sampled in each interval.Click here for fileTable 4: Comparison of RMSE by using the adaptive method and global method with real data by a different sampling method. The procedure to generate the data was described in the paper and the sampling rate was shown in Table 3 see .Click here for filepn, pu, and po Derivation of joint distributions of  for Click here for file


Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone.2 antiplasmin, prevented collagen dissolution. During ECM degradation breast cancer cell lines expressed urokinase-type plasminogen activator (u-PA) and uPA receptor, and MMPs-1, -3, -9,-13, and -14. The normal breast epithelial cell line expressed low levels of MMPs-1, and -3, uPA and uPA receptor. Inhibitors of both the PAS (aprotinin and PA inhibitor-1) and MMPs  blocked collagen degradation, demonstrating the requirement of both plasminogen activation and MMP activity for degradation. The activation of MMP-13 in human breast cancer cells was prevented by plasminogen activator inhibitor-1 but not by tissue inhibitor of metalloproteinase-1, suggesting that plasmin activates MMP-13 directly.The 3 breast cancer cell lines all produced significant degradation of the 3 collagenous extracellular matrices (ECMs) whilst the normal breast cell line was without effect. Breast cancer cells displayed an absolute requirement for serum to dissolve collagen. Degradation of collagen was abolished in plasminogen-depleted serum and could be restored by the addition of exogenous plasminogen. Localization of plasmin activity to the cell surface was critical for the degradation process as aprotinin, but not αThese data demonstrate that breast cancer cells dissolve type I collagen and that there is an absolute requirement for plasminogen activation and MMP activity in the degradation process. Breast cancer is the most frequent cancer in the female population of industrialized countries. Metastasis of breast cancer cells to the skeleton occur in >70% of patients with progressive disease, resulting in debilitating symptoms such as severe bone pain, fractures, hypercalcaemia and spinal cord or nerve compressions due to extensive bone loss and tumour cell growth and expansion. Such bone loss occurs as a result of increased bone matrix resorption but the mechanisms by which cancer cells mediate this increased degradation have not been fully elucidated. Obviously, tumour expansion in bone requires the removal of the extracellular matrix (ECM) that is particularly abundant in bone. Cancer cells express matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) -3 and thThe matrix metalloproteinases (MMPs) constitute a large family of structurally related matrix degrading proteases that have pivotal roles in development, tissue remodelling, and cancer -6. The gThe PAS comprises: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), their inhibitors, and receptors. T-PA is thought to be more important in fibrinolysis, due to its fibrin binding capacity, whilst u-PA, especially when it is bound to its specific cell surface receptor (u-PAR), is thought to be involved in tissue remodelling and cell migration processes . WhereasTo date, emphasis has focused on the ability of breast cancer cells to stimulate the formation and activity of osteoclasts, the cell primarily responsible for bone resorption under physiological conditions. The ability of osteoclasts to degrade bone lies in their ability to secrete protons and specialized collagenolytic proteinases, the cysteine proteinases in the acidic microenvironment that underlies osteoclasts during bone resorption .in vivo support the idea that they play an important role in metastasis of tumour cells  using plasminogen and S2251, the chromogenic substrate for plasmin.Differences between control and treatment groups were determined by the Mann Whitney U-test.


Two human coronaviruses are known since the 1960s: HCoV-229E and HCoV-OC43. SARS-CoV was discovered in the early spring of 2003, followed by the identification of HCoV-NL63, the fourth member of the coronaviridae family that infects humans. In this study, we describe the genome structure and the transcription strategy of HCoV-NL63 by experimental analysis of the viral subgenomic mRNAs.The genome of HCoV-NL63 has the following gene order: 1a-1b-S-ORF3-E-M-N. The GC content of the HCoV-NL63 genome is extremely low (34%) compared to other coronaviruses, and we therefore performed additional analysis of the nucleotide composition. Overall, the RNA genome is very low in C and high in U, and this is also reflected in the codon usage. Inspection of the nucleotide composition along the genome indicates that the C-count increases significantly in the last one-third of the genome at the expense of U and G. We document the production of subgenomic (sg) mRNAs coding for the S, ORF3, E, M and N proteins. We did not detect any additional sg mRNA. Furthermore, we sequenced the 5' end of all sg mRNAs, confirming the presence of an identical leader sequence in each sg mRNA. Northern blot analysis indicated that the expression level among the sg mRNAs differs significantly, with the sg mRNA encoding nucleocapsid (N) being the most abundant.The presented data give insight into the viral evolution and mutational patterns in coronaviral genome. Furthermore our data show that HCoV-NL63 employs the discontinuous replication strategy with generation of subgenomic mRNAs during the (-) strand synthesis. Because HCoV-NL63 has a low pathogenicity and is able to grow easily in cell culture, this virus can be a powerful tool to study SARS coronavirus pathogenesis. Until recently only two human coronaviruses were known – human coronavirus (HCoV) 229E and HCoV-OC43, representatives of the group 1 and 2 coronaviruses, respectively. Both were identified in 1960s and are generally considered as common cold viruses. An outbreak of severe acute respiratory syndrome (SARS) in the spring of 2003 led to the rapid identification of SARS-CoV . We idenHCoV-NL63 is a member of the coronaviridae family that clusters together with arteri-, toro- and roniviruses in the order of the nidovirales. Coronaviruses are enveloped viruses with a positive, single stranded RNA genome of approximately 27 to 32 kb. The 5' two-third of a coronavirus genome encodes a polyprotein that contains all enzymes necessary for RNA replication. The expression of the complete polyprotein requires a -1 ribosomal frameshift during translation that is triggered by a pseudoknot RNA structure ,9. The pCoronavirus replication is a complex, not yet fully understood mechanism ,14. The In this study, we analyzed the genome structure of HCoV-NL63. First, we focus on the unusual nucleotide composition of the RNA genome. We describe in detail the bias in the nucleotide composition and its influence on the codon usage of this virus. We provide a possible mechanistic explanation for a shift in nucleotide bias at two-third of the HCoV-NL63 genome that is based on the RNA replication mechanism. Second, we describe in detail the different sg mRNAs generated during HCoV-NL63 replication and their relative abundance.We described previously that the newly identified HCoV-NL63 virus has a typical coronavirus genome structure and gene order dCTP. Prehybridization and hybridization was done in ULTRAhyb buffer (Ambion) at 50°C for 1 and 12 hours, respectively. The membrane was then washed at room temperature with low-stringency buffer  and at 50°C in high stringency buffer . Images were obtained using the STORM 860 phosphorimager (Amersham Biosciences) and data analysis was performed with the ImageQuant software package. The size of sg mRNA fragments of HCoV-NL63 were estimated from their migration on the Northern blot using the sg mRNA of MHV as size marker.Gel electrophoresis of viral RNA was performed on a 1% agarose gel with 7% of formaldehyde at 100 Volt in 1×MOPS buffer . Transfer onto a positively charged nylon membrane (Boehringer Mannheim) was done overnight by means of capillary force. RNA was linked to the membrane in a UV crosslinker (Stratagene). For generation of the HCoV-NL63 probe, the RT-PCR product was further amplified with 5' primer N5PCR1 (CTG TTA CTT TGG CTT TAA AGA ACT TAG G) and 3' primer N3PCR1 (CTC ACT ATC AAA GAA TAA CGC AGC CTG). Similarly, the MHV probe was amplified with 5' primer MHV_UTR-B5' (GAT GAA GTA GAT AAT GTA AGC GT) and 3' primer MHV_UTR-B3' (TGC CAC AAC CTT CTC TAT CTG TTA T). Labeling of the probes was done in a standard PCR reaction with specific 3' primers (N3PCR1 and MHV_UTR-B3') in presence of [α-The L/body TRS junctions were PCR-amplified from an HCoV-NL63 cDNA bank. We performed 35 cycle PCR with the 5' L primer (L5 – TAA AGA ATT TTT CTA TCT ATA GAT AG) and gene specific 3' primers . The PCR products were analyzed by electrophoresis on a 0.8% agarose gel and products of discrete size were used for sequencing using the BigDye terminator kit (ABI) and ABI Prism 377 sequencer (Perkin Elmer). Sequence analysis was performed by Sequence Navigator and AutoAssembler 2.1 software.The complete genome sequence of HCoV-NL63 [The authors declare that they have no competing interests.KP carried out the viral RNA isolation, RT-PCR, sequencing of sg mRNAs, Northern blot evaluation and all computer analysis done in this study; MFJ carried out the full genome sequencing; all authors participated in writing the manuscript. Lv/dH and BB are the principal investigators


How much is the aggression we observe in nonhuman primates the result of culture, and will the answer provide insights into our own violent behaviour? Dances with Wolves (the 1990 movie with Kevin Costner) is violent, people look at you as if you are crazy. They see an idyllic, sentimental movie, with beautiful landscapes, showing a rare white man who respects American Indians. The bloody scenes barely register.Upon arrival from Europe, now more than two decades ago, I was taken aback by the level of violence in the American media. I do not just mean the daily news, even though it is hard getting used to multiple murders per day in any large city. No, I mean sitcoms, comedies, drama series, and movies. Staying away from Schwarzenegger and Stallone does not do it; almost any American movie features violence. Inevitably, desensitization sets in. If you say, for example, that Saturday Night Live for its inside commentary on peculiarly American phenomena, such as cheerleaders, televangelists, and celebrity lawyers. But SNL is incomplete without at least one sketch in which someone's car explodes or head gets blown off. Characters such as Hans and Franz (“We're going to pump you up!”) appeal to me for their names alone , but when their free weights are so heavy that their arms get torn off, I am baffled. The spouting blood gets a big laugh from the audience, but I fail to see the humor.Comedy is no different. I love, for example, Did I grow up in a land of sissies? Perhaps, but I am not mentioning this to decide whether violence in the media and our ability to grow immune to it—as I also have over the years—is desirable, or not. I simply wish to draw attention to the cultural fissures in how violence is portrayed, how we teach conflict resolution, and whether harmony is valued over competitiveness. This is the problem with the human species. Somewhere in all of this resides a human nature, but it is molded and stretched into so many different directions that it is hard to say if we are naturally competitive or naturally community-builders. In fact, we are both, but each society reaches its own balance between the two. In America, the squeaky wheel gets the grease. In Japan, the nail that stands out gets pounded into the ground.Pan troglodytes) being far more violent than that equally close relative of ours, the bonobo (P. paniscus)  . But alsThere was a time when no review of human nature would be complete without assertions about our inborn aggressiveness. The first scientist to bring up this issue, not coincidentally after World War II, was Seville Statement on Violence  and the rich array of tool use by wild chimpanzees, orangutans (Pongo pymaeus), and capuchin monkeys (Cebus spp.)  . HoweverSocial culture is obviously harder to document than tool use. In human culture, for instance, it is easy to tell if people eat with knife and fork or with chopsticks, but to notice if a culture is egalitarian or hierarchical, warm or distant, collectivistic or individualistic takes time and is difficult to capture in behavioral measures. A well-documented monkey example of social culture is the inheritance of rank positions in macaque and baboon societies. The future position in the hierarchy of a newborn female can be predicted with almost one hundred percent certainty on the basis of her mother's rank. Females with relatives in high places are born with a silver spoon in their mouth, so to speak, whereas those of lowly origin will spend their life at the bottom. Despite its stability, the system depends on learning. Early in life, the young monkey finds out against which opponents it can expect help from her mother and sisters. When sparring with peer A she may utter screams that recruit massive support to defeat A. But against peer B she can scream her lungs out and nothing happens. Consequently, she will come to dominate A but not B. Experiments manipulating the presence of family members have found that when support dwindles dominant females are unable to maintain their positions . In otheMacaca mulatta), known as quarrelsome and violent, were housed with the more tolerant and easy-going stumptail monkeys (M. arctoides)  . Stumptactoides) . PrimatePLoS Biology, we now have the first field evidence that primates can go the flower power route (Papio anubis) on the plains of the Masai Mara, in Kenya, wage wars of nerves, compromising their rivals' immune systems and pushing up the level of their blood cortisol (Not unlike rhesus monkeys, baboons have a reputation for fierce competition and nasty fights. With the study by Robert Sapolsky and Lisa Share published in this issue of er route . Wild bacortisol . An acciIt is somewhat ironic that at a time when researchers on human aggression are increasingly attracted, albeit with a far more sophisticated approach, to the Lorenzian idea of a biological basis of aggression , studentLet us hope this applies to humanity as well.


Understanding the factors that affect patients' utilisation of health services is important for health service provision and effective patient management. This study aimed to investigate the specific morbidity and demographic factors related to the frequency with which general practice patients visit a general practitioner/family physician (GP) in Australia.A sub-study was undertaken as part of an ongoing national study of general practice activity in Australia. A cluster sample of 10,755 general practice patients were surveyed through a random sample of 379 general practitioners. The patient reported the number of times he/she had visited a general practitioner in the previous twelve months. The GP recorded all the patient's major health problems, including those managed at the current consultation.Patients reported an average of 8.8 visits to a general practitioner per year. After adjusting for other patient demographics and number of health problems, concession health care card holders made on average 2.6 more visits per year to a general practitioner than did non-card holders (p < .001). After adjustment, patients from remote/very remote locations made 2.3 fewer visits per year than patients from locations where services were highly accessible (p < .001). After adjustment for patient demographics, patients with diagnosed anxiety made on average 2.7 more visits per year (p = 0.003), those with diagnosed depression 2.2 more visits than average (p < .0001), and those with back problems 2.4 more visits (p = 0.009) than patients without the respective disorders.Anxiety, back pain and depression are associated with greater patient demand for general practice services than other health problems. The effect of sociodemographic factors on patient utilisation of general practice services is complex. Equity of access to general practice services remains an issue for patients from remote areas, while concession health care card holders are attending general practice more frequently than other patients relative to their number of health problems. The frequency of patient visits to general/family practice is affected by a range of factors including patient characteristics, physician/practice factors and broader issues such as access to services. A major contributor to the variance in visit rates to general/family practitioners is the complexity of health problems experienced by the patient. Those patients who attend general practice frequently report poorer health than those who attend less frequently ,2. Not aNumerous studies have found that for a subset of patients, the presence of psychological health problems is associated with frequent attendance at medical services -6. HigheThe general practitioner (GP) also influences patients' attendance rates through doctor-initiated visits and some practitioners attract a larger proportion of frequent attenders than others . RemunerPositive aspects to increasing general practice attendance includes recalling patients for chronic conditions as part of a program of structured care ,12. PatiThe available evidence on visit frequency to general/family practice comes from a range of settings in several countries. There are differences in general practice between countries that may affect patterns of patient visits and some studies are based on a limited number of practices, which may affect the generalisability of the findings ,15. AlthThe study reported here is part of the Bettering the Evaluation and Care of Health (BEACH) program, a national study of general practice activity in Australia. The BEACH method has been described in detail elsewhere . In summThis paper is based on a sub-sample of GPs who participated in BEACH between August 2001 and March 2002. For a subset of 30 out of the 100 encounters, the GP recorded, in addition to the problems managed at the encounter, any other major health problems of the patient that had not been managed at the current encounter. GPs were instructed to include chronic illnesses requiring ongoing care, past problems that affect present and future care and social problems that influence health. As many as 12 extra problems could be recorded per patient and when added to the maximum of four problems managed at the encounter this allowed up to 16 health problems to be recorded per patient. The GP recorded problems in free text, which were secondarily classified according to the International Classification of Primary Care (ICPC-2) using an extended vocabulary of terms ,18. ICPCMental health (particularly depression), asthma, diabetes, osteoarthritis and cardiovascular disease are five National Health Priority Areas that are major contributors to mortality, morbidity and health service costs in Australia . They arThe GP asked each patient in the sub-sample to recall how many times he/she had visited a GP in the previous 12 months.Commonwealth concession health care cards are available to people on limited incomes and entitle the holder to health services at greatly reduced cost. In 1998, around one third of Australians aged 15 years and over held a concession health care card . ConcessThe Remote Index of Australia (ARIA) was used to classify patients according to geographical remoteness and accessibility of services . PatientThe patient sample was a single stage cluster design with the GP as the primary sampling unit. Observations recorded by the same GP were therefore not independent and the statistical analysis adjusted for the correlation between patients within each cluster. We used procedures in SAS software V8.2 that adjust the standard error for the intra-cluster correlation  and all Number of annual visits to a GP was the main outcome of interest. After checking the linear relationship between number of visits and other ordinal variables , multiple regression was performed in two stages to identify predictors of visit frequency.1) Demographic predictorsWe performed multiple regression, with self-reported annual GP visits as an ordinal outcome to identify the independent demographic predictors of the frequency of visits to a GP after controlling for morbidity. We fitted the morbidity covariate as the total number of major health problems recorded for the patient.2) Morbidity predictorsWe fitted separate regression models to estimate the effect of specific morbidity on visit frequency, after adjusting for patient age, sex, other significant demographic predictors and the number of other health problems of the patient. The morbidities of interest were chronic problems most commonly managed in general practice, specifically depression, anxiety, back problems, diabetes, hypertension, asthma, ischaemic heart disease, and osteoarthritis . We inteThree hundred and seventy nine GPs participated in the sub-study. Six hundred and eighteen patients (5.4%) did not answer the question on visit frequency, giving a final sample of 10,755 respondents. Patient respondents and non-respondents were not significantly different in mean age, sex distribution or mean number of health problems recorded.While all states of Australia were well represented in the GP sample, the sample had a smaller proportion of GPs aged less than 35 years and was somewhat more urban than the population of Australian GPs Table .Patients aged less than 25 years were under-represented in the sample relative to all patients who had visited a GP in Australia at least once in 2001, while patients aged 65 years and older were over-represented (Table The majority of patients in the sample (83.3%) were from locations where services were "highly accessible", and 8.3% spoke a language other than English at home.The mean patient self-reported visit rate for the sample was 8.8 (95% CI: 8.3–9.2) visits to a GP per year. On average the GPs recorded 2.9 morbidities per patient, and half (51.1%) of the recorded problems were being managed by the GP at the current consultation (results not tabled).There was no difference in self-reported visit frequency between the sexes (p = 0.11) (results not tabled). The mean visit rate for children aged less than 5 was 5.8 visits per year, which fell to 4.3 for children 10–14 years of age, then increased linearly after age 15 years (p < .0001). There was a simple linear increase in frequency of visits with increasing number of recorded health problems (p < .0001). Therefore age was fitted as a categorical variable and number of problems as a numeric variable in the multiple regression models.After adjusting for age and sex, there was no difference in visit frequency according to language spoken at home Table . After aThe mean annual visit rates for patients with common chronic disorders were well above average Table . After aAs would be expected the number of health problems recorded for the patient was a major predictor of frequency of attendance at general practice. However this study demonstrated that specific chronic disorders had differential effects on visit frequency. Anxiety, back complaints, and depression had the greatest effects on increasing patient annual visits to a GP.The relationship between psychological health problems and increased medical visits has been demonstrated across many settings -3,5. TheOther research has reported a link between unexplained back pain and frequent attendance at medical services . In the Other research indicates that patients with diabetes make more general practice visits than average  and struAs expected, patients from remote areas reported visiting general practice less frequently relative to their health needs, confirming that geographic access to services remains a significant barrier to meeting the health care needs of patients from remote locations ,14.Concession health care card holders have more chronic and psychological health problems managed in general practice relative to the rest of the Australian general practice population . In thisThis study had the advantage of randomly sampling clusters of patients across a wide range of practices in all states and regions of Australia. A further advantage was having a qualified medical practitioner to record patient morbidity and the use of the medical record to help validate patient recall of GP visits.The methodology of this study was limited by using patient self-report of visit frequency and by sampling patients at the point of consultation. The average number of GP visits among Australians who visit a general practitioner at least once in a 12 month period is known to be six visits per year, increasing with increasing age , while the self-reported visit rate in the current study was 8.8 visits per year. Because the patients were sampled at the consultation, older patients who attend general practice more frequently had a greater chance of being sampled and this was reflected in the older age distribution of the patient sample. Even allowing for this selection bias, the mean annual self-reported visits was somewhat higher than expected given the sample's age structure, indicating some overestimation by patients in their recall of the number of GP visits. One Australian study that validated patient self-report against actual Medicare benefit claims found that patients who overestimate recent use of medical services are "telescoping" real but less recent events into the nominated time period and are in fact higher users of health services over the long term . In the This study has demonstrated that number of visits to a GP by patients in Australia is largely explained by the number of health problems the patient is experiencing. However psychological health problems and sociodemographic factors have differential effects on visit rates. GPs need to be alert to the particular psychological and social needs of patients who are attending frequently with multiple health problems. Equity of access, in terms of equal utilisation of services, continues to be an issue for patients living in remote locations.None declared.SK designed and performed the statistical analysis, researched and wrote the main draft. HB directed the study, participated in the design and assisted in writing and reviewing the final article.The pre-publication history for this paper can be accessed here: