Drosophila genes wingless (wg) and decapentaplegic (dpp) comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda.The Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g. endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative.All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions. Both genes encode secreted morphogens that generate combinatorial gradients across the developing imaginal leg discs , dachshund (dac)) (e.g. [wg and dpp are key factors involved in the early events of PD axis formation.The genes cs (e.g. ). These )) e.g. ,3). Thus. Thuswinwg and dpp at the top level, or whether the different branches are patterned through branch-specific mechanisms.In recent years several comparative studies in other arthropod species have suggested that the action of the leg-gap genes in PD patterning is evolutionarily conserved -9. Tus, wg and dpp expression during appendage formation to date mainly focus on insects . Onl Athalia ,13. HereGlomeris gene that shows sequence similarity to members of the TGF-beta gene family was isolated. In order to establish the orthology of this fragment, I performed a phylogenetic analysis incorporating a selection of TGF-beta proteins from Drosophila, other arthropods and a variety of deuterostome taxa. The resulting phylogenetic tree distinguishes two groups of proteins. One group comprises the arthropod dpp genes and their deuterostome homologs, the BMP2/4 genes. The second group consists of the Drosophila TGF-beta genes screw (scw) and glass bottom boat (gbb), and the remaining deuterostome BMP genes with TGF-beta homology, including zebrafish anti dorsalizing morphogenetic protein (ADMP). The Glomeris fragment resides in the dpp/BMP2/4 group and is therefore designated as Gm-dpp  and BMP2/4 from the yellow acorn worm (Ptychodera flava). However, support for this grouping is not statistically significant . A higher resolution of the dpp/BMP2/4 group may be achieved in the future by the aquisition of additional sequence information.A fragment of a dpp Fig. . The resGlomeris marginata [Gm-dpp is the weakest. A specific in situ hybridization signal is observed after approximately six hours of staining, whereas the normal staining interval of other genes ranges between 15 and 30 minutes. This extended staining time is responsible for the intense artificial background that is visible in the preparations displayed in this paper  weakly expressing Gm-dpp  has shown that the stripe of Gm-wg expression abuts the parasegment border and, thus, is located in cells of the anterior segmental compartment [Gm-wg stripes. Preparations of complete hemisegments of stage 3 embryos show, that expression of Gm-wg extends more or less contiguously across the neuroectoderm into the forming limb buds in all four different appendage types .The maxilla displays a rather dynamic expression profile of lla Fig. . Three dIn the mandible, a similar fragmentation of the initial mostly homogeneous expression pattern takes place. In the external lobe the expression is strong throughout and is separated from the expression domain in the internal lobe by an area of very weak expression Fig. . The expGm-wg in the maxilla is reminiscent of the pattern described for Gm-Dll [Gm-Dll gene is expressed in the primordia of the maxillary sensory organs. Expression of the Gm-wg gene, however, is at least partially complementary to the pattern of Gm-Dll. In older stages, strong expression of Gm-wg is not detected within the primordia of the sensory organs, but rather it surrounds the primordia  domains is more extensive than the more restricted expression pattern of Gm-Dll in two (later three) well-defined stripes . TherefGm-Dll has been described in the mandible [Gm-wg and Gm-Dll appear to overlap completely in the mandible. In preparations of mandibles labeled with a cocktail of probes against both genes no significant difference to the pattern of Gm-wg alone is observed  and most other arthropods  [In de (e.g. ). Togethds e.g. ,9,10,13),10,13Droar e.g. ,18,19). ,19. Drostgrowth) .Glomeris presented here may be interpreted in favor of this hypothesis. The Gm-wg gene is expressed along the ventral side in the legs and Gm-dpp is expressed most strongly in the leg tips. Taking these expression loci as the sources of Gm-WG and Gm-DPP protein, the resulting hypothetical protein gradients would facilitate PD patterning events similar to the ones in the Drosophila leg discs .The data from g. 11 in ). Howevedpp expression in arthropods is that the dorsal sector in Drosophila seems to be an exception rather than the rule. The hypothesis proposed by Prpic et al. [Drosophila and most other arthropod species. However, according to their hypothesis, the presence of combinatorial protein gradients is conserved. It should be pointed out in this context that the existence of a DPP gradient (or a WG gradient for that matter) has yet to be demonstrated in an arthropod other than Drosophila. Thus, although the expression data may be interpreted as the PD axis patterning using WG/DPP signaling being conserved among arthropods, it is obvious that comparative expression analyses alone cannot answer the question satisfactorily. It must now be considered whether experiments capable of demonstrating WG/DPP signaling during leg development in arthropods other than Drosophila may be conceived.In any case, the picture emerging from the available data on c et al.  attemptsAside from the primary PD axis, many arthropods have limbs with additional branches (rami). It has been proposed that these additional rami are patterned in the same way as the main branch, simply by duplications of the WG/DPP signaling system . Recent Glomeris develop ventral lobes that are very reminiscent of the endite lobes of the embryonic mouthparts in insects. The exclusive ventral origin of these lobes is further corroborated by the lack of expression of the dorsal marker optomotor-blind [Glomeris lobes possess Dll-positive sensory organs, which is typical of arthropod endites [The presence of endites in the mouthparts of myriapods is unclear, mainly because of the modified morphology of the adult gnathalia. Certain elements of the centipede mandible and first maxilla are probably derived from endites e.g. ,22) and  and 22])or-blind . Further endites ,24-26. T endites ), these Glomeris mandible develops two lobes, the internal and external mandibular lobe. Both lobes express Gm-dpp, but not at a position suggestive of a role in PD axis formation . These two sensory structures grow from the internal side of the stipes . The poGlomeris appear to utilize conventional WG/DPP signaling to organize PD growth. Similar results have been obtained recently for the endites in the grasshopper Schistocerca and the beetle Tribolium [Schistocerca at least one endite  grows without dpp expression  [omb as dorsal instructor is evolutionarily conserved, but H15 does not seem to be a general ventralizing factor in all arthropods. Thus, the dorsal, but not the ventral developmental mechanisms seem to be conserved. It is interesting that the expression data of wg and dpp suggest that the opposite is true. The wg expression on the ventral side is highly conserved among the arthropods, but the dpp patterns differ between species and in most part expression is not localized to the entire dorsal side. This paradox clearly demonstrates the limited understanding of the evolution of DV axis formation in arthropod appendages.A second role of the legs ,29. wg, s salei) ,17. The Glomeris has several sensory organs. Recent studies have identified the genes Dll, dac and H15, which show a restricted expression pattern in the primordia of the maxillary sensory organs [Dll and H15, are known from Drosophila to be activated upon signaling through the wingless pathway [Gm-wg surrounds the sensory primordia in the Glomeris maxilla. It may therefore be the case that cells expressing Gm-wg in the surrounding of the primordia signal to their neighbors within the primordia and stimulate them to activate Gm-Dll and Gm-H15. Minimally the activation of Dll appears to be a general feature of appendicular sensory organs in arthropods since Dll expression has been observed in appendicular sensory organs in chelicerates, crustaceans, myriapods and insects  [The maxilla of y organs ,17. Two  pathway ,30. It its e.g. ,25,31). ,31. Glomanlagen) ,33.Gm-wg and Gm-dpp during appendage development indicates a role for both genes in guiding this process. Involvement of wg and dpp in appendage development appears to be conserved among all extant arthropod classes including myriapods. The data from Glomeris and other arthropods suggest that the WG/DPP morphogen signaling system as it is known from Drosophila leg discs is present in all arthropods. However, this morphogen system apparently functions in only the main branch of the appendages, the so-called telopodite [Glomeris mouthparts supports this notion. Aside from the role in PD axis formation, the expression profile of Gm-wg suggests an additional role for this gene in patterning appendicular sensory organs.The expression of lopodite . Limb tyAnimals were collected during Spring 2003 in beech forests in the vicinity of Cologne, Germany and near Kranenburg, Germany. They have been treated as described before ,14. The Glomeris embryos of all developmental stages up to stage 6.1 (see [dpp-like gene fragments, the primers dpp-fw-1 (GAY GTN GGN TGG GAY GAY TGG) and dpp-bw-1 (CKR CAN CCR CAN CCN CAN AC) were used in the initial PCR, and the primers dpp-fw-2 (GGN TAY GAY GCN TAY TAY TG) and dpp-bw-1 were used in the nested PCR. Additional sequence information was gained by RACE PCR. No full-length fragment could be obtained and several artificial clones were encountered, probably representing chimeric products resulting from jumping PCR between different TGF-beta-like cDNAs. Using species specific primers, artificial and genuine fragments were identified. A confirmed genuine fragment of almost 360 bp was isolated and cloned. This fragment was used for sequence analysis and probe synthesis. The isolation of Gm-wg has been previously reported [Gm-wg (AJ616907); Gm-dpp (AJ843875).The cloning assays were based on cDNA transcribed from polyA-RNA extracted from selected 6.1 (see ,35 for areported . The GenPairwise alignments of aminoacid sequences were performed by searching GenBank  using thIn situ hybridization with digoxigenin-labeled RNA probes has been performed as previously described . Whole-m


Administrative health care databases may be particularly useful for injury surveillance, given that they are population-based, readily available, and relatively complete. Surveillance based on administrative data, though, is often restricted to injuries that result in hospitalization. Adding physician billing data to administrative data-based surveillance efforts may improve comprehensiveness, but the feasibility of such an approach has rarely been examined. It is also not clear how injury surveillance information obtained using administrative health care databases compares with that obtained using self-report surveys. This study explored the value of using physician billing data along with hospitalization data for the surveillance of adolescent injuries in Ontario, Canada. We aimed i) to document the burden of adolescent injury using administrative health care data, focusing on the relative contribution of physician billing information; and ii) to explore data quality issues by directly comparing adolescent injuries identified in administrative and self-report data.The sample included adolescents aged 12 to 19 years who participated in the 1996–1997 cross-sectional Ontario Health Survey, and whose survey responses were linked to administrative health care datasets (N = 2067). Descriptive analysis was used to document the burden of injuries as a proportion of all physician care by gender and location of care, and to examine the distribution of both administratively-defined and self-reported activity-limiting injuries according to demographic characteristics. Administratively-defined and self-reported injuries were also directly compared at the individual level.Approximately 10% of physician care for the sample was identified as injury-related. While 18.8% of adolescents had self-reported injury in the previous year, 25.0% had documented administratively-defined injury. The distribution of injuries according to demographic characteristics was similar across data sources, but congruence was low at the individual level. Possible reasons for discrepancies between the data sources included recall errors in the survey data and errors in the physician billing data algorithm.If further validated, physician billing data could be used along with hospital inpatient data to make an important and unique contribution to adolescent injury surveillance. The limitations inherent in different datasets highlight the need to continue rely on multiple information sources for complete injury surveillance information. The contribution of surveillance systems in providing valuable information for injury prevention and control is widely recognized; for example, surveillance data can be used to highlight the burden of injury, set priorities for prevention, and evaluate preventive strategies ,2. EstimAdministrative health care databases, due to their presumed near complete coverage of injuries requiring medical care and their lack of reliance on self-reports, may be particularly useful for injury surveillance. Such databases allow for local or regional estimates of the burden of injury, which has been identified as an important goal ,6,7, andIn Ontario, Canada, administrative health care databases that may provide information on the incidence of non-fatal injuries include hospital discharge and physician billing data. Although hospital discharge data have been widely used in Canada to study injury, the feasibility of using physician billing information for injury surveillance has rarely been investigated . These dIt is not clear how the injury information provided by administrative databases compares with that obtained from population-based surveys. A study of adolescent injuries we conducted using data from the Ontario Health Survey (OHS)  presenteThe overall purpose of this study was to explore the feasibility and value of using physician billing data for Ontario, Canada, along with hospitalization data, for the surveillance of adolescent injuries. The first objective was to document the burden of adolescent injury based on administrative health care data, focusing on the relative contribution of physician billing information and comparing overall estimates with surveillance information from survey data. The second objective was to examine data quality issues, by directly comparing adolescent injuries identified using administrative health care databases ("administratively-defined injuries") with those identified using self-report survey data ("self-reported injuries").The study sample included adolescents aged 12 to 19 years who participated in the health component of the 1996–1997 OHS (N = 3331), which was part of the National Population Health Survey (NPHS) . Survey Inpatient hospitalizations for injuries were identified using the Discharge Abstract Database (DAD) of the Canadian Institute for Health Information (CIHI) . All OntOHS respondents were asked a series of questions related to acute injuries in the past 12 months that were, from the perspective of the respondent, serious enough to limit normal activities (examples given by the interviewer included "...a broken bone, a bad cut or burn, a sprain, or a poisoning") . Particith revision [Inpatient hospitalizations for injury were identified for the 365-day period prior to the OHS interview, for each adolescent. An adolescent was considered to have an injury-related hospitalization if, during the one-year period, he or she had at least one documented hospital discharge with an External Cause of Injury Code (E Code) in the range 800–999 , based on the International Classification of Disease (ICD), 9revision .The OHIP physician claims database does not contain codes representing causes of injury. Rather, injuries were identified based on codes that reflect billable services ("procedure codes"), and the diagnoses associated with such services ("diagnostic codes"). To improve sensitivity, a combination of both diagnostic and procedure codes from the database was used to create an injury algorithm that would identify physician care for injury during the one-year study period, based on the methods of Tamblyn and colleagues . The devAdolescents who had at least one documented injury in either the hospitalization or the physician billing database were considered to have an administratively-defined injury. Adolescents with documented injury-related physician care at a physician's office or outpatient facility were considered to have a physicians' office visit for injury. Adolescents with documented injury-related physician care at an emergency department were considered to have an emergency visit for injury.Two alternative injury outcomes were created to examine the impact of decisions made in developing the physician billing algorithm. These outcomes were based on diagnoses that were relatively common and were captured only as possible injuries in the algorithm, including non-specific conditions of the musculoskeletal system and adverse effects of drugs and medications.First, the injury algorithm was used with the full sample of 2067 adolescents, resulting in estimates of adolescent injury-related physician care by gender and location of care. Descriptive analysis was then carried out for adolescents with non-missing data on important OHS variables (N = 2047). In addition to examining differences in the overall observed burden of adolescent injury between administrative and survey data, we also explored whether there were differences within demographic subsets of the sample where the types of injuries experienced or the types of injury care received were likely to differ. Thus, we examined the proportion of adolescents with administratively-defined and self-reported injuries separately by gender, age group, and rural versus urban residence. Since numerous comparisons were possible within the results related to the burden of injury, we chose to report 95% confidence intervals around each proportion, rather than presenting statistical tests. Because the OHS used a complex sampling design to yield a provincially representative sample, weighted proportions were calculated. Variance estimates were adjusted using bootstrap replicate weights to account for clustering within the sample .Two analyses were conducted to explore data quality. First, as a sensitivity analysis for the physician billing database, we examined the impact of re-classifying two common "possible injury" diagnoses as actual injuries in the physician billing database. These diagnoses included i) non-specific musculoskeletal system diagnoses, and ii) adverse reactions to drugs and/or medications. These diagnoses were viewed as potentially problematic because they were not specific enough to injuries to warrant inclusion as "definite" injuries, but we believed that they might be commonly used by physicians providing injury-related care.Secondly, we directly compared self-reported and administratively-defined injuries at an individual level, to provide further insight into data quality. Not all self-reported injuries identified using the survey data would be expected to have led to medical treatment, and conversely, it is possible that some medically treated injuries may not have led to a restriction in normal activity. Some overlap between the survey data and administrative data was expected, though, in terms of injuries identified. Thus, we examined discrepancies between the injury variables at the level of the individual adolescent . Odds ratios, based on two-way data tables, were used as a measure of association for these direct comparisons of injury variables across data sources. We also explored possible reasons for the discrepancies observed, including potential recall error in the survey data, and potential error in both datasets resulting from overlap between acute injuries and repetitive strain injuries. These exploratory analyses involved, where appropriate, descriptive statistics  or odds ratios (as a measure of association for two dichotomous variables). All of the data quality analyses were used to examine within-sample methodologic issues. Therefore, as we did not wish to generalize the results from these analyses to a target population, unweighted analyses were conducted, and confidence intervals were not included.During the one-year period prior to the OHS interview, there were a total of 13501 physician visits for any cause among the 2067 adolescents in the initial sample , based on the physician billing data. Of these, 1390 visits were identified as being related to injury, representing 10.3% of all physician care. The proportion of visits due to injury varied across locations of care. For example, 8.6% of physicians' office visits (n = 604) and 45.6% of emergency department visits (n = 402) were identified as injury-related. A greater proportion of physician care was injury-related among males (14.2%), relative to females (7.3%).Within the final sample (N = 2047), while 18.8% of adolescents self-reported an activity-limiting injury in the past year, 25.0% had at least one administratively-defined injury, based on the hospitalization and physician billing databases .The results of the within-sample analysis used to compare injuries identified using different data sources are shown in Table In order to investigate the possibility that recall error may have led to underreporting in the survey, we investigated the relationship to recall time , and to explore whether the algorithm used with the physician billing database may have led to misclassification of some repetitive strain injuries as acute injuries, we examined the relationship to self-reported repetitive strain injuries. Both self-reported acute injury and administratively-defined acute injury appeared to be related to self-reported repetitive strain injury  to capture a broader spectrum of injury severity compared with the administrative data . The higher proportion of adolescents with administratively-defined injury, though, suggests that there may also be a subset of medically treated injuries that do not in fact limit normal activities; in other words, perhaps the definition of injuries used in the survey was actually more restrictive. Although neither data source can be viewed as a "gold standard", these results suggest that administrative health care data may actually provide a more sensitive means of ascertaining injuries, relative to self-reported survey data. Injuries identified as medically treated using administrative data may also be viewed as representing the health concerns of the person seeking care, and they have an impact on the health care system.These findings highlight the potential importance of administrative databases as a source of population-based injury information that can be used for affordable ongoing surveillance and for examining health care system issues such as patterns of service delivery. Despite these advantages, a limitation of many claims datasets, including the OHIP database, is a lack of detail on the circumstances surrounding the occurrence of injuries. The billing data contained no external cause information, such that description of injuries by mechanism and intent was not possible.Although the distribution of injuries, particularly for gender and age, was fairly similar for self-reported and administratively-defined injuries Table , congrueIn the survey data, we found some evidence of recall errors , and the unique sampling weights created for this sub-sample may have improved representativeness. Finally, because this study capitalized on an opportunity presented by a larger study on youth injuries, we focused specifically on adolescents. Further research could examine the generalizability of both the approach and the findings to other age groups, where the types of injuries experienced and care-seeking patterns may differ. Studies in jurisdictions with similar medical claims databases would also help in assessing the generalizability of our research.Collectively, our findings allow us to draw two main conclusions. First, the results suggest that there is potential value in using physician billing data along with other administrative health care databases for the surveillance of injuries among adolescents. Although they are lacking in details about the circumstances surrounding injuries, comprehensive administrative injury datasets may be particularly useful for describing the overall occurrence of injury at local or regional levels, and for describing the economic implications of injury for the health care system. Secondly, we identified data quality concerns in both the survey and administrative databases that suggest a need for improvement and further study; for example, further research could help to identify appropriate recall periods and question wording for minimizing errors in survey data, and to determine the level of detail needed to accurately identify injuries in administrative databases. Because various sources of data are susceptible to different limitations, it remains important to consult multiple sources of information to fully document the burden of injury ,4,7.The author(s) declare that they have no competing interests.BKP participated in the design and coordination of the study and the development of the physician billing algorithm, carried out all statistical analyses, and drafted the manuscript. DM participated in the design, coordination, and supervision of the study and development of the physician billing algorithm, as well as revisions to the manuscript. KNS participated in the design, coordination, and supervision of the study, as well as revisions to the manuscript. IAG participated in the design of the study and development of the physician billing algorithm, as well as revisions to the manuscript. MKC participated in the design of the study and revisions to the manuscript. JJK participated in the design of the study and revisions to the manuscript, and provided statistical guidance. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Several studies have linked low birth weight to adverse levels of cardiovascular risk factors and related diseases. However, information is sparse at a community level in the U.S. general population regarding the effects of low birth weight on the longitudinal trends in cardiovascular risk factor variables measured concurrently from childhood to adolescence.th percentile; n = 123) and control  groups.Longitudinal analysis was performed retrospectively on data collected from the Bogalusa Heart Study cohort  followed from childhood to adolescence by repeated surveys between 1973 and 1996. Subjects were categorized into low birth weight (below the race-specific 10Low birth weight group vs control group had lower mean HDL cholesterol (p = 0.05) and higher LDL cholesterol (p = 0.05) during childhood (ages 4–11 years); higher glucose (p = 0.02) during adolescence. Yearly rates of change from childhood to adolescence in systolic blood pressure (p = 0.02), LDL cholesterol (p = 0.05), and glucose (p = 0.07) were faster, and body mass index (p = 0.03) slower among the low birth weight group. In a multivariate analysis, low birth weight was related independently and adversely to longitudinal trends in systolic blood pressure (p = 0.004), triglycerides (p = 0.03), and glucose (p = 0.07), regardless of race or gender. These adverse associations became amplified with age.Low birth weight is characterized by adverse developmental trends in metabolic and hemodynamic variables during childhood and adolescence; and thus, it may be an early risk factor in this regard. The growth of a fetus in an undernourished intrauterine environment is considered to result in adaptive fetal programming or metabolic imprinting with pathophysiologic consequences later in life -4. It isSeveral studies including our own have examined the association between low birth weight and selected cardiovascular risk factor variables in childhood and adolescence -27. HoweBetween 1973 and 1996, 7 cross-sectional surveys of children and adolescents were conducted in the community (65% white, 35% black) of Bogalusa, LA. This panel design, based on repeated cross-sectional examinations performed every 3 to 4 years, resulted in serial observations required for the longitudinal analysis. For the present report, two sets of data were merged as described previously : 1) singth percentile  were categorized as low birth weight group; those (n = 296) in the upper normal range of 50–75th percentile  as control group. Birth weights above the 75th percentile were not included in the control group because of the u-shaped associations between birth weight and risk factors or disease [Low birth weight and control groups were selected from the eligible cohort, according to birth weight percentile cut points . Subject disease . Race-sp disease .Identical protocols were used by trained examiners across all surveys . SubjectHeight and weight were measured 2 times; subscapular skinfold thickness 3 times. Body mass index (BMI) calculated as weight in kg divided by the square of the height in meters was used as a measure of overall adiposity; subscapular skinfold for truncal fatness. The reproducibility in terms of intraclass (intra-observer) correlation coefficient was greater than 0.99 for weight and height, and greater than 0.97 for subscapular skinfold.Blood pressure levels were measured in 6 replicates by 2 randomly assigned nurses on the right arm of subjects in a relaxed, sitting position. Systolic and diastolic blood pressures were recorded at the first, fourth, and fifth Korotkoff phases using mercury sphygmomanometer. For this analysis fourth phase was used for diastolic blood pressure because in our experience the fourth phase is more reliably measured in children and more predictive of adult hypertension .From 1973 to 1986 cholesterol and triglyceride levels in serum were measured using chemical procedures on Technicon Autoanalyzer II (Technician Instrument Corp., Tarrytown, NY). Since then these measurements were done using enzymatic procedures on Abbott VP instrument (Abbott laboratories, North Chicago, IL). Serum lipoprotein cholesterols were analyzed by a combination of heparin-calcium precipitation and agar-agarose gel electrophoresis procedures . Both chPlasma immunoreactive insulin levels were measured by a commercial radioimmunoassay kit (Phadebas, Pharmacia Diagnostics, Piscataway, NJ). Plasma glucose was measured by a glucose oxidase method either using a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA) or as part of a multichemistry (SMA20) profile. The intraclass correlation coefficient between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose. An index of insulin resistance was calculated according to the homeostasis assessment model formula : HOMA-IR2, race and gender and their interaction with birth weight (low vs control). A backward stepwise method was used to remove nonsignificant terms.For test of significance glucose and insulin were logarithmically transformed to approach normality. The average of multiple measurements for subjects within age groups 4 to 11 and 12 to 18 years corresponding to childhood and adolescence periods was used to calculate mean levels of risk variables by birth weight status and age groups. Mean levels of risk variables within each age group were compared between low birth weight and control group by a general linear model, adjusting for age, race, and gender. The longitudinal rates of change in risk variables was assessed by the generalized estimation equation (GEE) method  with ageMean levels of cardiovascular risk variables during childhood ages 4–11 years) and adolescence (ages 12–18 years) periods are shown in table 1 years aLongitudinal rates of change in cardiovascular risk variables from childhood to adolescence, adjusted for race and gender, are presented in table In a multivariate analysis, low birth weight was retained as an independent predictor variable for adverse longitudinal trends in systolic blood pressure, triglycerides, and glucose  from childhood to adolescence, regardless of race or gender table . FurtherInformation is sparse at a community level in the U.S. general population regarding the effects of low birth weight on the longitudinal trends in C-V risk factor variables measured serially and concurrently from childhood to adolescence. The present community-based study demonstrates the adverse effects of low birth weight on the longitudinal  trends in systolic blood pressure, triglycerides, and glucose during childhood and adolescence, regardless of race or gender. These observations are in accord with the emerging evidence supporting the concept of intrauterine imprinting and its pathophysiologic consequences enunciated by the fetal origin or thrifty phenotype hypothesis .Many, but not all, previous studies in children and adolescents have found adverse associations between birth weight and levels of cardiovascular risk factor variables -27. In tAlthough observational studies like the present one can not establish causality, several putative mechanisms link low birth weight to adverse trends in risk factor variables. It has been suggested that insulin resistance may be one mechanism by which intrauterine events may program disease risk . UndernuThis study has certain limitations. The lack of information on the duration of gestation precluded us from adjusting the birth weight for gestational age, a potential confounder. However, earlier studies have found that adverse effects of low birth weight on cardiovascular risk factor variables were independent of gestation period ,17,49. FLow birth is characterized by adverse developmental trends in metabolic and hemodynamic variables during childhood and adolescence, especially as the children get older. These observations in conjunction with earlier findings support the view that low birth weight, albeit a crude marker of prenatal growth and physiological environment, is a potential early risk factor for the emergence of metabolic and hemodynamic disorders and related diseases ,50.BMI, body mass index; LDL, low-density lipoprotein; HDL, high-density lipoproteinThe author(s) declare that they have no competing interests.MGF participated in study design, data analysis and manuscript preparation. SRS and GSB contributed to study concept and design, data collection, acquisition of funding and manuscript preparation. JX was involved in measurements of laboratory variables. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


NeuroEngineering and Rehabilitation (JNER) is intended to fill this gap and foster cross-fertilizations among these disciplines. By making readily available to clinicians selected studies with potential impact on physical medicine & rehabilitation, JNER is anticipated to foster the development of novel and more effective rehabilitation strategies. Conversely, by presenting clinical problems to a readership of neuroscientists and engineers, JNER is expected to generate innovative work in neuroscience and biomedical engineering with future applications to physical medicine & rehabilitation. JNER will leverage on Open Access as a means to guarantee that its content is readily available to scientists, clinicians, and the general public thus promoting scientific and technological advances that are relevant to rehabilitation. JNER is an Open Access initiative. Open Access assures dissemination to the widest possible audience and is seen by many as essential for publicly funded research. BioMed Central offers an outstanding platform to make JNER possible and allow neuroscientists, biomedical engineers, and clinicians to see their work published in a timely manner and thus make an immediate impact in the field of rehabilitation. JNER will focus on innovative work with higher likelihood of a dramatic impact on rehabilitation. Thus, priority will be given to outstanding and visionary scientific reports, i.e. those proposing exceptionally innovative concepts with great potential in the field.Advances in neuroscience and biomedical engineering deeply affect the clinical practice of physical medicine & rehabilitation. New research findings and engineering tools are continuously made available that have the potential of dramatically enhancing the ability of clinicians to design effective rehabilitation interventions. This quickly evolving research field is difficult to track because related literature appears in a wide range of scientific journals. There is a need for a scientific journal that offers to its readership a forum at the intersection of neuroscience, biomedical engineering, and physical medicine & rehabilitation. The Journal of  During the past decade, we have witnessed profound changes in physical medicine & rehabilitation originated by advances in neuroscience and biomedical engineering. For example, imaging and neurological assessment methods have dramatically improved the management of patients with motor impairments; robotics and artificial muscle research have generated revolutionary concepts in orthotics and prosthetics; and advances in cortical recordings and the understanding of central nervous system mechanisms have changed the way clinicians look at movement disorders. These techniques and others have brought about, and will continue to give rise in the future to, dramatic advances in physical medicine & rehabilitation.Journal of NeuroEngineering and Rehabilitation (JNER) aims to provide such an outlet, hosting the introduction of new methods and the discussion of their clinical implications, and offering an opportunity to publish, in a timely manner, articles relevant to the cross-fertilization of neuroscience, biomedical engineering, and physical medicine & rehabilitation.As advances in neuroscience and biomedical engineering continue to generate new techniques, with tremendous impact in the field of physical medicine & rehabilitation, it becomes apparent that there is an urgent need for establishing an outlet for the intersection of these three research fields. JNER's editorial board [al board  demonstrJNER's Open Access policy changes the way in which articles are made available to the scientific community. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [JNER's articles are archived in PubMed Central [troduced . Third,  Central , the US  Central  in Germa Central  in Franc Central , the NatOpen Access has four broad benefits for science and the general public. First, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access their work. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability . Second,JNER will contribute to make the latest advances in neuroscience and biomedical engineering, which have the potential to impact on the clinical practice of physical medicine & rehabilitation, readily available to scientists, clinicians, and the general public. Because of its inherent interdisciplinary nature, JNER will foster further advances in the field thanks to the cross-fertilization among science, technology, and clinical practice. Science and technology are expected to offer new tools to design clinical interventions and, vice versa, clinical problems are anticipated to foster basic research in neuroscience and the development of new technologies. Besides, increased awareness of the way science and technology can improve clinical outcomes will lead to better quality of healthcare in rehabilitation. Changes currently occurring in this field are so dramatic that we expect, in a few years, that modernized rehabilitation inpatient and outpatient units will be completely different from what is the state-of-the-art today. For instance, we envision that continuous monitoring of patient status will be performed via miniature, wireless, wearable sensors which not only allow clinicians to monitor vital signs, but also track motor activities and provide a means to analyze motor patterns associated with recovery. Furthermore, robotic devices will be used to enhance physical therapy, ad hoc protocols will be designed for each patient, and augmented and virtual reality tools will enhance rehabilitation by becoming part of routine exercise protocols.Open Access will increasingly become an accepted way to disseminate information to the scientific community and the public at large. By becoming part of the movement for Open Access, JNER intends to provide a forum for ideas and concepts that could make a difference in physical medicine & rehabilitation by innovating the design of clinical interventions.Open Access to outstanding and visionary scientific reports appears to be a tremendous tool to increase the speed at which clinical practice changes as a result of advances in neuroscience and biomedical engineering. By prioritizing outstanding and visionary publications, JNER is free for the first 6 months following the launch of the journal. Manuscripts submitted after this period will be subject to an article-processing charge on acceptance. Waiver requests will be considered on a case-by-case basis, by the Editor-in-Chief. Authors can circumvent the charge by getting their institution to become a 'member' of BioMed Central, whereby the annual membership fee covers the article processing charges for authors publishing in any of the BioMed Central journals. Current members include NHS England, the World Health Organization, the US National Institutes of Health, Harvard, Princeton and Yale universities, and all UK universities [Publication in ersities . No charersities .JNER's editorial board will be solely used to further promote the journal and to continuously increase the scientific quality of JNER's articles.The article-processing charges pay for efficient and thorough peer review, for the article to be freely and universally accessible in various formats online, and for the processes required for inclusion in PubMed and archiving in PubMed Central, e-Depot, Potsdam and INIST. Funding available to JNER to high quality, prioritizing visionary work, and focusing on research that has the potential of a great impact on physical medicine & rehabilitation. Forthcoming articles will further prove such commitment. Topics of interest to come in the next few months are virtual and augmented reality in rehabilitation, wearable technology in rehabilitation, methods for the analysis of movement, and robotics applied to rehabilitation. These are all topics of great relevance for the research at the intersection of neuroscience, biomedical engineering, and physical medicine & rehabilitation.The first articles published in the journal demonstrate the commitment of JNER as well as to the authors who submit their manuscripts in the future and support our journal and the Open Access initiative. Members of the editorial board and reviewers have done some excellent work; special thanks to them and the Managing Editor, Sara Midwood, for their contributions to JNER.Special thanks to the authors of the first articles published in


This work has investigated under what conditions confidence intervals around the differences in mean costs from a cluster RCT are suitable for estimation using a commonly used cluster-adjusted bootstrap in preference to methods that utilise the Huber-White robust estimator of variance. The bootstrap's main advantage is in dealing with skewed data, which often characterise patient costs. However, it is insufficiently well recognised that one method of adjusting the bootstrap to deal with clustered data is only valid in large samples. In particular, the requirement that the number of clusters randomised should be large would not be satisfied in many cluster RCTs performed to date.The performances of confidence intervals for simple differences in mean costs utilising a robust (cluster-adjusted) standard error and from two cluster-adjusted bootstrap procedures were compared in terms of confidence interval coverage in a large number of simulations. Parameters varied included the intracluster correlation coefficient, the sample size and the distributions used to generate the data.The bootstrap's advantage in dealing with skewed data was found to be outweighed by its poor confidence interval coverage when the number of clusters was at the level frequently found in cluster RCTs in practice. Simulations showed that confidence intervals based on robust methods of standard error estimation achieved coverage rates between 93.5% and 94.8% for a 95% nominal level whereas those for the bootstrap ranged between 86.4% and 93.8%.In general, 24 clusters per treatment arm is probably the minimum number for which one would even begin to consider the bootstrap in preference to traditional robust methods, for the parameter combinations investigated here. At least this number of clusters and extremely skewed data would be necessary for the bootstrap to be considered in favour of the robust method. There is a need for further investigation of more complex bootstrap procedures if economic data from cluster RCTs are to be analysed appropriately. The greater complexity of cluster randomised controlled trials (RCTs) compared with their individually randomised counterparts has led to much methodological work concerning their design and analysis. Howevert-test are fairly robust to moderate violations of the assumptions of normality and homogeneity of variances [with replacement from this 'population' and calculation of a parameter of interest builds up a picture, the 'empirical distribution' of this parameter, based on so-called 'B bootstrap estimates' of the parameter of interest. This can be used to construct directly the required confidence interval by, for instance, reading off the 2.5% and 97.5% percentiles of the distribution. Bootstrapped confidence intervals may, therefore, be asymmetric and be better able to deal with skewed data.The first is that many trials randomise only a small number of clusters. This can sometimes produce inadequate randomisations where, for example, all clusters with a characteristic related to outcome are allocated to one treatment arm . It may ariances . The sitariances , first pariances . This reThe bootstrap approach is flexible but does assume that the data are independently and identically distributed ,8. When single outcome model. This single outcome model was primarily conceptualised as a model for cost data and the term cost is therefore used as short-hand. However, some of the scenarios presented may be equally applicable to clinical data.This paper details the results of simulation studies to evaluate how clustered cost data might be analysed. Small numbers of clusters together with skewed data were utilised to ascertain how the bootstrap performed against a method of analysis commonly used in clustered clinical data. Thus it details the generation and analysis of a , which was considered acceptable. Over 20,000 simulations per parameter set, then, the following were noted:The performances of confidence intervals for simple differences in mean costs utilising a robust (cluster-adjusted) standard error and from two cluster-adjusted non-parametric bootstrap procedures were compared in terms of confidence interval coverage. Specifically, the comparison was of the percentage of 20,000 simulations for which the estimated 95% confidence interval contained the true value of the treatment effect. If the observed coverage were to be 95% on average across the simulations, then 20,000 simulations would from simple binomial theory give a margin of error of approximately 1. The mean value  of the estimated treatment effect and the confidence limits under each of the three procedures,and whose lower/upper limit was greater/less than this true value,2. The percentages of simulations for which the estimated confidence interval did not contain the true value of the treatment effect (zero) 3. The percentage of simulations for which the estimated confidence interval contained the true value for the treatment effect. This figure was simply 100 minus the sum of the two percentages in 2 above.Thus ideally the two figures in 2 should each be 2.5% whilst that for 3 should be 95% . It was decided to split observed non-coverage rates according to whether there was spurious positive treatment effect or a spurious negative one because skewed distributions were expected to have different implications for each of these.n individuals in each of 2k clusters. Half of these were randomised to a hypothesised intervention group, whilst the other half were randomised to a control group. A random effects model incorporating a treatment dummy variable was used:The data were constructed by assuming there were Ehij = αhi + βTh + εhij     h = 0,1; i = 1,...k;j = 1,...n;E(αhi) = 0; E(εhij) = 0jth individual in the ith cluster was randomised to receive treatment h. Each individual's outcome, Ehij, comprised three elements: the effect of treatment, βTh, the cluster-specific effect, αhi, and the individual-specific effect, εhij. In order to inform the values of these parameters, it was necessary to undertake some exposition of how cost data might be distributed in a cluster RCT.Thus the In attempting to construct realistic scenarios for cost data from a cluster RCT, three main factors were considered:1. What is the nature of the distribution of individual patient costs expected to be in the population of patients normally eligible for treatment?2. How representative of this population distribution are the cost distributions within clusters likely to be? This has implications for the ICC and the assumptions regarding the distributions.3. How might the introduction of an intervention affect 2 above?As detailed previously, the existence of one unrepresentative cluster  in one arm may affect the ICC, independently of treatment, or the treatment could directly change the ICC and the distribution .This section formally sets out the parameters which, when varied in the simulation model, attempted to capture two potential extreme scenarios as well as situations in between. Under the first scenario, all clusters are equally representative of the population, leading to a high degree of skewness within most, if not all, clusters. Under such a scenario, the ICC is likely to be small and results would be expected to be robust to any incorrect assumptions made regarding the between-cluster distribution. Under the second scenario, within-cluster costs are expected to be more homogenous and much of the skewness in the cost data at the population level is attributable to differences in the cluster mean costs. As a result, the ICC would be expected to be much larger, and the between-cluster distribution might be expected to exhibit considerable skewness. Six factors were varied:1. The between-cluster distributions,2. The within-cluster distributions,3. The ICC in the control group,4. The ICC in the intervention group,5. The number of clusters in each intervention arm,6. The number of individuals in each cluster.There were two distributional assumptions used for the cluster means. The first was the normal distribution and the second was the lognormal distribution, which, for a given mean and variance, exhibits higher kurtosis and skewness than the gamma distribution, the main alternative skewed distribution.For each of the ICC combinations given below, there were three possible combinations of the between-cluster distributions:1. The cluster means in both the control and intervention groups were normally distributed2. The cluster means in both the control and intervention groups were lognormally distributed3. The cluster means in the  control group were normally distributed, whilst in the  intervention group they were lognormally distributed.The same distribution was used for individual level data in both treatment arms in order not to further increase the number of parameter combinations possible. Lognormal data were used in order to provide a scenario applicable to individual level cost data.Given that the likely range of cost ICCs is largely unknown, values between zero and 0.5 were used. While high, the upper value is consistent with the one published source of cost ICCs . The ICCFor a given value of the ICC in the control group, the intervention ICC can remain the same or it can change in a number of ways. In particular, the intervention ICC:1. Remained the same as the control ICC,2. Doubled, as a result of an appropriate increase in the between-cluster variance,3. Doubled, as a result of an appropriate decrease in the within-cluster variance,4. Halved, as a result of an appropriate decrease in the between-cluster variance,5. Halved, as a result of an appropriate increase in the within-cluster variance.Although it is only the between-cluster variance or the within-cluster variance that changes at any one time (not both), the changes involved are large ones. Hence these extreme scenarios should cover a range of findings.The number of clusters in each group was 6, 12 or 24. These figures reflect the small numbers of clusters recruited in many cluster RCTs and, coupled with the cluster sizes given below, they allowed alternative combinations of cluster size and number of clusters to be investigated for a given total trial size.The cluster size was 25, 50 or 100. Mean cluster sizes between 50 and 100 are not unusual in health services research trials, but there is enormous variation in cluster size, depending upon treatment area and type of cluster[k clusters with replacement from the treatment group and selecting k clusters with replacement from the control group – in other words, resampling of clusters was stratified by intervention group. Under method 1 all individuals within a resampled cluster were then selected. Under method 2 a second level of bootstrap was performed on individuals within clusters selected at level one. The difference between the two randomisation group means was then calculated for each method. This was repeated to give 1000 bootstrap estimates (estimates performed on the resampled data) of the treatment effect. A bias-corrected and accelerated (BCa) confidence interval was then estimated at the same nominal 95% level as for the robust method [a method, the resulting confidence interval need not be symmetric. The bootstrap methods are described in more detail below.The first method utilises a standard procedure, where 'standard' has been taken to mean a 95% confidence interval quoted for continuous data in packages such as Stata , utilisit method . Given tyij, such thatUnder this procedure (BS1), clusters are bootstrapped and each resampled cluster is kept intact. This method is utilised by Stata  when theyij = αi + εij     i = 1,...k;j = 1,...n.αis are sampled randomly from the distribution Fα and the εijs are independently sampled randomly from the distribution Fε.The αi) = 0;     (1)E(It can be shown that for the bootstrap estimates (with superscript asterisks):When compared with (2) and (3) it should be noted that the expected variance and covariance of the resampled outcome data are slightly biased downwards. However, an estimator such as the sample mean is strongly consistent ; the level of bias is small unless the number of clusters becomes very small. (see page 102 of their book):An alternative method (BS2) involving resampling individuals as well as resampling whole clusters was also considered. This uses a first stage bootstrap applied to the estimated cluster means (sampling with replacement). The second stage, in which individuals are bootstrapped, involves resampling the deviations from the estimated cluster means. However, the estimated cluster means incorporate both within and between-cluster variability and any analysis that restricts itself to the cluster means will over-estimate the variance in these means . By incowhere c is given by; if the right hand side is negative, it is reset to zero., is then .The variance of the adjusted cluster means, The deviations from the estimated cluster means were also standardised toFinally, for all resampled clusters, the 'shrunken' mean is added to the standardised deviation for each resampled individual:This method, with the rescaling procedures will in future be referred to as the BS2 method, or double bootstrap.The primary focus of this work was on confidence interval coverage and rejection rates; the estimated confidence limits are, therefore, not presented but general inferences regarding the relative width of various confidence intervals can be made easily from the coverage and rejection rates presented below.As a summary, Tables Issues specific to variance or distribution combinations are presented below. In the three tables, within each box the first number represents the coverage of the robust confidence interval, the second represents the coverage of the BS1 method whilst the third figure represents that of the BS2 method. A number of results are immediately apparent:• All three methods produce coverage of less than 95%, the nominal level,• All three methods appear to be consistent with respect to the impact of the number of clusters per arm. In other words, as the number of clusters per arm increases, the observed coverage approaches 95% for each method,• The coverage of the robust method is within approximately 1.5% of 95%,• The BS1 method is always outperformed by the other two methods. In other words the other two methods always achieve coverage that is closer to 95%,• The BS2 method performs much better than the BS1 method but never as well as the robust method,• As the ICC in the control group increases (that is, across tables) the robust method performs slightly worse whilst the performances of the bootstrap methods are noticeably poorer,• When examining numbers along the diagonal in each figure , the bootstrap methods perform much better for a large number of clusters and small cluster size compared with vice-versa. This is probably due to the slight downward bias in the second moments; the degree of bias is an inverse function of the number of clusters.From the results in these tables there does not appear to be much to commend the bootstrap, since both bootstrap methods are always outperformed by the robust method. Moreover, when examining the confidence interval coverage results split by distribution and variance combination (results not shown), there was only one parameter combination for which the robust method was outperformed and this was by an amount that was consistent with Monte Carlo sampling error. However, the rejection rates for confidence intervals were split to ascertain if any method was noticeably better at taking account of the skewness in the data.When examining the rejection rates for six clusters of size 25 (results not shown), those of both bootstrap methods virtually always exceeded those under the robust method. The exceptions were for a control ICC of 0.01 when the within-cluster variance increased as a result of the intervention for two of the distributional combinations. For the third distributional combination the rejection rates were the same. Since the individual level data were always lognormally distributed , an increase in the within-cluster variance for lognormally distributed data is likely to have a large effect on skewness, provided the ICC is not too large (which would dilute the effect of within-cluster factors).Thus, if the desired criterion is ability to match the nominal 2.5% rejection rate in any given direction, there are occasions when one or both bootstrap methods outperform the robust method in terms of the proportion of lower rejections. In particular, this appeared to happen when the ICC was moderate to large, together with an extremely skewed distribution of the treatment effect, typically achieved by a large change in the ICC and something other than a normal, normal (N, N) distribution combination. Under these circumstances the distribution of the treatment effect is most skewed, since the cluster means in the intervention group are exhibiting large skewness by way of a lognormal distribution whose  variance has doubled. However, this must be balanced against the poor performance of the bootstrap methods in terms of the proportion of upper rejections, which tended to be particularly high compared with those from the robust method.With a cluster size of 50 or 100 (results not shown), similar results were seen to those above, in that a large difference in the absolute value of the ICC together with extremely skewed distributions were required for the double bootstrap to achieve a rejection rate closer to the nominal level than the robust method. In particular, increases in the between-cluster variance accompanied by skewed distributions at the between-cluster level typically caused the double bootstrap to give better lower rejection rates.For 12 clusters of size 25 per arm (results not shown), all three methods performed better than for an equivalent sample size with fewer clusters per arm (6 clusters of size 50). There were very few instances in which the rejection rate exceeded that for 6 clusters of size 50. Even the BS1 method appeared to perform more consistently than when there were only six clusters per arm. Although it was still always outperformed by the BS2 and robust methods, its maximum rejection rate was 10.26%, compared with 14.24%. As before, when the ICC was very small (that is, most of the variability was within clusters), the double bootstrap method typically only outperformed the robust method when the within-cluster variance increased. In addition, for larger values of the ICC, changes in the between-cluster variance or the within-cluster variance could result in the double bootstrap outperforming the robust method, confirming the results obtained for six clusters of size 100. Lastly, normal distributions at the between-cluster level in both arms were sufficient to ensure that the robust method always performed better than the bootstrap.All the trends identified in previous sections were replicated for 24 clusters of size 25  data. Whilst this is of interest, policymakers are more often interested in cost-effectiveness data. These have usually involved ratio statistics, which often cause major problems for traditional estimators. Future work should, therefore, investigate how well the cluster bootstrap deals with cost-effectiveness data and hence address the question of whether the disadvantages of the cluster bootstrap identified here are outweighed by its potential advantages in dealing with ratio statistics.The author(s) declare that they have no competing interests.TNF conceived of the study, wrote and conducted the simulation programs and drafted the manuscript.TJP participated in the design and coordination of the study, read, critically reviewed and contributed to drafts of the manuscript.The pre-publication history for this paper can be accessed here:


Austrofundulus limnaeus). In stenothermal Antarctic notothenioid fish, losses in capacity for temperature-mediated gene expression, including the absence of a heat-shock response, may reduce the abilities of these species to acclimate to increased temperatures. Differences among species in thermal tolerance limits and in the capacities to adjust these limits may determine how organisms are affected by climate change.Temperature-adaptive physiological variation plays important roles in latitudinal biogeographic patterning and in setting vertical distributions along subtidal-to-intertidal gradients in coastal marine ecosystems. Comparisons of congeneric marine invertebrates reveal that the most warm-adapted species may live closer to their thermal tolerance limits and have lower abilities to increase heat tolerance through acclimation than more cold-adapted species. In crabs and snails, heart function may be of critical importance in establishing thermal tolerance limits. Temperature-mediated shifts in gene expression may be critical in thermal acclimation. Transcriptional changes, monitored using cDNA microarrays, have been shown to differ between steady-state thermal acclimation and diurnal temperature cycling in a eurythermal teleost fish ( Understanding the roles played by physiological adaptation to temperature in governing the distribution patterns of species has taken on a new urgency because of the potential effects of global warming on both aquatic and terrestrial ecosystems. There is already compelling evidence for widespread changes in ecosystems due to climate change. Recent meta-analyses have shown that species' distribution patterns, the structures of ecosystems, and the timing of annual events (phenology) such as migration and reproduction have changed markedly during the past century, when average global temperatures rose by approximately 0.6°C -4. If thIn this brief review, I discuss studies performed with a taxonomically varied group of aquatic ectotherms that shed light on several questions related to the roles played by physiological adaptations in setting species' distribution patterns. The more clearly we understand the mechanistic basis of biogeography, the better prepared we will be to predict the effects of climate change on distribution patterns and ecosystem structure. The questions I address include the following. First, how do thermal tolerance limits differ among species adapted to different temperatures? Which species are most likely to be threatened by increases in habitat temperatures, i.e., which species currently live closest to their thermal tolerance limits? Second, what are the "weak links" in physiological systems that appear most likely to set thermal tolerance limits? Can we identify physiological, biochemical, and molecular level effects that account for thermal tolerance ranges? Third, how do capacities for acclimation to changes in habitat temperature differ among species? Which species have the greatest – or the least – ability to acclimate to increases in temperature? Mechanistically speaking, what types of acclimatory changes are needed to adapt physiological systems to permit tolerance of new thermal conditions? Fourth, what types of changes in gene expression are needed to achieve thermal acclimation? How does gene expression during acclimation to rapid, diurnal change in temperature differ from the response to slower, seasonal time-scale change in temperature? How do stenothermal and eurythermal species differ in their capacities for altering gene expression in temperature-adaptive manners? Have extreme stenotherms like Antarctic animals that have evolved for many millions of years under highly stable, cold temperatures lost key abilities to acclimate to increasing temperatures?To address these questions, I focus primarily on studies that our laboratory has performed on aquatic ectothermic species that have been chosen for study because they seemed to fit well the August Krogh Principle, which can be paraphrased as follows: For any question a biologist asks, Nature can provide a most appropriate study organism. Through study of such "Kroghian" species, it has been possible to obtain at least initial answers to the several questions raised above.Petrolisthes) from the eastern Pacific Ocean [Petrolisthes are abundant (46 species occur in the eastern Pacific), found over wide ranges of latitude, and occur in subtidal and intertidal habitats. Among these congeners, body temperatures extend from slightly below 0°C to over 40°C; for some eurythermal species, the range of body temperatures across different seasons can be over 32°C [One of the challenges facing a comparative or evolutionary physiologist wishing to elucidate adaptive differences among species is the need to separate effects due to phylogeny from true adaptive variation. One means of achieving this end is to study groups of closely related organisms from widely different habitat conditions, for which a comprehensive phylogeny exists. A group of species that is especially "Kroghian" from this standpoint are porcelain crabs  in Fig. Petrolisthes to increase thermal tolerance through acclimation to increased temperature are less in intertidal species than in more cold-adapted subtidal congeners [The thermal tolerance limits of field-acclimatized temperate , subtropical  and tropical (Panama) congeners of habitats . The preund Fig. : the mosongeners . Thus, wversus reciprocal temperature (K)) occurs. Once the ABT is exceeded, heart function of porcelain crabs does not recover from heat stress. ABT values for the temperate congeners P. cinctipes  and P. eriomerus  are 31.5°C and 26.5°C, respectively. The maximal habitat temperatures for the two species are approximately 31°C and 16°C, respectively [50, and ABT for heart function are essentially the same, 31–32°C. It bears emphasizing that ABT values for other physiological processes, for example, mitochondrial respiration [One physiological system that is a "weak link" in the thermal tolerance of these species is heart function. As shown in Fig. ectively ,10. Thuspiration ,12 and epiration , may be 50  of P. eriomerus rose by 2.3°C when acclimation temperature was increased by 10°C, but the most warm-adapted species, P. gracilis, increased ABT (CTmax) by only 0.5°C. The eurythermal intertidal species did exhibit greater capacities than the subtidal species to acclimate to lower temperatures, however, as shown by their greater ability to lower the temperature at which heart beat ceased (CTmin)  Fig. . DespiteTegula [Tegula congeners exhibit significant differences in thermal limits of protein synthesis and onset temperatures for production of heat-shock proteins that reflect their vertical distributions [Tegula congeners had significantly different thermal limits of heart function: the low- to mid-intertidal congener, T. funebralis, had a higher ABT of heart function (31.0°C) than two subtidal congeners, T. brunnea and T. montereyi (ABTs of 25.0°C and 24.2°C, respectively) [T. funebralis approximated the highest body temperature recorded for this species in the field, 32°C [T. funebralis had a lower ability to increase ABT during warm acclimation than the subtidal species [The differences in thermal tolerance and capacities for warm acclimation noted with porcelain crabs were mirrored in studies done with another set of congeneric marine invertebrates found at different vertical positions along the subtidal to intertidal gradient, turban snails of the genus Tegula . Intertiibutions ,16. In actively) . The ABTld, 32°C . Body teld, 32°C , so thesld, 32°C , T. fune species .Petrolisthes eriomerus has an upper lethal temperature approximately 6°C below the upper limit of body temperature recorded for the intertidal species P. cinctipes. Interspecific variations in LT50 and ABT values also mirror the biogeographic patterning found across latitude. For instance, north temperate congeners of Petrolisthes would be unable to survive under the habitat conditions found in the northern Gulf of California or Panama. The close agreement among LT50, ABT, and maximal habitat temperatures for intertidal species suggests that further increases in habitat temperature could have strong impacts on the persistence of these species in their habitats, especially when the limits of acclimatory ability are reached. Because the greatest thermal stress occurs during emersion periods during the warmest time of the day, and because solar heating is more critical than ambient water temperature, the precise consequences of global warming are difficult to predict. Furthermore, because of latitudinal variation in the timing of tidal cycles, these and other intertidal species may face greater threats from climate change at mid-latitudes than at lower latitudes [These interspecific differences in thermal tolerance limits for whole organism survival and maintenance of heart function provide important lessons concerning biogeographic and vertical patterning and the potential effects of climate change. First, the differences in thermal tolerance between subtidal and intertidal species indicate that the former species would be unable to persist at the upper habitat temperatures commonly found in the intertidal zone during daytime low tides in warm seasons. For example, the temperate subtidal species atitudes . DespiteThe abilities of ectotherms to cope satisfactorily with increases in habitat temperature are based on several factors, including the proximity of habitat temperatures to the edges of the thermal tolerance range and the abilities to shift the tolerance range through acclimatization. In the case of porcelain crabs and turban snails, acclimatory ability was shown to vary among species. This finding that even closely related congeneric species differ in acclimatory ability raises questions about the mechanistic basis of phenotypic plasticity and the roles of evolutionary thermal history in establishing this plasticity.The ability to modify the phenotype in response to a change in body temperature is certain to depend strongly on a capacity for modulating gene expression in an adaptive manner. Although comprehensive surveys of temperature-induced shifts in gene expression have not been done for any marine organisms, studies of model species, defined here as species for which the genome has been sequenced and relatively well annotated, are beginning to reveal the pervasiveness of stress-induced shifts in gene expression. The studies of Gasch and colleagues  on yeastTrematomus bernacchii, the lack of capacity to acclimate to elevated temperatures may stem in part from deficiencies in its ability to alter gene expression as its body temperature changes. Unlike virtually all other species, T. bernacchii is unable to increase the synthesis of any class of heat-shock protein following thermal stress [T. bernacchii may be unable to effect adequate chaperone-mediated restoration of the native structures of heat-denatured proteins. A build-up of aggregates of denatured proteins is likely to be cytotoxic and lead to the eventual death of cells [Despite the likely occurrence among species of generally similar requirements for temperature-adaptive shifts in gene expression, species that fall into different regions of the stenotherm to eurytherm spectrum may have distinctly different capacities for acclimatory regulation of transcription. In the context of effects of climate change, one of the most important interspecific differences relates to the ability of extreme stenotherms, which may have evolved for millions of years under stable thermal conditions, to alter gene expression in the face of temperature change. The Antarctic notothenioid fishes are a primary case in point. These species have evolved for 10–14 million years in a thermally stable "ice bath," in which annual temperature variation is usually less than 1–2°C . McMurdol stress . Lackingof cells . The absof cells , suggestof cells  or myoglof cells  is anothof cells ,28. In gAustrofundulus limnaeus, revealed wide-scale shifts in gene expression, as noted earlier for yeast [versus intermittent heat stress. Although both acclimation regimes led to changes in transcript abundance for proteins associated with alterations in lipid composition, differences between cycling and steady-state acclimation were noted in the types of lipids that appear to be produced. For steady-state acclimation, the expected changes in transcripts for enzymes involved in acyl chain double bond content (saturation) were observed, consistent with many previous studies of homeoviscous acclimation in membranes [Even among eurythermal ectotherms, there may be a variety of capacities for modulating gene expression to compensate for changes in body temperature. Although few data are currently available to test this point, acclimatory responses to rapid, diurnal variations in body temperature may require different shifts in gene expression from those that characterize slower, e.g., seasonal time-scale, responses to temperature change. A recent study  of tempeor yeast . A totalor yeast . Under sembranes . During Austrofundulus limnaeus was for the message encoding high mobility group b1 protein (HMGB1)Fig. . High moGB1)Fig.  and duriAustrofundulus limnaeus and in a recent study of carp [The changes in gene expression noted in  of carp  represen50s) and the more limited abilities to acclimate to increases in temperature noted for intertidal species. The physiological determinants of upper thermal limits are certain to be multifarious, but heart function stands out as a key contributor to these limits. In porcelain crabs and turban snails, upper lethal temperatures coincide closely with temperatures of heart failure. Such cardiac effects also could contribute to a shift from aerobic to anaerobic processes of ATP generation, which has been proposed as one important mechanism of death from high and low temperatures [Ectothermic species differ widely in thermal tolerance limits and in their abilities to adjust these limits in temperature-adaptive manners. Comparisons of congeneric species from different latitudes and different positions along the subtidal to intertidal gradient have provided important insights into adaptive variation and the threats posed by increased habitat temperature. In what at first view may seem paradoxical, warm-adapted congeners may be more threatened by increased temperatures than their cold-adapted subtidal relatives. This difference stems from two factors: the proximity of current habitat temperatures to upper lethal temperatures (LTeratures ,33. Accl


An earthquake measuring 6.3 on the Richter scale struck the city of Bam in Iran on the 26th of December 2003 at 5.26 A.M. It was devastating, and left over 40,000 dead and around 30,000 injured. The profound tragedy of thousands killed has caused emotional and psychological trauma for tens of thousands of people who have survived. A study was carried out to assess psychological distress among Bam earthquake survivors and factors associated with severe mental health in those who survived the tragedy.This was a population-based study measuring psychological distress among the survivors of Bam earthquake in Iran. Using a multi-stage stratified sampling method a random sample of individuals aged 15 years and over living in Bam were interviewed. Psychological distress was measured using the 12-item General Health Questionnaire (GHQ-12).In all 916 survivors were interviewed. The mean age of the respondents was 32.9 years (SD = 12.4), mostly were males (53%), married (66%) and had secondary school education (50%). Forty-one percent reported they lost 3 to 5 members of their family in the earthquake. In addition the findings showed that 58% of the respondents suffered from severe mental health as measured by the GHQ-12 and this was three times higher than reported psychological distress among the general population. There were significant differences between sub-groups of the study sample with regard to their psychological distress. The results of the logistic regression analysis also indicated that female gender; lower education, unemployment, and loss of family members were associated with severe psychological distress among earthquake victims.The study findings indicated that the amount of psychological distress among earthquake survivors was high and there is an urgent need to deliver mental health care to disaster victims in local medical settings and to reduce negative health impacts of the earthquake adequate psychological counseling is needed for those who survived the tragedy. An earthquake measuring 6.3 on the Richter scale struck the city of Bam in Iran on the 26th of December 2003 at 5.26 A.M. It was devastating, the city was destroyed, left over 40,000 dead and around 30,000 injured, as well as destroying approximately 20,000 homes, leaving more than 45,000 people homeless . The proThis was a population-based study of psychological distress among Bam earthquake survivors aged 15 years and over at five months after the event. The pre-earthquake population of Bam was reported to be 97,000 and surprisingly it was increased to around 120,000 at the post-earthquake period (listing from the authorities). This was due to the fact that because of worry many people living in rural areas were moved to the city of Bam after earthquake. At the time of screening about 80% of people stayed inside their homes and the remaining 20% were moved to four specially designated sites with around 4000 tents. However, to select a representative sample of individuals living in Bam a stratified multi-stage area sampling was applied. The stratification was used because people living in the southern region of the city were less affluent and exposed to more damage as compared to those living in the northern part of the city. Every household or tent within the city had the same probability to be sampled. For the purposes of the first stage, units (census sections) were randomly selected after stratifying by region and size of residence. Then, within each census section the homes and tents to be sampled were selected using the procedure of random routes. Finally the last stage sampling units  were selected randomly from all persons living in the same home or tent. Random sampling was based on a list of all households (N = 23000) derived from the electricity bills. A sample of 1150 households (5%) was selected as explained. However, we were actually able to screen 1000 households, giving a success rate of 87%. The main reason for the failed attempts for contact was due to the fact that the targeted house was uninhabited because the residents had moved out and there was no possibility to trace the survivors. In addition considering logistic difficulties we did not replace them with other households.A team of trained interviewers collected data in a week and all participants were interviewed in their homes or designated camps. However, the data were collected in a disorganized environment and thus data gathering strategy was based on offering help to survivors. Since there was no validated measure of PTSD in the Iranian language, only psychological distress was measured using the Iranian version of the 12-item General Health Questionnaire, GHQ-12 . The scaData were collected from 1000 randomly selected households (600 homes and 400 tents). In total 999 survivors were interviewed, and data for 916 respondents were complete, giving a response rate of 91.7%. The mean age of the respondents was 32.9 years (SD = 12.4), and mostly were male (53%), married (66%) and had secondary school education (50%). The mean score of the survivors on the GHQ-12 was 8.7 (SD = 3.2). The findings showed that 58% of the respondents suffered from severe psychological distress as measured by the GHQ-12. Forty-one percent of the respondents reported that they lost three to five members of their family in the earthquake. The characteristics of the respondents are shown in Table To examine differences in psychological distress among sub-groups of the study sample Student's t-test and one-way analysis of variance were performed. The findings showed that there were significant differences between people with different characteristics. People with old age, females, less educated individuals, divorced or widowed and unemployed respondents, and those with loss of more family members in the earthquake reported more severe psychological distress. The results are shown in Table To investigate factors associated with severe psychological distress all the significant findings from the univariate analysis were entered into the logistic regression analysis. The analysis indicated that being female (OR = 2.73, 95% CI 1.19–6.26, P = 0.02), illiterate (OR = 3.36, 95% CI 1.11–10.2, P = 0.03), unemployed (OR = 4.39, 95% CI 1.56–12.4, P = 0.005), and the loss of more family members (OR for loss of more than five family members = 2.11, 95% CI 1.22–3.61, P = 0.007) were associated with more severe psychological distress. The results are shown in Table This was a population-based study measuring psychological distress among survivors of the Bam earthquake in Iran. Psychological morbidity was higher among females, less educated respondents and unemployed individuals. Perhaps such observation might relate to the fact that these groups of people were exposed to a relatively homogenous set of psychological stressors, leading to relatively homogenous mental health outcomes ,11. In aMost notably it was found that people who lost family members reported significant severe psychological distress compared to those who did not. Also there was a distinct pattern of psychological distress among those who lost their family members in the earthquake showing a dose-response relationship between loss and the risk of more psychological distress. One might argue that the relationship between loss of relatives and psychological distress may be a reflection of the relationship between severity of trauma exposure and psychopathology. Since no other variables reflecting trauma-exposure (e.g. fear during the earthquake, level of damage to home, injury, etc.) were entered in the regression equation, 'loss of relatives' was the only variable tapping that, as there is high correlation between loss of first degree relatives (with whom survivors often share the same roof) and exposure to serious damage or collapse of the house. In addition studies have indicated that loss is a strong determinant of PTSD among earthquake survivors and thus it is argued the observation that the risk of PTSD is linked to the amount of loss is an important issue that needs to be incorporated in the development of any effective preventive strategy . As discPsychological distress among earthquake survivors alongside experience of other problems could be considered a serious issue for people's health status living in such difficult conditions. Evidence suggests that severe earthquakes even can cause long-standing morbidity . HoweverIt is recommended that the mean GHQ score for the whole population of respondents provide a rough guide to the best cut-off threshold . Thus coThe results reported here is an estimate for the overall population experiencing the earthquake. Studies have shown that there is variation in psychological morbidity among earthquake survivors by epicenter proximity and rate of property damage ,18. HoweThe present study had certain methodological limitations. Firstly, the results were based on the GHQ-12 scores and we did not measure PTSD symptoms. In this respect also it is important to acknowledge that the GHQ is a general measure of mental health and it is not a measure of diagnostic depression. Thus, since depression is also common in earthquake survivors, our study is limited in not including a validated diagnostic measure of depression. Secondly, no information was obtained on important trauma-exposure variables such as extent of fear or perceived life-threat during the earthquake, rubble experience, disability or injury, et cetera. Thirdly, data on important demographic variables such as past psychiatric illness, and family psychiatric illness were not collected. However in spite of these limitations, the results from this study are useful. This study is the only paper addressing reports on the psychological consequences of the most disastrous earthquakes of the last 50 years. In addition, to our knowledge the international literature does not contain any study on the psychological status of Iranian earthquake survivors, despite the fact that Iran is an earthquake-prone country. It seems that the future research should carefully assess the psychological distress and disruption experiences of the survivors in order to implement necessary interventions. Given that Iran is a country that suffers catastrophic earthquakes relatively frequently, there is need to develop and validate standard instruments such as PTSD measures to include in the future research.In conclusion the findings from this study indicated that the survivors of Bam earthquake suffer from psychological distress three times higher than the normal population. In addition female gender, lower education, unemployment, and loss of family members were found to be associated with more severe psychological morbidity among survivors. This suggests that to reduce negative health impacts of the earthquake adequate psychological counseling is needed for those who survived the tragedy.GHQ-12: The 12-item General Health Questionnaire; PTSD: Post-traumatic stress disorder; SD: Standard deviation; OR: Odds ratio, CI: Confidence interval.The author(s) declare that they have no competing interests.AM was the principle investigator and contributed to the study design, the data analysis and wrote the paper. All other investigators contributed to the study design, the data collection and analysis. All authors reviewed and contributed to the final manuscript.The pre-publication history for this paper can be accessed here:


Traditional two dimensional journal publishing may no longer be enough, and may be hindering, the interpretation of the complex datasets and analyses generated by microarray experiments in medicine Statistical Applications in Genetics and Molecular Biology (3: article 37), discuss a paradigm for the presentation of complex data—in this case, from microarray analyses. The title of the article, “A Compendium to Ensure Reproducibility in High-Dimensional Classification Tasks,” may not lend itself easily to a clinical audience, but the underlying message to clinicians could not be more important: that, currently, studies involving large datasets, especially ones that have a clinical outcome, are so poorly reported (or possibly so poorly done) that many are not reproducible. This problem was also the topic of a recent meeting in Heidelberg, “Best Practice in Microarray Studies” (http://www.biometrie.uni-heidelberg.de/workshops/bestpractice/index.htm).“Publishing results in traditional paper based way in a journal hides too much information.” This is the verdict of Markus Ruschhaupt and colleagues who, in a paper in As microarrays have become mainstream research tools in biology and medicine, the large datasets and complex analyses from these studies have presented challenges: for authors in analyzing the data, for reviewers and editors in deciding on the suitability of papers for publication, for journals in determining how much data needs to be presented within the paper itself, for other researchers in reproducing the data, and, finally, for readers in deciding how to assess the data presented. The results from several high-profile papers have already proved difficult to reproduce, even by those with sufficient time and computing expertise.Where do such analyses leave the new science of molecular pathology? Ruschhaupt and colleagues comment that “the literature on the induction of prognostic profiles from microarray studies is a methodological wasteland.” Much the same could be said of other applications of molecular biology to clinical samples. A systematic review of molecular and biological tumor markers in neuroblastoma (Clin Cancer Res 10: 4–12) found that its conclusions were limited by “small sample sizes, poor statistical reporting, large heterogeneity across studies…and publication bias.” John Ioannidis and colleagues (Lancet 362: 1439–1444) did a similar analysis of 30 microarray studies with major clinical outcomes in cancer. They showed that the studies were small—median sample size was 25 patients, and validation was incomplete in most studies. They recommended that molecular prognostic studies be classified as phase 1 (early exploratory probing associations), phase 2 , or phase 3 (large confirmatory studies with pre-stated hypotheses and precise quantification of the magnitude of the effect), and that only studies that had undergone phase 3 testing should be considered robust enough for use in clinical practice. Most current studies should be considered as phase 1 or, at best, phase 2.So, despite considerable hype, the published studies are far from the level of evidence that would be accepted for virtually any other medical test. In a review in 2003 (Hematology [Am Soc Hematol Educ Program] 2003: 279–293), Rita Braziel and colleagues concluded, “rapid identification and neutralization of spurious results is essential to prevent them from becoming accepted facts.”But these problems are not new in medical research. In 1994 (BMJ 308: 283–284), Doug Altman, who was instrumental in developing the CONSORT guidelines for reporting of clinical trials, said that “huge sums of money are spent annually on research that is seriously flawed through the use of inappropriate designs, unrepresentative samples, small samples, incorrect methods of analysis, and faulty interpretation,” and “that quality control needs to be built in from the start rather than the failures being discarded.”PLoS Medicine is keen to work with authors towards making such reporting possible. But although the time might have gone when the two-dimensional journal article could suffice for complex papers, clinicians should nonetheless apply the same critical assessment that they would for any other clinical tool. If a result is too good to be true, it probably is.So how can we ensure that the wealth of data pouring out of microarray and other molecular diagnostic studies is turned into meaningful knowledge? The Microarray Gene Expression Data Society has proposed a set of guidelines (MIAME) for the reporting of microarray data, and that all such data should be deposited in public databases. But as Ruschhaupt and others have shown, disclosure of results and data is not enough, since there is little consensus on the appropriate statistical analyses and many are developed on a case by case basis, which may not be reproducible, even by the authors. Some researchers advocate the use of standard statistical packages, which allows the reader to repeat an entire analysis quickly and, hence, assess the robustness of the results. Some authors have produced a transcript of their statistical analyses as a supplement to their articles (e.g., Nucleic Acids Res 32: e50). At the very least authors should have a protocol with a prespecified plan for patient selection and statistical analysis—accepted practice for clinical trials, but not yet for other medical research. An ultimate aim for reporting would be the type of compendium discussed by Ruschhaupt and colleagues—“an interactive document that bundles primary data, statistical processing methods, figures, and derived data together with the textual documentation and conclusions.” One such compendium is illustrated in a paper by Robert Gentleman (Stat Appl Genet Mol Biol 4: article 2).